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Sample records for acid lna antisense

  1. Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

    PubMed

    Moschos, Sterghios A; Frick, Manfred; Taylor, Bruce; Turnpenny, Paul; Graves, Helen; Spink, Karen G; Brady, Kevin; Lamb, David; Collins, David; Rockel, Thomas D; Weber, Markus; Lazari, Ovadia; Perez-Tosar, Luis; Fancy, Sally A; Lapthorn, Chris; Green, Martin X; Evans, Steve; Selby, Matthew; Jones, Gareth; Jones, Lyn; Kearney, Sarah; Mechiche, Houria; Gikunju, Diana; Subramanian, Romesh; Uhlmann, Eugen; Jurk, Marion; Vollmer, Jörg; Ciaramella, Giuseppe; Yeadon, Michael

    2011-12-01

    Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

  2. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    PubMed

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  3. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.

    PubMed

    Seth, Punit P; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R; Bhat, Balkrishen; Swayze, Eric E

    2012-01-01

    We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012. PMID:23344239

  4. PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

    PubMed

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

    2012-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

  5. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  6. X-ray Crystal Structure of a Locked Nucleic Acid (LNA) Duplex Composed of a Palindromic 10-mer DNA Strand Containing One LNA Thymine Monomer

    SciTech Connect

    Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J.

    2010-03-05

    Locked nucleic acid (LNA), a recently introduced nucleic acid analogue with a bicyclic 2'-O,4'-C-methylene linked furanose sugar, exhibits enhanced affinities for DNA and RNA relative to the corresponding oligodeoxyribonucleotides and oligoribonucleotides; we report the first crystal structure of an LNA unit incorporated in an oligonucleotide duplex. The structure at 1.4 {angstrom} resolution of the DNA-LNA decamer duplex with one LNA thymine monomer per strand provides a detailed view of the conformation and hydration of locked nucleic acid residues in a duplex A-form. Our study provides a first look at the conformational properties of LNA in a crystal structure at relatively high resolution. The main characteristics of the structure are the standard A-type conformation induced by LNA residues and the capacity of the 2'-oxygen that is part of the bicyclic sugar framework to engage in a least two hydrogen bonds to water molecules. Circular dichroism spectra (CD) of LNA-LNA duplexes in solution indicated that such duplexes appear to adopt a conformation that closely resembles the A-form geometry of RNA-RNA duplexes. However, these spectra also manifested subtle differences between the two species (data not shown). The present analysis of a duplex with only a single LNA residue per strand does not provide any insight into potential conformational differences between LNA and RNA duplexes. Attempts to determine a crystal structure of a completely modified LNA-LNA duplex are underway.

  7. Chemical modification study of antisense gapmers.

    PubMed

    Stanton, Robert; Sciabola, Simone; Salatto, Christopher; Weng, Yan; Moshinsky, Debra; Little, Jeremy; Walters, Evan; Kreeger, John; DiMattia, Debra; Chen, Tracy; Clark, Tracey; Liu, Mei; Qian, Jessie; Roy, Marc; Dullea, Robert

    2012-10-01

    A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic--using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.

  8. Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

    PubMed

    Vilas Boas, Diana; Almeida, Carina; Sillankorva, Sanna; Nicolau, Ana; Azeredo, Joana; Azevedo, Nuno F

    2016-01-01

    Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations. PMID:26813295

  9. Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

    PubMed

    Vilas Boas, Diana; Almeida, Carina; Sillankorva, Sanna; Nicolau, Ana; Azeredo, Joana; Azevedo, Nuno F

    2016-01-01

    Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

  10. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    PubMed

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  11. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

    PubMed Central

    Kasuya, Takeshi; Hori, Shin-ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  12. Optimization of an AMBER Force Field for the Artificial Nucleic Acid, LNA, and Benchmarking with NMR of L(CAAU)

    PubMed Central

    2013-01-01

    Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced. PMID:24377321

  13. Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

    PubMed

    Poongavanam, Vasanthanathan; Madala, Praveen K; Højland, Torben; Veedu, Rakesh N

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  14. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

    PubMed Central

    Moreno, Pedro M. D.; Geny, Sylvain; Pabon, Y. Vladimir; Bergquist, Helen; Zaghloul, Eman M.; Rocha, Cristina S. J.; Oprea, Iulian I.; Bestas, Burcu; Andaloussi, Samir EL; Jørgensen, Per T.; Pedersen, Erik B.; Lundin, Karin E.; Zain, Rula; Wengel, Jesper; Smith, C. I. Edvard

    2013-01-01

    In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes. PMID:23345620

  15. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  16. Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

    PubMed Central

    Poongavanam, Vasanthanathan; Madala, Praveen K.; Højland, Torben; Veedu, Rakesh N.

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  17. An Exocyclic Methylene Group Acts As a Bioisostere of the 2′-Oxygen Atom in LNA

    SciTech Connect

    Seth, Punit P.; Allerson, Charles R.; Berdeja, Andres; Siwkowski, Andrew; Pallan, Pradeep S.; Gaus, Hans; Prakash, Thazha P.; Watt, Andrew T.; Egli, Martin; Swayze, Eric E.

    2010-12-07

    We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2{prime}-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2{prime}-position and a propynyl group at the C-4{prime} position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2{prime}-deoxyribose residue of the 5{prime}-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2{prime}-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

  18. Synthesis of selenomethylene-locked nucleic acid (SeLNA)-modified oligonucleotides by polymerases.

    PubMed

    Wheeler, Megan; Chardon, Antoine; Goubet, Astrid; Morihiro, Kunihiko; Tsan, Sze Yee; Edwards, Stacey L; Kodama, Tetsuya; Obika, Satoshi; Veedu, Rakesh N

    2012-11-18

    Enzymatic recognition of SeLNA nucleotides was investigated. KOD XL DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of SeLNA-modified DNA templates was also efficiently achieved by Phusion and KOD XL DNA polymerases. PMID:23042489

  19. Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents

    PubMed Central

    Torres, Adrian G.; Threlfall, Richard N.

    2011-01-01

    Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics. PMID:22567190

  20. Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses.

    PubMed

    Chen, Ru; Gao, Xiao-Bo; Yu, Xiao-Lu; Song, Chang-Xu; Qiu, Yang

    2016-02-01

    A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay. PMID:26615807

  1. Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

    PubMed

    Højland, Torben; Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2012-01-01

    We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide. PMID:22679529

  2. Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

    PubMed Central

    Geny, Sylvain; Moreno, Pedro M. D.; Krzywkowski, Tomasz; Gissberg, Olof; Andersen, Nicolai K.; Isse, Abdirisaq J.; El-Madani, Amro M.; Lou, Chenguang; Pabon, Y. Vladimir; Anderson, Brooke A.; Zaghloul, Eman M.; Zain, Rula; Hrdlicka, Patrick J.; Jørgensen, Per T.; Nilsson, Mats; Lundin, Karin E.; Pedersen, Erik B.; Wengel, Jesper; Smith, C. I. Edvard

    2016-01-01

    Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson–Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson–Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2′-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context. PMID:26857548

  3. Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes.

    PubMed

    Geny, Sylvain; Moreno, Pedro M D; Krzywkowski, Tomasz; Gissberg, Olof; Andersen, Nicolai K; Isse, Abdirisaq J; El-Madani, Amro M; Lou, Chenguang; Pabon, Y Vladimir; Anderson, Brooke A; Zaghloul, Eman M; Zain, Rula; Hrdlicka, Patrick J; Jørgensen, Per T; Nilsson, Mats; Lundin, Karin E; Pedersen, Erik B; Wengel, Jesper; Smith, C I Edvard

    2016-03-18

    Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context. PMID:26857548

  4. Quantum mechanical studies of DNA and LNA.

    PubMed

    Koch, Troels; Shim, Irene; Lindow, Morten; Ørum, Henrik; Bohr, Henrik G

    2014-04-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs.

  5. Quantum Mechanical Studies of DNA and LNA

    PubMed Central

    Shim, Irene; Lindow, Morten; Ørum, Henrik

    2014-01-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs. PMID:24491259

  6. Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes.

    PubMed

    Palmano, Sabrina; Mulholland, Vincent; Kenyon, David; Saddler, Gerry S; Jeffries, Colin

    2015-01-01

    Phytoplasma infections are regularly reported worldwide, and concerns about their threats on agricultural production, especially in relation to global climate change, are increasing. Sensitive and reliable detection methods are important to ensure that propagation material is free of phytoplasma infection and for epidemiological studies that may provide information to limit the extent of phytoplasma diseases and to prevent large-scale crop losses. The detection method described here uses LNA chemistry in real-time PCR. It has been developed and validated for use on potatoes, and its sensitivity and specificity make it suitable for use in postentry potato quarantine and initiation of potato nuclear stocks to ensure that material is phytoplasma-free. PMID:25981250

  7. [Control of gene expression by antisense nucleic acids].

    PubMed

    Lebleu, B; Clarenc, J P; Degols, G; Leonetti, J P; Milhaud, P

    1992-01-01

    The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.

  8. A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    PubMed

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  9. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    PubMed

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice. PMID:24336348

  10. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    PubMed

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

  11. Application of Locked Nucleic Acid (LNA) Oligonucleotide–PCR Clamping Technique to Selectively PCR Amplify the SSU rRNA Genes of Bacteria in Investigating the Plant-Associated Community Structures

    PubMed Central

    Ikenaga, Makoto; Sakai, Masao

    2014-01-01

    The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide–PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3′ end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide–PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes. PMID:25030190

  12. Application of Locked Nucleic Acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures.

    PubMed

    Ikenaga, Makoto; Sakai, Masao

    2014-09-17

    The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide-PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3' end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide-PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes.

  13. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

    PubMed Central

    Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

    2016-01-01

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

  14. Potent Antibacterial Antisense Peptide–Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    PubMed Central

    Ghosal, Anubrata

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce antisense peptide–peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide–PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)4-Ahx-βala or the H-(R-Ahx)6-βala peptide exhibited complete growth inhibition of P. aeruginosa strains PA01, PA14, and LESB58 at 1–2 μM concentrations without any indication of bacterial membrane disruption (even at 20 μM), and resulted in specific reduction of the targeted mRNA levels. One of the four compounds showed clear bactericidal activity while the other significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections. PMID:23030590

  15. PEI-complexed LNA antiseeds as miRNA inhibitors

    PubMed Central

    Thomas, Maren; Lange-Grünweller, Kerstin; Dayyoub, Eyas; Bakowsky, Udo; Weirauch, Ulrike; Aigner, Achim; Hartmann, Roland K.; Grünweller, Arnold

    2012-01-01

    Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications. PMID:22894918

  16. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

    Andersen, Nicolai K.; Anderson, Brooke A.; Wengel, Jesper

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

  17. Poly(propylacrylic acid) enhances cationic lipid mediated delivery of antisense oligonucleotides

    PubMed Central

    Lee, Li Kim; Williams, Charity L.; Devore, David; Roth, Charles M.

    2008-01-01

    The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved two- to threefold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides. PMID:16677032

  18. An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)

    PubMed Central

    Mehiri, Mohamed; Upert, Gregory; Tripathi, Snehlata; Di Giorgio, Audrey; Condom, Roger

    2008-01-01

    With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA–TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC50 value of 1 μM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test. PMID:18707540

  19. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    PubMed

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  20. Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

    PubMed Central

    Zhang, Ling; Yang, Xiang-dong; Zhang, Yuan-yu; Yang, Jing; Qi, Guang-xun; Guo, Dong-quan; Xing, Guo-jie; Yao, Yao; Xu, Wen-jing; Li, Hai-yun; Li, Qi-yun; Dong, Ying-shan

    2014-01-01

    The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts. PMID:25197629

  1. Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.

    PubMed

    Zhang, Ling; Yang, Xiang-Dong; Zhang, Yuan-Yu; Yang, Jing; Qi, Guang-Xun; Guo, Dong-Quan; Xing, Guo-Jie; Yao, Yao; Xu, Wen-Jing; Li, Hai-Yun; Li, Qi-Yun; Dong, Ying-Shan

    2014-01-01

    The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

  2. Non-covalent complexes of folic acid and oleic acid conjugated polyethylenimine: An efficient vehicle for antisense oligonucleotide delivery

    PubMed Central

    Yang, Shuang; Yang, Xuewei; Liu, Yan; Zheng, Bin; Meng, Lingjun; Lee, Robert J.; Xie, Jing; Teng, Lesheng

    2016-01-01

    Polyethylenimine (PEI) was conjugated to oleic acid (PEI-OA) and evaluated as a delivery agent for LOR-2501, an antisense oligonucleotide against ribonucleotide reductase R1 subunit. PEI-OA/LOR-2501 complexes were further coated with folic acid (FA/PEI-OA/LOR-2501) and evaluated in tumor cells. The level of cellular uptake of FA/PEI-OA/LOR-2501 was more than double that of PEI/LOR-2501 complexes, and was not affected by the expression level of folate receptor (FR) on the cell surface. Efficient delivery was seen in several cell lines. Furthermore, pathway specific cellular internalization inhibitors and markers were used to reveal the principal mechanism of cellular uptake. FA/PEI-OA/LOR-2501 significantly induced the downregulation of R1 mRNA and R1 protein. This novel formulation of FA/PEI-OA provides a reliable and highly efficient method for delivery of oligonucleotide and warrants further investigation. PMID:26263216

  3. Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes.

    PubMed Central

    Hagenbuch, B; Scharschmidt, B F; Meier, P J

    1996-01-01

    A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver. PMID:8670169

  4. Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development.

    PubMed

    Yu, Xiyan; Wang, Xiufeng; Zhang, Wenqian; Qian, Tingting; Tang, Guimin; Guo, Yankui; Zheng, Chengchao

    2008-01-01

    To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.

  5. Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy

    PubMed Central

    Wojtkowiak-Szlachcic, Agnieszka; Taylor, Katarzyna; Stepniak-Konieczna, Ewa; Sznajder, Lukasz J.; Mykowska, Agnieszka; Sroka, Joanna; Thornton, Charles A.; Sobczak, Krzysztof

    2015-01-01

    Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3′-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUGexp) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUGexp/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUGexp foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUGexp-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1. PMID:25753670

  6. Sequence motifs associated with hepatotoxicity of locked nucleic acid—modified antisense oligonucleotides

    PubMed Central

    Burdick, Andrew D.; Sciabola, Simone; Mantena, Srinivasa R.; Hollingshead, Brett D.; Stanton, Robert; Warneke, James A.; Zeng, Ming; Martsen, Elena; Medvedev, Alexander; Makarov, Sergei S.; Reed, Lori A.; Davis, John W.; Whiteley, Laurence O.

    2014-01-01

    Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing. PMID:24550163

  7. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    PubMed

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  8. In vitro incorporation of LNA nucleotides.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  9. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  10. Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves.

    PubMed

    Fan, L; Zheng, S; Wang, X

    1997-12-01

    Membrane disruption has been proposed to be a key event in plant senescence, and phospholipase D (PLD; EC 3.1.4.4) has been thought to play an important role in membrane deterioration. We recently cloned and biochemically characterized three different PLDs from Arabidopsis. In this study, we investigated the role of the most prevalent phospholipid-hydrolyzing enzyme, PLD alpha, in membrane degradation and senescence in Arabidopsis. The expression of PLD alpha was suppressed by introducing a PLD alpha antisense cDNA fragment into Arabidopsis. When incubated with abscisic acid and ethylene, leaves detached from the PLD alpha-deficient transgenic plants showed a slower rate of senescence than did those from wild-type and transgenic control plants. The retardation of senescence was demonstrated by delayed leaf yellowing, lower ion leakage, greater photosynthetic activity, and higher content of chlorophyll and phospholipids in the PLD alpha antisense leaves than in those of the wild type. Treatment of detached leaves with abscisic acid and ethylene stimulated PLD alpha expression, as indicated by increases in PLD alpha mRNA, protein, and activity. In the absence of abscisic acid and ethylene, however, detached leaves from the PLD alpha-deficient and wild-type plants showed a similar rate of senescence. In addition, the suppression of PLD alpha did not alter natural plant growth and development. These data suggest that PLD alpha is an important mediator in phytohormone-promoted senescence in detached leaves but is not a direct promoter of natural senescence. The physiological relevance of these findings is discussed.

  11. The LNA VPH characterization experiment

    NASA Astrophysics Data System (ADS)

    Ribeiro, Flávio F.; Katime-Santrich, Orlando J.; Gneiding, Clemens D.; Castilho, Bruno V.; Campos, Rodrigo P.; Nicolau, Rogério A.

    2008-07-01

    The use of Volume Phase Holographic (VPH) gratings in astronomy is increasing worldwide due to its high efficiency, flexibility in manufacturing and lower costs. For example 3 of 4 SOAR Telescope spectrographs are based on VPH gratings. Following the growth in this technology use, tools are needed to characterize these gratings for their physical and diffraction efficiency properties. We developed, at Laboratorio Nacional de Astrofisica / MCT (LNA), Brazil, an assembly for characterization of VPH gratings. The relative efficiency of the gratings can be measured for specific angles or scanned through the grating operation angles. Furthermore surface flatness and mounting stress effects are measured using interferometric techniques. We present the experiment design and characteristics, describe the measurement procedures and show the characterization results for some gratings of the SOAR Telescope spectrograph STELES.

  12. Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates

    PubMed Central

    Gallo Cantafio, Maria Eugenia; Nielsen, Boye Schnack; Mignogna, Chiara; Arbitrio, Mariamena; Botta, Cirino; Frandsen, Niels M; Rolfo, Christian; Tagliaferri, Pierosandro; Tassone, Pierfrancesco; Di Martino, Maria Teresa

    2016-01-01

    Locked nucleic acid (LNA) oligonucleotides have been successfully used to efficiently inhibit endogenous small noncoding RNAs in vitro and in vivo. We previously demonstrated that the direct miR-221 inhibition by the novel 13-mer LNA-i-miR-221 induces significant antimyeloma activity and upregulates canonical miR-221 targets in vitro and in vivo. To evaluate the LNA-i-miR-221 pharmacokinetics and pharmacodynamics, novel assays for oligonucleotides quantification in NOD.SCID mice and Cynomolgus monkeys (Macaca fascicularis) plasma, urine and tissues were developed. To this aim, a liquid chromatography/mass spectrometry method, after solid-phase extraction, was used for the detection of LNA-i-miR-221 in plasma and urine, while a specific in situ hybridization assay for tissue uptake analysis was designed. Our analysis revealed short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. Up to 3 weeks, LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, together with p27 target upregulation. Importantly, no toxicity in the pilot monkey study was observed. Overall, our findings indicate the suitability of LNA-i-miR-221 for clinical use and we provide here pilot data for safety analysis and further development of LNA-miRNA-based therapeutics for human cancer. PMID:27327137

  13. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  14. Sequence-Specific Peptide Nucleic Acid-Based Antisense Inhibitors of TEM-1 β-Lactamase and Mechanism of Adaptive Resistance.

    PubMed

    Courtney, Colleen M; Chatterjee, Anushree

    2015-06-12

    The recent surge of drug-resistant superbugs and shrinking antibiotic pipeline are serious challenges to global health. In particular, the emergence of β-lactamases has caused extensive resistance against the most frequently prescribed class of β-lactam antibiotics. Here, we develop novel synthetic peptide nucleic acid-based antisense inhibitors that target the start codon and ribosomal binding site of the TEM-1 β-lactamase transcript and act via translation inhibition mechanism. We show that these antisense inhibitors are capable of resensitizing drug-resistant Escherichia coli to β-lactam antibiotics exhibiting 10-fold reduction in the minimum inhibitory concentration (MIC). To study the mechanism of resistance, we adapted E. coli at MIC levels of the β-lactam/antisense inhibitor combination and observed a nonmutational, bet-hedging based adaptive antibiotic resistance response as evidenced by phenotypic heterogeneity as well as heterogeneous expression of key stress response genes. Our data show that both the development of new antimicrobials and an understanding of cellular response during the development of tolerance could aid in mitigating the impending antibiotic crisis. PMID:27622741

  15. Viral escape from antisense RNA.

    PubMed

    Bull, J J; Jacobson, A; Badgett, M R; Molineux, I J

    1998-05-01

    RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition. PMID:9643550

  16. Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting

    PubMed Central

    Kumar, T. Santhosh; Madsen, Andreas S.; Østergaard, Michael E.; Sau, Sujay P.; Wengel, Jesper; Hrdlicka, Patrick J.

    2010-01-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting. PMID:19108636

  17. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

    PubMed Central

    Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé

    2015-01-01

    Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni. PMID:26452552

  18. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium.

    PubMed

    Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé; Grandvalet, Cosette

    2015-10-09

    Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.

  19. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  20. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    PubMed Central

    Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

    2002-01-01

    α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

  1. Gibberellic acid, synthetic auxins, and ethylene differentially modulate alpha-L-Arabinofuranosidase activities in antisense 1-aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs.

    PubMed

    Sozzi, Gabriel O; Greve, L Carl; Prody, Gerry A; Labavitch, John M

    2002-07-01

    Alpha-L-Arabinofuranosidases (alpha-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different alpha-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. alpha-Af I and II are active throughout fruit ontogeny. alpha-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. alpha-Af II activity accounts for over 80% of the total alpha-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, alpha-Af III is ethylene dependent and specifically active during ripening. alpha-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas alpha-Af II and III acted on Na(2)CO(3)-soluble pectins. Different alpha-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. alpha-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only alpha-Af III activity. Results suggest that tomato alpha-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.

  2. Enzymatic polymerisation involving 2'-amino-LNA nucleotides.

    PubMed

    Johannsen, Marie W; Veedu, Rakesh N; Madsen, Andreas Stahl; Wengel, Jesper

    2012-05-15

    The triphosphate of the thymine derivative of 2'-amino-LNA (2'-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2'-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2'-Amino-LNA-T in the template and 2'-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2'-amino-LNA-T nucleotides. PMID:22503454

  3. Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice

    PubMed Central

    Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Nakatani, Moeka; Wada, Shunsuke; Yasuhara, Hidenori; Narukawa, Keisuke; Sasaki, Kiyomi; Shibata, Masa-Aki; Torigoe, Hidetaka; Yamaoka, Tetsuji; Imanishi, Takeshi; Obika, Satoshi

    2012-01-01

    Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia. PMID:23344002

  4. Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3;#8242;-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

    SciTech Connect

    Egli, Martin; Pallan, Pradeep S.; Allerson, Charles R.; Prakash, Thazha P.; Berdeja, Andres; Yu, Jinghua; Lee, Sam; Watt, Andrew; Gaus, Hans; Bhat, Balkrishen; Swayze, Eric E.; Seth, Punit P.

    2012-03-16

    The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F {hor_ellipsis} H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py) {hor_ellipsis} H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

  5. In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides.

    PubMed

    Chen, Ying-Shan; Alany, Raid G; Young, Simon A; Green, Colin R; Rupenthal, Ilva D

    2011-01-01

    The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.

  6. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

    PubMed

    Darfeuille, Fabien; Hansen, Jens Bo; Orum, Henrik; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2004-01-01

    One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets. PMID:15181175

  7. Upping the Antisense Ante.

    ERIC Educational Resources Information Center

    Weiss, Rick

    1991-01-01

    Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)

  8. Polymerase-directed synthesis of C5-ethynyl locked nucleic acids.

    PubMed

    Veedu, Rakesh N; Burri, Harsha V; Kumar, Pawan; Sharma, Pawan K; Hrdlicka, Patrick J; Vester, Birte; Wengel, Jesper

    2010-11-15

    Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5'-triphosphate as substrate for RNA transcripts. PMID:20932755

  9. Antisense therapeutics in oncology: current status

    PubMed Central

    Farooqi, Ammad Ahmad; Rehman, Zia ur; Muntane, Jordi

    2014-01-01

    There is increasing progress in translational oncology and tremendous breakthroughs have been made as evidenced by preclinical and clinical trials. Data obtained from high-throughput technologies are deepening our understanding about the molecular and gene network in cancer cells and rapidly emerging in vitro and in vivo evidence is highlighting the role of antisense agents as specific inhibitors of the expression of target genes, thus modulating the response of cancer cells to different therapeutic strategies. Much information is continuously being added into various facets of molecular oncology and it is now understood that overexpression of antiapoptotic proteins, oncogenes, oncogenic microRNAs (miRNA), and fusion proteins make cancer cells difficult to target. Delivery of antisense oligonucleotides has remained a challenge and technological developments have helped in overcoming hurdles by improving the ability to penetrate cells, effective and targeted binding to gene sequences, and downregulation of target gene function. Different delivery systems, including stable nucleic acid lipid particles, have shown potential in enhancing the delivery of cargo to the target site. In this review, we attempt to summarize the current progress in the development of antisense therapeutics and their potential in medical research. We partition this multicomponent review into introductory aspects about recent breakthroughs in antisense therapeutics. We also discuss how antisense therapeutics have shown potential in resensitizing resistant cancer cells to apoptosis by targeted inhibition of antiapoptotic proteins, oncogenic miRNAs, and BCR-ABL. PMID:25395862

  10. Sense-, antisense- and RNAi-4CL1 regulate soluble phenolic acids, cell wall components and growth in transgenic Populus tomentosa Carr.

    PubMed

    Tian, Xiaoming; Xie, Jin; Zhao, Yanling; Lu, Hai; Liu, Shichang; Qu, Long; Li, Jianmei; Gai, Ying; Jiang, Xiangning

    2013-04-01

    Regulation of lignin biosynthesis affects plant growth and wood properties. Transgenic downregulation of 4-coumarate:coenzyme A ligase (4CL, EC 6.2.1.12) may reduce lignin content in cell walls, which could improve the qualities of pulp in papermaking and increase the efficiency of bioenergy applications. To determine the effects of Ptc4CL1 on lignin biosynthesis and plant growth, Populus tomentosa Carr. was transformed using sense-, antisense-, and RNAi-4CL1 genes. The growth properties, gene expression, enzyme activity, lignin content and composition and content of soluble phenolic acids were investigated in 1-year-old field-grown transgenic poplar trees. Transgenic up- and down-regulation of 4CL1 altered lignin content and composition in transgenic poplars, but there were no negative effects on the growth of transgenic plants. In addition, the severe changes in auxin observed in transgenic lines led to significantly enhanced growth performance. Furthermore, lignin content was tightly correlated with the alteration of 4CL1 enzymatic activity, which was correlated with 4CL1 gene expression. A significant increase in S units in lignin with a slight increase in sinapic acid was observed in 4CL1 down-regulated transgenic poplars. These results suggest that 4CL1 is a traffic control gene in monolignol biosynthesis and confirm that 4CL1 activity has been implicated with sinapoyl activation. Finally, our data demonstrate that there is cross-correlation among 4CL1 gene expression, 4CL1 enzyme activity, soluble phenolic acid, lignin monomer biosynthesis, and lignin content. PMID:23434928

  11. Making sense of antisense

    SciTech Connect

    Moffat, A.S.

    1991-08-02

    Out in the San Joaquin Valley of California grows a field of tomato plants that look like ordinary tomato plants. But these tomatoes are special - one of the first fruits of a new technology that may revolutionize not just commercial plant development but human medicine as well. The new technology uses novel RNAs, called antisense RNAs, to block the activity of specific genes. At first, researchers were mainly interested in antisense RNA as a tool for probing gene function. In the late 1970s, when the technology was first developed, molecular biologists didn't have a good way of mutating genes in the cells of higher organisms so that they could see what happens when the gene activity is lost. Antisense technology, in effect, provided a way of doing that. But the biotechnology industry soon recognized the immense practical potential of a technique that could be used to knock out the activity of bad genes. To make the tomato plants, for example, plant scientists used antisense RNAs to shut off the expression of the gene encoding an enzyme that makes tomatoes mushy, thereby yielding a product that may travel better and last longer on grocery shelves. Recent work by various labs suggests that it may be possible to design antisense compounds that inhibit the activity of viral genes or of the oncogenes thought to contribute to cancer development, without affecting normal cellular genes. That raises the possibility that the technology might aid in producing better, more selective drugs to treat viral diseases, including AIDS, and cancer.

  12. Interplay of LNA and 2'-O-methyl RNA in the structure and thermodynamics of RNA hybrid systems: a molecular dynamics study using the revised AMBER force field and comparison with experimental results.

    PubMed

    Yildirim, Ilyas; Kierzek, Elzbieta; Kierzek, Ryszard; Schatz, George C

    2014-12-11

    When used in nucleic acid duplexes, locked nucleic acid (LNA) and 2'-O-methyl RNA residues enhance the duplex stabilities, and this makes it possible to create much better RNA aptamers to target specific molecules in cells. Thus, LNA and 2'-O-methyl RNA residues are finding increasingly widespread use in RNA-based therapeutics. Herein, we utilize molecular dynamics (MD) simulations and UV melting experiments to investigate the structural and thermodynamic properties of 13 nucleic acid duplexes, including full DNA, RNA, LNA, and 2'-O-methyl RNA duplexes as well as hybrid systems such as LNA:RNA, 2'-O-methyl RNA:RNA, LNA/2'-O-methyl RNA:RNA, and RNA/2'-O-methyl RNA:RNA duplexes. The MD simulations are based on a version of the Amber force field revised specifically for RNA and LNA residues. Our results indicate that LNA and 2'-O-methyl RNA residues have two different hybridization mechanisms when included in hybrid duplexes with RNA wherein the former underwinds while the latter overwinds the duplexes. These computational predictions are supported by X-ray structures of LNA and 2'-O-methyl RNA duplexes that were recently presented by different groups, and there is also good agreement with the measured thermal stabilities of the duplexes. We find out that the "underwinding" phenomenon seen in LNA and LNA:RNA hybrid duplexes happens due to expansion of the major groove widths (Mgw) of the duplexes that is associated with decrease in the slide and twist values in base-pair steps. In contrast, 2'-O-methyl RNA residues in RNA duplexes slightly overwind the duplexes while the backbone is forced to stay in C3'-endo. Moreover, base-pair stacking in the LNA and LNA:RNA hybrid systems is gradually reduced with the inclusion of LNA residues in the duplexes while no such effect is seen in the 2'-O-methyl RNA systems. Our results show how competition between base stacking and structural rigidity in these RNA hybrid systems influences structures and stabilities. Even though both

  13. Synthesis and Antisense Properties of Fluoro Cyclohexenyl Nucleic Acid (F-CeNA) – A Nuclease Stable Mimic of 2′-Fluoro RNA

    PubMed Central

    Seth, Punit P.; Yu, Jinghua; Jazayeri, Ali; Pallan, Pradeep S.; Allerson, Charles R; Østergaard, Michael E.; Liu, Fengwu; Herdewijn, Piet; Egli, Martin; Swayze, Eric E.

    2013-01-01

    We report the design and synthesis of 2′-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2′-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3′-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2′-fluoro RNA (FRNA), 2′-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the 3H2 and 2H3 conformations, similar to the C3′-endo/C2′-endo conformation equilibrium seen in natural furanose nucleosides. In the 2H3 conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3′-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the 3H2 (C3′-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)]2. In an animal experiment, a 16-mer F

  14. Undetected antisense tRNAs in mitochondrial genomes?

    PubMed Central

    2010-01-01

    Background The hypothesis that both mitochondrial (mt) complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding) is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and Dr Andrei S Rodin (nominated

  15. Dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the marine finfish Larimichthys crocea: dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the large yellow croaker.

    PubMed

    Zuo, Rantao; Mai, Kangsen; Xu, Wei; Turchini, Giovanni M; Ai, Qinghui

    2015-02-01

    Whilst aquaculture feed is increasingly formulated with the inclusion of plant oils replacing fish oil, and increasing research effort has been invested in understanding the metabolic effects of reduced dietary n-3 long chain poly unsaturated fatty acids (n-3 LC-PUFA), relatively little information is available on the potential direct metabolic roles of dietary alpha-linolenic acid (ALA, 18:3n-3) and alpha-linolenic acid/linoleic acid (LNA, 18:2n-6) ratio in cultured marine finfish species. In this study, four plant oil based diets, with varying ALA/LNA ratio (0.0, 0.5, 1.0 and 1.5) were fed to juvenile large yellow croakers (Larimichthys crocea) and compared to a fish oil-based control diet (CD) to evaluate the resulting effects on growth, nonspecific immunity, anti-oxidant capacity and related gene expression. High dietary LNA negatively impacted fish growth performance, nonspecific immunity and antioxidant capacity, but growth and immunity were maintained to levels comparable to CD by increasing the ratio of dietary ALA/LNA. The over-expression of genes associated with inflammation (cyclooxygenase-2 and interleukin-1β) and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in croakers fed high concentrations of LNA were reduced to levels comparable to those fed CD by increasing dietary ALA/LNA. This study showed that dietary ALA, by increasing the overall n-3/n-6 PUFA ratio, exerts direct anti-inflammatory and antioxidant effects, similar to those exerted by dietary n-3 LC-PUFA.

  16. Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamer

    PubMed Central

    Förster, Charlotte; Oberthuer, Dominik; Gao, Jiang; Eichert, André; Quast, Frederick G.; Betzel, Christian; Nitsche, Andreas; Erdmann, Volker A.; Fürste, Jens P.

    2009-01-01

    Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as β-d-2′-O,4′-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The β-d-2′-O,4′-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-­mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an ‘all-locked’ LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl2, MgCl2, MnCl2, CaCl2, CoCl2 and ZnSO4. Preliminary X-ray diffraction data were collected and processed to 2.8 Å resolution. The LNA crystallized in space group P65, with unit-cell parameters a = 50.11, b = 50.11, c = 40.72 Å. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17 Å3 Da−1, which implies a solvent content of 70.15%. PMID:19724123

  17. The effect of linoleic acid on the whole body synthesis rates of polyunsaturated fatty acids from α-linolenic acid and linoleic acid in free-living rats.

    PubMed

    Domenichiello, Anthony F; Kitson, Alex P; Chen, Chuck T; Trépanier, Marc-Olivier; Stavro, P Mark; Bazinet, Richard P

    2016-04-01

    Docosahexaenoic acid (DHA) is thought to be important for brain function. The main dietary source of DHA is fish, however, DHA can also be synthesized from precursor omega-3 polyunsaturated fatty acids (n-3 PUFA), the most abundantly consumed being α-linolenic acid (ALA). The enzymes required to synthesize DHA from ALA are also used to synthesize longer chain omega-6 (n-6) PUFA from linoleic acid (LNA). The large increase in LNA consumption that has occurred over the last century has led to concern that LNA and other n-6 PUFA outcompete n-3 PUFA for enzymes involved in DHA synthesis, and therefore, decrease overall DHA synthesis. To assess this, rats were fed diets containing LNA at 53 (high LNA diet), 11 (medium LNA diet) or 1.5% (low LNA diet) of the fatty acids with ALA being constant across all diets (approximately 4% of the fatty acids). Rats were maintained on these diets from weaning for 8 weeks, at which point they were subjected to a steady-state infusion of labeled ALA and LNA to measure DHA and arachidonic acid (ARA) synthesis rates. DHA and ARA synthesis rates were generally highest in rats fed the medium and high LNA diets, while the plasma half-life of DHA was longer in rats fed the low LNA diet. Therefore, increasing dietary LNA, in rats, did not impair DHA synthesis; however, low dietary LNA led to a decrease in DHA synthesis with tissue concentrations of DHA possibly being maintained by a longer DHA half-life.

  18. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

    PubMed Central

    Bielinska, A; Kukowska-Latallo, J F; Johnson, J; Tomalia, D A; Baker, J R

    1996-01-01

    Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression. PMID:8668551

  19. Novel applications of locked nucleic acids.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion High Fidelity DNA polymerase and 9( degrees )Nm(TM) DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN(m) polymerase proved to be the best for amplification employing the LNA-A nucleotide. PMID:18029570

  20. Energetic aspects of locked nucleic acids quadruplex association and dissociation.

    PubMed

    Petraccone, Luigi; Erra, Eva; Randazzo, Antonio; Giancola, Concetta

    2006-12-15

    The design of modified nucleic acid aptamers is improved by considering thermodynamics and kinetics of their association/dissociation processes. Locked Nucleic Acids (LNA) is a promising class of nucleic acid analogs. In this work the thermodynamic and kinetic properties of a LNA quadruplex formed by the TGGGT sequence, containing only conformationally restricted LNA residues, are reported and compared to those of 2'-OMe-RNA (O-RNA) and DNA quadruplexes. The thermodynamic analysis indicates that the sugar-modified quadruplexes (LNA and O-RNA) are stabilized by entropic effects. The kinetic analysis shows that LNA and O-RNA quadruplexes are characterized by a slower dissociation and a faster association with respect to DNA quadruplex. Interestingly, the LNA quadruplex formation process shows a second-order kinetics with respect to single strand concentration and has a negative activation energy. To explain these data, a mechanism for tetramer formation with two intermediate states was proposed.

  1. Targeting Cancer with Antisense Oligomers

    SciTech Connect

    Hnatowich, DJ

    2008-10-28

    With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their native and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes

  2. Regulating the on-surface LNA probe density for the highest target recognition efficiency.

    PubMed

    Mishra, Sourav; Ghosh, Srabani; Mukhopadhyay, Rupa

    2014-09-01

    The recent emergence of on-surface LNA-based assays as potentially better alternatives over DNA-based approaches, due to enhanced sensitivity and target specificity, raises the need for the precise identification of the factors that control the performance of these assays. In this work, we investigated whether the probe density of fully modified ssLNA probes on the gold(111) surface could influence the target recognition capacity of the LNA sensing layer and illustrated simple means to control it, primarily by adjusting the salt concentration, nature of the cation, and pH of the immobilization buffer. It was observed that monovalent Na(+) could more effectively control the sensor probe density compared to bivalent Mg(2+), leading to better target recognition. Interestingly, unlike in the case of ssDNA sensor probes, the target recognition efficiency of the LNA layer at the optimum probe density was found to be almost spacer-independent, probably due to the rigidity of the LNA backbone. The optimized LNA sensor layer could discriminate single base mismatches, detect a minimum target DNA concentration of 5 nM, and sense a significant level of hybridization within a time scale of a few minutes. To our knowledge, for the first time, we identify the factors that control the on-surface LNA probe density for maximizing the performance of the LNA sensing layer.

  3. Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility.

    PubMed

    Thipyapong, Piyada; Hunt, Michelle D; Steffens, John C

    2004-11-01

    Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm(-2) than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm(-2) than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::beta-glucuronidase constructs when plants are challenged with P

  4. Neighboring Gene Regulation by Antisense Long Non-Coding RNAs

    PubMed Central

    Villegas, Victoria E.; Zaphiropoulos, Peter G.

    2015-01-01

    Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation—pretranscriptional, transcriptional and posttranscriptional—through DNA–RNA, RNA–RNA or protein–RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm. PMID:25654223

  5. NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

    PubMed Central

    Lebars, Isabelle; Richard, Tristan; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2007-01-01

    The trans-activating responsive (TAR) RNA element located in the 5′ untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605–1614]. These aptamers display a 5′-GUCCCAGA-3′ consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR–aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2′-O,4′-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR–LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G•A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson–Crick base pairing with a loop–loop conformation close to an A-type. PMID:17768146

  6. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  7. LNA with wide range of gain control and wideband interference rejection

    NASA Astrophysics Data System (ADS)

    Wang, Jhen-Ji; Chen, Duan-Yu

    2016-10-01

    This work presents a low-noise amplifier (LNA) design with a wide-range gain control characteristic that integrates adjustable current distribution and output impedance techniques. For a given gain characteristic, the proposed LNA provides better wideband interference rejection performance than conventional LNA. Moreover, the proposed LNA also has a wider gain control range than conventional LNA. Therefore, it is suitable for satellite communications systems. The simulation results demonstrate that the voltage gain control range is between 14.5 and 34.2 dB for such applications (2600 MHz); the input reflection coefficient is less than -18.9 dB; the noise figure (NF) is 1.25 dB; and the third-order intercept point (IIP3) is 4.52 dBm. The proposed LNA consumes 23.85-28.17 mW at a supply voltage of 1.8 V. It is implemented by using TSMC 0.18-um RF CMOS process technology.

  8. Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA.

    PubMed

    Hughesman, Curtis B; Turner, Robin F B; Haynes, Charles A

    2011-06-14

    Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° < 0) in the entropy gain accompanying the helix-to-coil transition, with the magnitude of the reduction dependent on the type of nucleobase and its base pairing properties. This knowledge and our average measured value for ΔC(p) of 42 ± 11 cal mol(-1) K(-1) bp(-1) are then used to derive a new model that accurately predicts melting thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.

  9. Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation.

    PubMed

    Igarashi, Miki; DeMar, James C; Ma, Kaizong; Chang, Lisa; Bell, Jane M; Rapoport, Stanley I

    2007-05-01

    Rates of conversion of alpha-linolenic acid (alpha-LNA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3) by the mammalian brain and the brain's ability to upregulate these rates during dietary deprivation of n-3 polyunsaturated fatty acids (PUFAs) are unknown. To answer these questions, we measured conversion coefficients and rates in post-weaning rats fed an n-3 PUFA deficient (0.2% alpha-LNA of total fatty acids, no DHA) or adequate (4.6% alpha-LNA, no DHA) diet for 15 weeks. Unanesthetized rats in each group were infused intravenously with [1-(14)C]alpha-LNA, and their arterial plasma and microwaved brains collected at 5 minutes were analyzed. The deficient compared with adequate diet reduced brain DHA by 37% and increased brain arachidonic (20:4n-6) and docosapentaenoic (22:5n-6) acids. Only 1% of plasma [1-(14)C]alpha-LNA entering brain was converted to DHA with the adequate diet, and conversion coefficients of alpha-LNA to DHA were unchanged by the deficient diet. In summary, the brain's ability to synthesize DHA from alpha-LNA is very low and is not altered by n-3 PUFA deprivation. Because the liver's reported ability is much higher, and can be upregulated by the deficient diet, DHA converted by the liver from circulating alphaLNA is the source of the brain's DHA when DHA is not in the diet.

  10. Ion trap collision-induced dissociation of locked nucleic acids.

    PubMed

    Huang, Teng-yi; Kharlamova, Anastasia; McLuckey, Scott A

    2010-01-01

    Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3'C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.

  11. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

  12. Nucleic acid programmed polymeric nanomaterials for biological communication

    NASA Astrophysics Data System (ADS)

    Rush, Anthony Michael

    A number of nucleic acid-polymer conjugates were synthesized, resulting in amphiphilic polymer-nucleic acid conjugates with the capability to self-assemble into a range of discrete nanoscale architectures. These nanomaterials, termed DNA-polymer amphiphile nanoparticles (DPA NPs), were studied with respect to their enzymatic processing by both endo- and exonucleases and further deployed as antisense genetic regulatory elements in live cultured human cells. DPA NPs were designed to act as substrates for both non sequence-specific exonucleases and a sequence-specific endonuclease. In all cases, nucleic acids arranged in the corona of spherical nanoparticles exhibited increased resistance to nucleolytic cleavage as compared to native single- or double-stranded analogues. For the exonucleases studied (Exonuclease III from E. Coli and phosphodiesterase I from Crotalus adamanteus), nanoparticle display retarded enzymatic processing by roughly a factor of five. For the endonuclease studied (Nt.CviPII), nanoparticle display prohibited virtually all enzyme activity on oligonucleotides within the nanoparticle shell. To test the ability of these materials to regulate mRNA levels in live cultured human cells, LPA (LNA-polymer amphiphile) NPs were designed to be perfectly complementary to a 20-base region of mRNA encoding the anti-apoptosis protein survivin. In this study two key observations were made. The first observation is that packaging LNA into spherical micellar nanoparticles serves to dramatically enhance cellular uptake of LNA based on flow cytometry and fluorescence microscopy data. The second observation is that LPA NPs are capable of regulating mRNA levels by what is hypothesized to be activation of target mRNA for catalytic RNase H-mediated degradation. These materials represent a unique class of DNA delivery system capable of rendering nucleic acids with natural backbone chemistry resistant to nuclease degradation and further serving to deliver DNA into cells to

  13. The Antisense Transcriptome and the Human Brain.

    PubMed

    Mills, James D; Chen, Bei Jun; Ueberham, Uwe; Arendt, Thomas; Janitz, Michael

    2016-01-01

    The transcriptome of a cell is made up of a varied array of RNA species, including protein-coding RNAs, long non-coding RNAs, short non-coding RNAs, and circular RNAs. The cellular transcriptome is dynamic and can change depending on environmental factors, disease state and cellular context. The human brain has perhaps the most diverse transcriptome profile that is enriched for many species of RNA, including antisense transcripts. Antisense transcripts are produced when both the plus and minus strand of the DNA helix are transcribed at a particular locus. This results in an RNA transcript that has a partial or complete overlap with an intronic or exonic region of the sense transcript. While antisense transcription is known to occur at some level in most organisms, this review focuses specifically on antisense transcription in the brain and how regulation of genes by antisense transcripts can contribute to functional aspects of the healthy and diseased brain. First, we discuss different techniques that can be used in the identification and quantification of antisense transcripts. This is followed by examples of antisense transcription and modes of regulatory function that have been identified in the brain.

  14. Locked nucleic acid molecular beacons.

    PubMed

    Wang, Lin; Yang, Chaoyong James; Medley, Colin D; Benner, Steven A; Tan, Weihong

    2005-11-16

    A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.

  15. NTZIP antisense plants show reduced chlorophyll levels.

    PubMed

    Liu, Ning; Yang, Yu-Tao; Liu, Han-Hua; Yang, Guo-Dong; Zhang, Nai-Hua; Zheng, Cheng Chao

    2004-04-01

    We have isolated and characterized a new photosynthetic tissue-specific gene NTZIP (Nicotiana tabacum leucine zipper) from tobacco (N. tabacum). Its deduced amino acid sequence has two highly conserved regions, leucine zipper and [EX(n)DEXRH](2) motifs, which are related to the gene's biochemical functions. NTZIP was expressed in leaves and stems, but was not detected in roots or flowers, suggesting that its physiological functions might be associated with photosynthesis. Northern blot analysis showed that NTZIP mRNA accumulation was induced by light signals, increased greatly under low temperatures and was repressed by strong light illumination. Furthermore, a number of homologs of NTZIP were isolated from cucumber (Cucumis sativus), rape (Brassica napus), clover (Trifolium repens), willow (Salix babylonica), rosebush (Rusa dovurica), wheat (Triticum aestivum) and spinach (Spinacia oleracea), proving the ubiquitous existence of the NTZIP-like genes in higher plants. Transgenic tobaccos constitutively expressing antisense RNA to NTZIP displayed chlorosis and a lack of ability to turn green even under normal growth conditions. The chlorophyll deficiency was further confirmed by chlorophyll content determination and gas exchange analysis. Based on these observations, we propose that NTZIP may be involved in chlorophyll biosynthesis, and might define a novel family of evolutionarily conserved proteins with its homologs in other plant species.

  16. Development of Antisense Drugs for Dyslipidemia.

    PubMed

    Yamamoto, Tsuyoshi; Wada, Fumito; Harada-Shiba, Mariko

    2016-09-01

    Abnormal elevation of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins in plasma as well as dysfunction of anti-atherogenic high-density lipoprotein (HDL) have both been recognized as essential components of the pathogenesis of atherosclerosis and are classified as dyslipidemia. This review describes the arc of development of antisense oligonucleotides for the treatment of dyslipidemia. Chemically-armed antisense candidates can act on various kinds of transcripts, including mRNA and miRNA, via several different endogenous antisense mechanisms, and have exhibited potent systemic anti-dyslipidemic effects. Here, we present specific cutting-edge technologies have recently been brought into antisense strategies, and describe how they have improved the potency of antisense drugs in regard to pharmacokinetics and pharmacodynamics. In addition, we discuss perspectives for the use of armed antisense oligonucleotides as new clinical options for dyslipidemia, in the light of outcomes of recent clinical trials and safety concerns indicated by several clinical and preclinical studies. PMID:27466159

  17. Suppression of endo B cytokeratin by its antisense RNA inhibits the normal coexpression of endo A cytokeratin.

    PubMed Central

    Trevor, K; Linney, E; Oshima, R G

    1987-01-01

    Antisense endo B cytokeratin RNA encoded by a retrovirus vector was expressed in a derivative of the F9 embryonal carcinoma cell line. Two G418-resistant clones were selected that expressed a colinear transcript containing both neomycin and antisense endo B cytokeratin sequences. Expression of a 5-fold excess of antisense endo B RNA over endogenous, retinoic acid-induced endo B RNA resulted in suppression of endo B cytokeratin protein expression. In addition, the normal induction of endo A protein, the type II cytokeratin that polymerizes with endo B, was suppressed at the RNA and protein levels. Revertant clones, which synthesize little if any neo or antisense endo B RNA, regain the ability to express the affected gene products in response to retinoic acid. These results indicate that the suppression of endo B cytokeratin protein synthesis influences the stable levels of endo A mRNA. Images PMID:2434948

  18. Interaction of α-melanocortin and its pentapeptide antisense LVKAT: effects on hepatoprotection in male CBA mice.

    PubMed

    Houra, Karlo; Turčić, Petra; Gabričević, Mario; Weitner, Tin; Konjevoda, Paško; Stambuk, Nikola

    2011-01-01

    The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of continuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3'→5' direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo. PMID:21873934

  19. Functionalization of an Antisense Small RNA

    PubMed Central

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  20. Development of antisense oligodeoxynucleotides for transplantation.

    PubMed

    Stepkowski, S M

    2000-06-01

    Over last ten years antisense technology has been improved to provide powerful tools to selectively inhibit production of different mRNAs. This technology has been applied in transplantation to prolong the survival of organ allografts and to prevent development of ischemic/reperfusion injury in grafts. The present review describes technological progress in chemical modifications from antisense phosphodiester oligonucleotides to phosphorothioate oligonucleotides and the most advanced chimeric oligonucleotides with methoxyethyl groups attached at both ends or at one end of the oligonucleotide. Results indicate that phosphorothioate oligonucleotides, designed to block intercellular adhesion molecule-1 (ICAM-1), extended the survival of heart and kidney allografts when administered to donors or recipients. Combination of ICAM-1 antisense phosphorothioate oligonucleotide and cyclosporine (CsA) produced a potent synergistic interaction on allograft survival in comparison with each drug alone. The same ICAM-1 phosphorothioate oligonucleotide used for perfusion of kidney grafts prevented development of ischemic/reperfusion injury. We also compared the effect of c-raf mRNA inhibition on heart allograft survival by phosphorothioate oligonucleotide or phosphorothioate/methoxyethyl oligonucleotide used alone or in combination with CsA or sirolimus (SRL). The results documented that addition of methoxyethyl modifications at both ends or at one end of oligonucleotides significantly improved the in vivo antisense activity. Combined therapy with c-raf antisense phosphorothioate/methoxyethyl oligonucleotide and SRL synergistically extended the survival of heart allografts. Thus, antisense technology may provide not only tools to examine the effects of selective inhibition of different molecules involved in allograft rejection but also act as potential therapeutic agents.

  1. Does Active Learning through an Antisense Jigsaw Make Sense?

    NASA Astrophysics Data System (ADS)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

    Three journal articles on nucleic acid antisense modification strategies were assigned to 12 students as part of an active learning "jigsaw" exercise for a graduate-level chemistry course on nucleic acids. Each student was required to read one of the three articles. This assignment was preceded by an hour-long lecture on the basic concepts in antisense antigene technology. On the day of the jigsaw, the students with the same article (three groups of four students) discussed their article briefly, and then formed four new groups where no one had read the same article. Each student spent about five minutes teaching his or her article to the other group members, using specific questions provided to guide the discussion. This exercise laid the foundation for bringing the discussion to the entire class, where most of the students actively participated. To test the students' comprehension of the reading materials, a problem set was designed that required not only an understanding of the three articles, but also application of the concepts learned. The effectiveness of this active learning strategy and its applicability to other topics are discussed in this article.

  2. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

    PubMed

    Liu, Jie; Hao, Zhenyu; Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  3. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

    PubMed Central

    Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  4. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

    PubMed

    Liu, Jie; Hao, Zhenyu; Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

  5. Nd:LNA laser optical pumping of He-4 - Application to space magnetometers

    NASA Astrophysics Data System (ADS)

    Slocum, R. E.; Schearer, L. D.; Tin, P.; Marquedant, R.

    1988-12-01

    Results obtained from laser pumping in a helium magnetometer sensor, using a tunable Nd:LNA laser pumped with a high-power diode laser, are reported. It is shown that it was possible to observe both the Hanle signals and the n = 0, p = 1 parametric resonance by monitoring the pumping radiation passing through the cell. As the diode laser-pumped Nd:LNA laser was tuned through the D0, D1, and D2 transitions, three distinct resonance signals were produced. A comparison of the slope of lamp-pumped signals and laser-pumped D1 signals showed that, under otherwise identical conditions, the slope of the D1 laser signal was 45 times greater than the lamp-pumped signal.

  6. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  7. Antisense DNAs as multisite genomic modulators identified by DNA microarray

    PubMed Central

    Cho, Yee Sook; Kim, Meyoung-Kon; Cheadle, Chris; Neary, Catherine; Becker, Kevin G.; Cho-Chung, Yoon S.

    2001-01-01

    Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIα alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation. PMID:11481453

  8. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    PubMed Central

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  9. Inhibition of Gastric Tumor Cell Growth Using Seed-targeting LNA as Specific, Long-lasting MicroRNA Inhibitors.

    PubMed

    Staedel, Cathy; Varon, Christine; Nguyen, Phu Hung; Vialet, Brune; Chambonnier, Lucie; Rousseau, Benoît; Soubeyran, Isabelle; Evrard, Serge; Couillaud, Franck; Darfeuille, Fabien

    2015-07-07

    MicroRNAs regulate eukaryotic gene expression upon pairing onto target mRNAs. This targeting is influenced by the complementarity between the microRNA "seed" sequence at its 5' end and the seed-matching sequences in the mRNA. Here, we assess the efficiency and specificity of 8-mer locked nucleic acid (LNA)-modified oligonucleotides raised against the seeds of miR-372 and miR-373, two embryonic stem cell-specific microRNAs prominently expressed in the human gastric adenocarcinoma AGS cell line. Provided that the pairing is perfect over all the eight nucleotides of the seed and starts at nucleotide 2 or 1 at the microRNA 5' end, these short LNAs inhibit miR-372/373 functions and derepress their common target, the cell cycle regulator LATS2. They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations. Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation. Their therapeutic potential is confirmed in fast-growing, miR-372-positive, primary human gastric adenocarcinoma xenografts in mice. Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

  10. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  11. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  12. Scaffolding along nucleic acid duplexes using 2'-amino-locked nucleic acids.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2014-06-17

    CONSPECTUS: Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2'-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2'-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2'-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2'-amino- and isomeric 2'-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2'-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable

  13. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

    PubMed

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-03-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

  14. Chemosensitization by antisense oligonucleotides targeting MDM2.

    PubMed

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  15. Using both strands: The fundamental nature of antisense transcription.

    PubMed

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  16. Diversification of antisense research and development: review of the Ringberg meeting, April 1994. Mechanisms of antisense-mediated gene silencing.

    PubMed

    Hawkins, J W; Nellen, W

    1994-01-01

    Antisense technology has established itself as a new and vibrant entrant into the discipline of molecular biology. As such, it has contributed to basic research by providing tools for the molecular dissection of diverse experimental systems. In applied research, antisense approaches have contributed to development of agricultural products (D. Grierson) now coming to market and to the design of a number of oligonucleotide drugs, now in clinical trials. However, few activities to date have focused on the study of antisense per se. Further, few conceptual perspectives have regarded antisense as an integral part of cellular function and genetic regulation. The Ringberg conference showcased a number of systems that would seem unrelated if we regard antisense as a superficial tool to be imposed on nature. On the other hand, if we want to begin to regard antisense as a field of its own with deeper biological and genetic rationales, the Ringberg meeting provided much tantalizing evidence to do so.

  17. Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

    PubMed

    Roh, Young Hoon; Lee, Jong Bum; Shopsowitz, Kevin E; Dreaden, Erik C; Morton, Stephen W; Poon, Zhiyong; Hong, Jinkee; Yamin, Inbar; Bonner, Daniel K; Hammond, Paula T

    2014-10-28

    Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

  18. A Differential CMOS Common-Gate LNA Linearized by Cross-Coupled Post Distortion Technique

    NASA Astrophysics Data System (ADS)

    Guo, Benqing; Yang, Guomin; Bin, Xiexian

    2014-05-01

    A linearized differential common-gate CMOS low noise amplifier is proposed. The linearity is improved by a cross-coupled post distortion technique, employing auxiliary PMOS transistors in weak inversion region to cancel the third-order nonlinear currents of common-gate LNA and impair the second-order nonlinear currents of that. The negative conductance characteristic of cross-coupled auxiliary PMOS transistors improves the gain while the resulted NF is little affected. Furthermore, noise contribution and linearity deterioration from the cascode stage is eliminated by an inductor resonating with the parasitic capacitance observed at the source net of the cascode transistor. The LNA implemented in a 0.18 μm CMOS technology demonstrates that IIP3 and gain have about 8.2 dB and 1.4 dB improvements in the designed frequency band, respectively. The noise figure of 3.4 dB is obtained with a power dissipation of 6.8 mW under a 1.8 V power supply.

  19. EGFR Mutation Analysis of Circulating Tumor DNA Using an Improved PNA-LNA PCR Clamp Method

    PubMed Central

    Watanabe, Kana; Fukuhara, Tatsuro; Tsukita, Yoko; Morita, Mami; Suzuki, Aya; Tanaka, Nobuyuki; Terasaki, Hiroshi; Nukiwa, Toshihiro

    2016-01-01

    Introduction. Rebiopsies have become more crucial in non-small cell lung cancer (NSCLC). Instead of invasive biopsies, development of collecting biological data of the tumor from blood samples is expected. We conducted a prospective study to assess the feasibility of detection of epidermal growth factor receptor (EGFR) mutation in plasma samples. Method. NSCLC patients harboring EGFR activating mutations, who were going to receive EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatment, were enrolled in this study. Plasma EGFR activating mutations and the T790M resistance mutation were analyzed by an improved PNA-LNA PCR clamp method, characterized by a 10-fold or more sensitivity compared with the original methods. Result. Six patients with wild-type EGFR and 24 patients with EGFR mutations were enrolled in this study. Pretreatment plasma samples achieved sensitivity of 79%. The 6 patients with wild-type EGFR were all negative for plasma EGFR mutations. At the time of disease progression, plasma T790M mutation was detected in 8 of 16 cases. Absence of T790M before and during TKI treatment and disappearance of activating mutations during TKI treatment were considered as predictors of EGFR-TKIs efficacy. Conclusion. We were able to detect EGFR mutations in plasma samples by using an improved PNA-LNA PCR clamp method. PMID:27478396

  20. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  1. Antisense transcription as a tool to tune gene expression.

    PubMed

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  2. Elucidation of gene function using C-5 propyne antisense oligonucleotides.

    PubMed

    Flanagan, W M; Su, L L; Wagner, R W

    1996-09-01

    Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells. PMID:9631067

  3. IGF-1 Antisense Strategies for Cancer Treatment.

    PubMed

    Pan, Y X; Anthony, D D

    2000-01-01

    The technical approaches to gene therapy for cancer utilize ex vivo and in vivo gene-transfer methodology. This chapter focuses on applicability and use of an ex vivo approach using an IGF-1 antisense RNA strategy of treatment. Insulin-like growth factor 1 (IGF-1) and IGF-2 have pivotal roles in cell proliferation and development (for review, see 1-6). The preponderance of peptide synthesis and activity occur during fetal development, and protein synthesis is downregulated in most mature tissues except for adult liver. Further modulating the activities of these proteins are the levels of their respective cell-surface receptors and ligand-receptor interactions (3,5,6).

  4. Antisense oligonucleotides, microRNAs, and antibodies.

    PubMed

    Dávalos, Alberto; Chroni, Angeliki

    2015-01-01

    The specificity of Watson-Crick base pairing and the development of several chemical modifications to oligonucleotides have enabled the development of novel drug classes for the treatment of different human diseases. This review focuses on promising results of recent preclinical or clinical studies on targeting HDL metabolism and function by antisense oligonucleotides and miRNA-based therapies. Although many hurdles regarding basic mechanism of action, delivery, specificity, and toxicity need to be overcome, promising results from recent clinical trials and recent approval of these types of therapy to treat dyslipidemia suggest that the treatment of HDL dysfunction will benefit from these unique clinical opportunities. Moreover, an overview of monoclonal antibodies (mAbs) developed for the treatment of dyslipidemia and cardiovascular disease and currently being tested in clinical studies is provided. Initial studies have shown that these compounds are generally safe and well tolerated, but ongoing large clinical studies will assess their long-term safety and efficacy.

  5. Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen; Chatterjee, Anushree

    2014-03-01

    ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

  6. VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

    PubMed Central

    Gu, Zhong-Ping; Wang, Yun-Jie; Li, Jin-Ge; Zhou, Yong-An

    2002-01-01

    AIM: To investigate the effect of antisense RNA to vascular endothelial growth factor165 (VEGF165) on human esophageal squamous cell carcinoma cell line EC109 and the feasibility of gene therapy for esophageal carcinoma. METHODS: By using subclone technique, the full length of VEGF165 amino acid cDNA, which was cut from pGEM-3Zf(+), was cloned inversely into the eukaryotic expression vector pCEP4.The recombinant plasmid pCEP-AVEGF165 was transfected into EC109 cell with lipofectamine. After a stable transfection, dot blot, enzyme-linked immunosorbent assay (ELISA), laser confocal imaging system analysis, transmission electron microscopy and flow cytometry were performed to determine the biological characteristics of EC109 cell line before and after transfection in vitro and whether there was a reversion in the tumorigenic properties of the EC109 cell in vivo. RESULTS: The eukaryotic expression vector pCEP-AVEGF165 was successfully constructed and transfected into EC109 cells. The expression of VEGF165 was significantly decreased in the transfected cells while the biological characteristics of the cells were not influenced by the expression of antisense gene. The tumorigenic and angiogenic capabilities were greatly reduced in nude mice, as demonstrated by reduced tumor end volume (820 ± 112.5) mm3 vs (7930 ± 1035) mm3 and (7850 ± 950) mm3,P£¼0.01£½ and microvessel density(8.5 ± 1.2) mm-2 vs (44.3 ± 9.4) mm-2 and (46.4 ± 12.6) mm-2,P < 0.01) in comparison between experimental groups empty vector transfected group and control group. CONCLUSION: The angiogenesis and tumorigenicity of human esophageal squamous cell carcinoma were effectively inhibited by VEGF165 antisense RNA. Antisense RNA to VEGF165 can potentially be used as an adjuvant therapy for solid tumors. PMID:11833069

  7. RNA therapeutics: RNAi and antisense mechanisms and clinical applications

    PubMed Central

    Chery, Jessica

    2016-01-01

    RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology1. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials2. Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs. PMID:27570789

  8. Proteomic analysis of mature barley grains from C-hordein antisense lines.

    PubMed

    Schmidt, Daiana; Gaziola, Salete Aparecida; Boaretto, Luis Felipe; Azevedo, Ricardo Antunes

    2016-05-01

    Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.

  9. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  10. Biodistribution of antisense nanoparticles in mammary carcinoma rat model.

    PubMed

    Elazar, Victoria; Adwan, Hassan; Rohekar, Keren; Zepp, Michael; Lifshitz-Shovali, Rinat; Berger, Martin R; Golomb, Gershon

    2010-08-01

    Efficient and specific delivery of antisenses (ASs) and protection of the sequences from degradation are critical factors for effective therapy. Sustained release nanoparticles (NP) offer increased resistance to nuclease degradation, increased amounts of AS uptake, and the possibility of control in dosing and sustained duration of AS administration. The biodegradable and biocompatible poly(D,L-lactic-co-glycolic acid) copolymer (PLGA) was utilized to encapsulate AS directed against osteopontin (OPN), which is a promising therapeutic target in mammary carcinoma. Whole body biodistribution of OPN AS NP was evaluated in comparison to naked AS, in intact and mammary carcinoma metastasis model bearing rats. Naked and NP encapsulated AS exhibited different biodistribution profiles. AS NP, in contrast to naked AS, tended to accumulate mostly in the spleen, liver, and at the tumor inoculation site. Drug levels in intact organs were negligible. The elimination of naked AS was faster, due to rapid degradation of the unprotected sequence. It is concluded that AS NP protect the AS from degradation, provide efficient AS delivery to the tumor tissue, and minimize AS accumulation in intact organs due to the AS sustained release profile as well as the favorable NP physicochemical properties.

  11. Antisense properties of duplex- and triplex-forming PNAs.

    PubMed Central

    Knudsen, H; Nielsen, P E

    1996-01-01

    The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA. PMID:8602363

  12. Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe.

    PubMed

    Yoon, Jee-Hyun; Nam, Ji-Sun; Kim, Kyung-Jin; Ro, Young-Tae

    2013-03-01

    Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1 pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates.

  13. A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue.

    PubMed

    Vaughan, Roger A; Garrison, Richard L; Stamatikos, Alexis D; Kang, Minsung; Cooper, Jamie A; Paton, Chad M

    2015-11-01

    Recently, the pro-inflammatory effects of linoleic acid (LNA) have been re-examined. It is now becoming clear that relatively few studies have adequately assessed the effects of LNA, independent of obesity. The purpose of this work was to compare the effects of several fat-enriched but non-obesigenic diets on inflammation to provide a more accurate assessment of LNA's ability to induce inflammation. Specifically, 8-week-old male C57Bl/6 mice were fed either saturated (SFA), monounsaturated (MUFA), LNA, or alpha-linolenic acid enriched diets (50 % Kcal from fat, 22 % wt/wt) for 4 weeks. Chow and high-fat, hyper-caloric diets were used as negative and positive controls, respectively. Expression of pro-inflammatory and pro-coagulant markers from epididymal fat, liver, and plasma were measured along with food intake and body weights. Mice fed the high SFA, MUFA, and high-fat diets exhibited increased pro-inflammatory markers in liver and adipose tissue; however, mice fed LNA for four weeks did not display significant changes in pro-inflammatory or pro-coagulant markers in epididymal fat, liver, or plasma. The present study demonstrates that LNA alone is insufficient to induce inflammation. Instead, it is more likely that hyper-caloric diets are responsible for diet-induced inflammation possibly due to adipose tissue remodeling.

  14. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers

    SciTech Connect

    Malgoyre, A.; Banzet, S.; Mouret, C.; Bigard, A.X.; Peinnequin, A. . E-mail: andrepeinnequin@crssa.net

    2007-03-02

    Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.

  15. Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.

    PubMed

    Lok, Chun-Nam; Viazovkina, Ekaterina; Min, Kyung-Lyum; Nagy, Eva; Wilds, Christopher J; Damha, Masad J; Parniak, Michael A

    2002-03-12

    Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially

  16. DESIGN OF 2.4 GHZ CMOS DIRECT CONVERSION LNA AND MIXER COMBINATION FOR WIRLESS DATA LINK TRANSCEIVER.

    SciTech Connect

    ZHAO, D.; OCONNOR, P.

    2002-04-10

    Three LNA and mixer combinations in 0.6{micro}m and 0.4{micro}m standard CMOS processes for direct-conversion receiver of 2.4GHz ISM band short-range wireless data-link applications are described in this paper. Taking low power dissipation as first consideration, these designs, employing differential common-source LNA and double balanced mixer architectures, achieve total conversion gain as high as 42.4dB, DSB noise figure as low as 9.5dB, output-referred IP3 as high as of 21.3dBm at about 4mA DC current consumption. This proves it is possible to apply standard CMOS process to implement receiver front-end with low power dissipation for this kind of application, but gain changeable LNA is needed to combat the dominant flicker noise of the mixer in order to achieve acceptable sensitivity and dynamic range at the same time.

  17. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic.

  18. Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

    PubMed

    Michikawa, Yuichi; Fujimoto, Kentaro; Kinoshita, Kenji; Kawai, Seiko; Sugahara, Keisuke; Suga, Tomo; Otsuka, Yoshimi; Fujiwara, Kazuhiko; Iwakawa, Mayumi; Imai, Takashi

    2006-12-01

    In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

  19. Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system.

    PubMed

    Yin, Huanshun; Zhou, Yunlei; Zhang, Haixia; Meng, Xiaomeng; Ai, Shiyun

    2012-03-15

    MicroRNAs (miRNAs), a kind of small, endogenous, noncoding RNAs (∼22 nucleotides), might play a crucial role in early cancer diagnose due to its abnormal expression in many solid tumors. As a result, label-free and PCR-amplification-free assay for miRNAs is of great significance. In this work, a highly sensitive biosensor for sequence specific miRNA-21 detection without miRNA-21 labeling and enrichment was constructed based on the substrate electrode of dendritic gold nanostructure (DenAu) and graphene nanosheets modified glassy carbon electrode. Sulfydryl functionalized locked nucleic acid (LNA) integrated hairpin molecule beacon (MB) probe was used as miRNA-21 capture probe. After hybridized with miRNA-21 and reported DNA loading in gold nanoparticles (AuNPs) and biotin multi-functionalized bio bar codes, streptavidin-HRP was brought to the electrode through the specific interaction with biotin to catalyze the chemical oxidation of hydroquinone by H(2)O(2) to form benzoquinone. The electrochemical reduction signal of benzoquinone was utilized to monitor the miRNA-21 hybridization event. The effect of experimental variables on the amperometric response was investigated and optimized. Based on the specific confirmation of probe and signal amplification, the biosensor showed excellent selectivity and high sensitivity with low detection limit of 0.06 pM. Successful attempts are made in miRNA-21 expression analysis of human hepatocarcinoma BEL-7402 cells and normal human hepatic L02 cells.

  20. Improvement in the quality of seed storage protein by transformation of Brassica napus with an antisense gene for cruciferin.

    PubMed

    Kohno-Murase, J; Murase, M; Ichikawa, H; Imamura, J

    1995-09-01

    The levels of certain essential amino acids, in particular cysteine, lysine and methionine, in the seed storage protein of a commercial spring variety of rape, Brassica napus, have been increased by the introduction of an antisense gene for cruciferin, which is the most abundant storage protein in rapeseed. The antisense construct contained part of the cruA gene in an inverted orientation, and the gene was driven by the 5' flanking region of the gene for napin such that antisense RNA was expressed in a seed-specific manner. The construct was introduced by Agrobacterium-mediated gene transfer. In self-pollinated seeds (T1 seeds) of transgenic plants there was a reduction in the levels of the α1β1 and α2/3β2/3 subunits of cruciferin, whereas the level of the α4β4 subunit was unchanged. The total protein and lipid contents of transgenic seeds did not differ significantly from that of normal seeds. Seeds with reduced amounts of cruciferin accumulated higher amounts of napin than non-transformed seeds, but the level of oleosin was unaffected. Amino-acid analysis of the seed storage protein revealed that T1 seeds with reduced amounts of cruciferin contained higher relative levels of three essential amino acids, namely, lysine, methionine and cysteine, with increases of 10%, 8% and 32% over the respective levels in non-transgenic seeds (B. napus cv Westar). PMID:24169890

  1. Inhibition of Hsp27 Radiosensitizes Head-and-Neck Cancer by Modulating Deoxyribonucleic Acid Repair

    SciTech Connect

    Guttmann, David M.; Hart, Lori; Du, Kevin; Seletsky, Andrew; Koumenis, Constantinos

    2013-09-01

    Purpose: To present a novel method of tumor radiosensitization through Hsp27 knockdown using locked nucleic acid (LNA) and to investigate the role of Hsp27 in DNA double strand break (DSB) repair. Methods and Materials: Clonogenic survival assays, immunoblotting, the proximity ligation assay, and γH2AX foci analysis were conducted in SQ20B and FaDu human head-and-neck cancer cell lines treated with Hsp27 LNA and Hsp27 short hairpin RNA (shRNA). Additionally, nude mice with FaDu flank tumors were treated with fractionated radiation therapy after pretreatment with Hsp27 LNA and monitored for tumor growth. Results: Hsp27 LNA and Hsp27 shRNA radiosensitized head-and-neck cancer cell lines in an Hsp27-dependent manner. Ataxia-Telangectasia Mutated-mediated DNA repair signaling was impaired in irradiated cells with Hsp27 knockdown. ATM kinase inhibition abrogated the radiosensitizing effect of Hsp27. Furthermore, Hsp27 LNA and shRNA both attenuated DNA repair kinetics after radiation, and Hsp27 was found to colocalize with ATM in both untreated and irradiated cells. Last, combined radiation and Hsp27 LNA treatment in tumor xenografts in nude mice suppressed tumor growth compared with either treatment alone. Conclusions: These results support a radiosensitizing property of Hsp27 LNA in vitro and in vivo, implicate Hsp27 in double strand break repair, and suggest that Hsp27 LNA might eventually serve as an effective clinical agent in the radiotherapy of head-and-neck cancer.

  2. Antisense therapy for cancer--the time of truth.

    PubMed

    Jansen, Burkhard; Zangemeister-Wittke, Uwe

    2002-11-01

    The recent acceleration in the identification and characterisation of new molecular targets for cancer and the limited effectiveness of conventional treatment strategies has focused considerable interest on the development of new types of anticancer agents. These new drugs are hoped to be highly specific for malignant cells with a favorable side-effect profile due to well-defined mechanisms of action. Antisense oligonucleotides are one such class of new agent--they are short, synthetic stretches of DNA which hybridise with specific mRNA strands that correspond to target genes. By binding to the mRNA, the antisense oligonucleotides prevent the sequence of the target gene being converted into a protein, thereby blocking the action of the gene. Several genes known to be important in the regulation of apoptosis, cell growth, metastasis, and angiogenesis, have been validated as molecular targets for antisense therapy. Furthermore, new targets are rapidly being uncovered through coordinated functional genomics and proteomics initiatives. Phosphorothioate oligonucleotides are the current gold standard for antisense therapy; they have acceptable physical and chemical properties and show reasonable resistance to nucleases. Recently, new generations of these phosphorothioate oligonucleotides that contain 2'-modified nucleoside building blocks to enhance RNA binding affinity and decrease indirect toxic effects have been developed. Antisense therapeutics are, after decades of difficulties, finally close to fulfilling their promise in the clinic.

  3. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic alpha-linolenic acid and eicosapentaenoic acid content in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Considerable research centers upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of a-linolenic (ALA; 18:3n3) metabolism; however, little data exist as to the impact of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) on ALA metabolism. We tested the hypothesi...

  4. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  5. RNA antisense purification (RAP) for mapping RNA interactions with chromatin.

    PubMed

    Engreitz, Jesse; Lander, Eric S; Guttman, Mitchell

    2015-01-01

    RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing.

  6. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    PubMed

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  7. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  8. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides.

    PubMed

    Sztainberg, Yehezkel; Chen, Hong-mei; Swann, John W; Hao, Shuang; Tang, Bin; Wu, Zhenyu; Tang, Jianrong; Wan, Ying-Wooi; Liu, Zhandong; Rigo, Frank; Zoghbi, Huda Y

    2015-12-01

    Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders. MECP2 duplication syndrome is one of the most common genomic rearrangements in males and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders, including loss of MeCP2 in Rett syndrome, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2-overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it, and have been successfully used to correct deficits in different mouse models. We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult

  9. Natural antisense transcripts associated with salinity response in alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...

  10. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation.

    PubMed

    Park, Jehyun; Seo, Ji Yeon; Ha, Hunjoo

    2010-12-01

    Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

  11. Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants.

    PubMed

    Zeh, M; Casazza, A P; Kreft, O; Roessner, U; Bieberich, K; Willmitzer, L; Hoefgen, R; Hesse, H

    2001-11-01

    Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality. PMID:11706163

  12. Antisense mRNA for NPY-Y1 receptor in the medial preoptic area increases prolactin secretion.

    PubMed

    Silveira, N A; Franci, C R

    1999-09-01

    We investigated the participation of neuropeptide Y-Y1 receptors within the medial preoptic area in luteinizing hormone, follicle-stimulating hormone and prolactin release. Four bilateral microinjections of sense (control) or antisense 18-base oligonucleotides of messenger ribonucleic acid (mRNA) (250 ng) corresponding to the NH2-terminus of the neuropeptide Y1 receptor were performed at 12-h intervals for two days into the medial preoptic area of ovariectomized Wistar rats (N = 16), weighing 180 to 200 g, treated with estrogen (50 microg) and progesterone (25 mg) two days before the experiments between 8.00 and 10:00 a.m. Blockade of Y1 receptor synthesis in the medial preoptic area by the antisense mRNA did not change plasma luteinizing hormone or follicle-stimulating hormone but did increase prolactin from 19.6 +/- 5.9 ng/ml in the sense group to 52.9 +/- 9.6 ng/ml in the antisense group. The plasma hormones were measured by radioimmunoassay and the values are reported as mean +/- SEM. These data suggest that endogenous neuropeptide Y in the medial preoptic area has an inhibitory action on prolactin secretion through Y1 receptors.

  13. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    PubMed Central

    Falzarano, Maria Sofia; Passarelli, Chiara

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782

  14. Cytotoxicity of food preservatives in cultured rat hepatocytes loaded with linolenic acid.

    PubMed

    Sugihara, N; Shimomichi, K; Furuno, K

    1997-06-01

    We investigated the ability of eight food preservatives to induce lipid peroxidation in normal and alpha-linolenic acid (LNA)-loaded cultured rat hepatocytes. On the addition of sodium dehydroacetate (DHA-Na), potassium sorbate (SA-K) or thiabendazole (TBZ) to the cell culture, lipid peroxidation, assessed in terms of the production of malondialdehyde (MDA), was induced in LNA-loaded cells, but not in normal cells. At the low concentrations, induction of lipid peroxidation in LNA-loaded cells was highest with TBZ, whereas at high concentrations DHA-Na greatly induced lipid peroxidation. The occurrence of lipid peroxidation in LNA-loaded cells was accompanied by a decrease in cellular GSH levels with the three preservatives and by a decrease in cellular protein-SH levels with DHA-Na and TBZ. Furthermore, cell injury, measured by the release of LDH, was produced in LNA-loaded cells exposed to DHA-Na and SA-K. The addition of TBZ caused substantial cell injury in normal cells, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD) almost completely prevented DHA-Na- and SA-K-induced cell injury, and reduced TBZ-induced cell injury. The addition of diphenyl (DP), o-phenylphenol (OPP) or butyl p-hydroxybenzoate (BHB) caused severe cell injury, in association with a marked decrease in cellular levels of both of GSH and protein-SH in both groups of cells. However, lipid peroxidation was not detectable in either group of cells exposed to these preservatives. Sodium propionate (PA-Na) and sodium benzoate (BA-Na) had little effect on any cytotoxic parameter in either group of cells.

  15. Bacterial antisense RNAs are mainly the product of transcriptional noise

    PubMed Central

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  16. Bacterial antisense RNAs are mainly the product of transcriptional noise.

    PubMed

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

    2016-03-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  17. Antisense oligonucleotides: is the glass half full or half empty?

    PubMed

    Bennett, C F

    1998-01-01

    Antisense oligonucleotides are widely used as tools to explore the pharmacological effects of inhibiting expression of a selected gene product. In addition, they are being investigated as therapeutic agents for the treatment of viral infections, cancers, and inflammatory disorders. Proof that the pharmacological effects produced by the oligonucleotides are attributable to an antisense mechanism of action requires careful experimentation. Central to this problem is the finding that oligonucleotides are capable of interacting with and modulating function of specific proteins in both a sequence-independent and -dependent manner. Despite these undesired interactions, it has been possible to demonstrate that oligonucleotides are capable of binding to a specific RNA in cultured cells, or within tissues, resulting in selective reduction of the targeted gene product and pharmacological activity. In general, these oligonucleotides were identified after a selection process in which multiple oligonucleotides targeting different regions on the RNA were evaluated for direct inhibition of targeted gene product, resulting in the identification of a potent and selective oligonucleotide. Similar to other drug-receptor interactions, selection of the most potent inhibitor results in an increase in the signal-to-noise ratio, yielding increased confidence that activity observed is the result of a desired effect of the inhibitor. With careful selection, proper controls, and careful dose-response curves it is possible to utilize antisense oligonucleotides as effective research tools and potentially as therapeutic agents. PMID:9413924

  18. Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

    PubMed

    Smith, Alvin W; Matson, David O; Stein, David A; Skilling, Douglas E; Kroeker, Andrew D; Berke, Tamas; Iversen, Patrick L

    2002-04-01

    The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases. PMID:12044040

  19. Autolysis of cell walls from polygalacturonase-antisense tomato fruit in simulated apoplastic solutions.

    PubMed

    Almeida, Domingos P F; Huber, Donald J

    2011-06-01

    Autolysis of cell walls from polygalacturonase (PG)-antisense tomato fruit was studied in a conventional buffer designed to maximize the catalytic activity of PG (30 mM sodium acetate, 150 mM NaCl, pH 4.5), and in solutions mimicking the pH and mineral composition of the fruit apoplast at the mature-green and ripe stages. Autolytic release of uronic acids was very limited under simulated apoplastic conditions compared with the conventional buffer, but minimal differences in the release of reducing groups were observed among the incubation conditions. Autolytic release of uronic acids from active walls was lower than solubilization from enzymically inactive walls. Uronic acids that remained ionically bound to the cell walls during autolysis were subsequently extracted and analyzed by size exclusion chromatography. The elution profiles of ionically bound uronic acids from cell walls incubated under optimal conditions were similar for all ripening stages. In solutions mimicking the pH and mineral composition of the apoplast of mature-green and ripe fruit, uronic acids extracted from pink and ripe fruit cell walls showed a decrease in average molecular mass compared with polymers from mature-green cell walls. The results suggest that the composition of the incubation solution exert strong influence on PG-independent cell wall autolysis and that enzymically active walls restrain PG-independent pectin solubilization.

  20. Sense and antisense transcription are associated with distinct chromatin architectures across genes.

    PubMed

    Murray, Struan C; Haenni, Simon; Howe, Françoise S; Fischl, Harry; Chocian, Karolina; Nair, Anitha; Mellor, Jane

    2015-09-18

    Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

  1. Monitoring HCV RNA viral load by locked nucleic acid molecular beacons real time PCR.

    PubMed

    Morandi, Luca; Ferrari, Daniela; Lombardo, Claudia; Pession, Annalisa; Tallini, Giovanni

    2007-03-01

    Locked nucleic acids (LNA) based real time PCR was used in particular situations where there are difficulties in primer design due to sequence complexity. In this study a new real time RT-PCR assay was developed using LNA modified primers and LNA molecular beacon probes to monitor hepatitis C virus (HCV) viral load in plasma and serum samples. The technique did not suffer from an heterogeneity of the HCV genome and, in addition, an internal RNA control was amplified in the same reaction tube with different short primers and beacon probe. Due to the short consensus LNA primers length, the PCR efficiency was close to 100% with no formation of hairpin loop structures. In summary a new LNA molecular beacon based real time RT-PCR assay was used successfully to measure quantitatively the total level of HCV RNA in both experimental and clinical specimens. The high sensitivity (50 IU/ml), the wide range of genotype detection, increased specificity and robustness obtained with this test are particularly useful for screening large number of specimens and measuring viral loads to monitor the progress of the disease.

  2. Linolenic acid grafted hyaluronan: Process development, structural characterization, biological assessing, and stability studies.

    PubMed

    Huerta-Angeles, Gloria; Brandejsová, Martina; Kulhánek, Jaromír; Pavlík, Vojtěch; Šmejkalová, Daniela; Vágnerová, Hana; Velebný, Vladimír

    2016-11-01

    In this study, hyaluronan (HA) was grafted with alpha-linolenic acidLNA) by benzoyl mixed anhydrides methodology, which allowed the derivatization of HA under mild reaction conditions. The reaction was optimized and transferred from laboratory to semi-scale production. The derivative revealed an unexpected cytotoxicity after oven drying and storage at 40°C. For this reason, the storage conditions of sodium linolenyl hyaluronate (αLNA-HA) were optimized in order to preserve the beneficial effect of the derivative. Oven, spray dried and lyophilized samples were prepared and stored at -20°C, 4°C and 25°C up to 6 months. A comprehensive material characterization including stability study of the derivative, as well as evaluation of possible changes on chemical structure and presence of peroxidation products were studied by Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), thermogravimetric analysis (TGA) and complemented with assessment of in vitro viability on mouse fibroblasts NIH-3T3. The most stable αLNA-HA derivative was obtained after spray drying and storage at ambient temperature under inert atmosphere. The choice of inert atmosphere is recommended to suppress oxidation of αLNA supporting the positive influence of the derivative on cell viability. The encapsulation of hydrophobic drugs of αLNA-HA were also demonstrated. PMID:27516333

  3. High-affinity DNA-targeting using Readily Accessible Mimics of N2′-Functionalized 2′-Amino-α-L-LNA

    PubMed Central

    Karmakar, Saswata; Anderson, Brooke A.; Rathje, Rie L.; Andersen, Sanne; Jensen, Troels B.; Nielsen, Poul; Hrdlicka, Patrick J.

    2011-01-01

    N2′-Pyrene-functionalized 2′-amino-α-L-LNAs (Locked Nucleic Acids) display extraordinary affinity toward complementary DNA targets due to favorable preorganization of the pyrene moieties for hybridization-induced intercalation. Unfortunately, the synthesis of these monomers is challenging (~20 steps, <3% overall yield), which has precluded full characterization of DNA-targeting applications based on these materials. Access to more readily accessible functional mimics would be highly desirable. Here we describe short synthetic routes toward a series of O2′-intercalator-functionalized uridine and N2′-intercalator-functionalized 2′-N-methyl-2′-aminouridine monomers and demonstrate – via thermal denaturation, UV-visible absorption and fluorescence spectroscopy experiments – that several of them mimic the DNA-hybridization properties of N2′-pyrene-functionalized 2′-amino-α-L-LNAs. For example, oligodeoxyribonucleotides (ONs) modified with 2′-O-(coronen-1-yl)methyluridine monomer Z, 2′-O-(pyren-1-yl)methyluridine monomer Y or 2′-N-(pyren-1-ylmethyl)-2′-N-methylaminouridine monomer Q, display prominent increases in thermal affinity toward complementary DNA relative to reference strands (average ΔTm/mod up to +12 °C), pronounced DNA-selectivity, and higher target specificity than 2′-amino-β-L-LNA benchmark probes. In contrast, ONs modified with 2′-O-(2-napthyl)uridine monomer W, 2′-O-(pyren-1-yl)uridine monomer X or 2′-N-(pyren-1-ylcarbonyl)-2′-N-methylaminouridine monomer S display very low affinity toward DNA targets. This demonstrates that even conservative alterations in linker chemistry, linker length and surface area of the appended intercalators have marked impact on DNA-hybridization characteristics. Straightforward access to high-affinity building blocks such as Q/Y/Z is likely to accelerate their use in DNA-targeting applications within nucleic acid based diagnostics, therapeutics, and material science. PMID:21827174

  4. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

  5. Antisense-Based Progerin Downregulation in HGPS-Like Patients’ Cells

    PubMed Central

    Harhouri, Karim; Navarro, Claire; Baquerre, Camille; Da Silva, Nathalie; Bartoli, Catherine; Casey, Frank; Mawuse, Guedenon Koffi; Doubaj, Yassamine; Lévy, Nicolas; De Sandre-Giovannoli, Annachiara

    2016-01-01

    Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named “HGPS-like” patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90) at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON) prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90) in HGPS-like patients’ cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A), was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients. PMID:27409638

  6. Cox-2 inhibitory effects of naturally occurring and modified fatty acids.

    PubMed

    Ringbom, T; Huss, U; Stenholm, A; Flock, S; Skattebøl, L; Perera, P; Bohlin, L

    2001-06-01

    In the search for new cyclooxygenase-2 (COX-2) selective inhibitors, the inhibitory effects of naturally occurring fatty acids and some of their structural derivatives on COX-2-catalyzed prostaglandin biosynthesis were investigated. Among these fatty acids, linoleic acid (LA), alpha-linolenic acid (alpha-LNA), myristic acid, and palmitic acid were isolated from a CH(2)Cl(2) extract of the plant Plantago major by bioassay-guided fractionation. Inhibitory effects of other natural, structurally related fatty acids were also investigated: stearic acid, oleic acid, pentadecanoic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Further, the inhibitory effects of these compounds on COX-2- and COX-1-catalyzed prostaglandin biosynthesis was compared with the inhibition of some synthesized analogues of EPA and DHA with ether or thioether functions. The most potent COX-2-catalyzed prostaglandin biosynthesis inhibitor was all-(Z)-5-thia-8,11,14,17-eicosatetraenoic acid (2), followed by EPA, DHA, alpha-LNA, LA, (7E,11Z,14Z,17Z)-5-thiaeicosa-7,11,14,17-tetraenoic acid, all-(Z)-3-thia-6,9,12,15-octadecatetraenoic acid, and (5E,9Z,12Z,15Z,18Z)-3-oxaheneicosa-5,9,12,15,18-pentaenoic acid, with IC(50) values ranging from 3.9 to180 microM. The modified compound 2 and alpha-LNA were most selective toward COX-2, with COX-2/COX-1 ratios of 0.2 and 0.1, respectively. This study shows that several of the natural fatty acids as well as all of the semisynthetic thioether-containing fatty acids inhibited COX-2-catalyzed prostaglandin biosynthesis, where alpha-LNA and compound 2 showed selectivity toward COX-2. PMID:11421736

  7. Pseudogenes as an alternative source of natural antisense transcripts

    PubMed Central

    2010-01-01

    Background Naturally occurring antisense transcripts (NATs) are non-coding RNAs that may regulate the activity of sense transcripts to which they bind because of complementarity. NATs that are not located in the gene they regulate (trans-NATs) have better chances to evolve than cis-NATs, which is evident when the sense strand of the cis-NAT is part of a protein coding gene. However, the generation of a trans-NAT requires the formation of a relatively large region of complementarity to the gene it regulates. Results Pseudogene formation may be one evolutionary mechanism that generates trans-NATs to the parental gene. For example, this could occur if the parental gene is regulated by a cis-NAT that is copied as a trans-NAT in the pseudogene. To support this we identified human pseudogenes with a trans-NAT to the parental gene in their antisense strand by analysis of the database of expressed sequence tags (ESTs). We found that the mutations that appeared in these trans-NATs after the pseudogene formation do not show the flat distribution that would be expected in a non functional transcript. Instead, we found higher similarity to the parental gene in a region nearby the 3' end of the trans-NATs. Conclusions Our results do not imply a functional relation of the trans-NAT arising from pseudogenes over their respective parental genes but add evidence for it and stress the importance of duplication mechanisms of genetic material in the generation of non-coding RNAs. We also provide a plausible explanation for the large transcripts that can be found in the antisense strand of some pseudogenes. PMID:21047404

  8. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    ERIC Educational Resources Information Center

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  9. Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: Role of norepinephrine and acetylcholine

    PubMed Central

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 μL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the β-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala. PMID:18772255

  10. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides.

    PubMed

    Bergsma, Atze J; In 't Groen, Stijn Lm; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, Wwm Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  11. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

    PubMed Central

    Bergsma, Atze J; in ‘t Groen, Stijn LM; Verheijen, Frans W; van der Ploeg, Ans T; Pijnappel, WWM Pim

    2016-01-01

    While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect. PMID:27623443

  12. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach.

    PubMed

    Bordoy, Antoni E; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  13. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach

    PubMed Central

    Bordoy, Antoni E.; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  14. Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat.

    PubMed

    Carrieri, Claudia; Cimatti, Laura; Biagioli, Marta; Beugnet, Anne; Zucchelli, Silvia; Fedele, Stefania; Pesce, Elisa; Ferrer, Isidre; Collavin, Licio; Santoro, Claudio; Forrest, Alistair R R; Carninci, Piero; Biffo, Stefano; Stupka, Elia; Gustincich, Stefano

    2012-11-15

    Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

  15. Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

    PubMed

    Wang, Tianjiao; Zuo, Rantao; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-11-01

    This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA. PMID:27378407

  16. Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

    PubMed

    Wang, Tianjiao; Zuo, Rantao; Mai, Kangsen; Xu, Wei; Ai, Qinghui

    2016-11-01

    This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA.

  17. COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures.

    PubMed

    Hawkes, Emily J; Hennelly, Scott P; Novikova, Irina V; Irwin, Judith A; Dean, Caroline; Sanbonmatsu, Karissa Y

    2016-09-20

    There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription. PMID:27653675

  18. MYCNOS functions as an antisense RNA regulating MYCN

    PubMed Central

    Vadie, Nadia; Saayman, Sheena; Lenox, Alexandra; Ackley, Amanda; Clemson, Mathew; Burdach, Jon; Hart, Jonathan; Vogt, Peter K; Morris, Kevin V

    2015-01-01

    Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci. PMID:26156430

  19. Antisense Reduction of Tau in Adult Mice Protects against Seizures

    PubMed Central

    DeVos, Sarah L.; Goncharoff, Dustin K.; Chen, Guo; Kebodeaux, Carey S.; Yamada, Kaoru; Stewart, Floy R.; Schuler, Dorothy R.; Maloney, Susan E.; Wozniak, David F.; Rigo, Frank; Bennett, C. Frank; Cirrito, John R.; Holtzman, David M.

    2013-01-01

    Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS—brain and spinal cord tissue, interstitial fluid, and CSF—while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability. PMID:23904623

  20. Photoregulating RNA digestion using azobenzene linked dumbbell antisense oligodeoxynucleotides.

    PubMed

    Wu, Li; He, Yujian; Tang, Xinjing

    2015-06-17

    Introduction of 4,4'-bis(hydroxymethyl)-azobenzene (azo) to dumbbell hairpin oligonucleotides at the loop position was able to reversibly control the stability of the whole hairpin structure via UV or visible light irradiation. Here, we designed and synthesized a series of azobenzene linked dumbbell antisense oligodeoxynucleotides (asODNs) containing two terminal hairpins that are composed of an asODN and a short inhibitory sense strand. Thermal melting studies of these azobenzene linked dumbbell asODNs indicated that efficient trans to cis photoisomerization of azobenzene moieties induced large difference in thermal stability (ΔTm = 12.1-21.3 °C). In addition, photomodulation of their RNA binding abilities and RNA digestion by RNase H was investigated. The trans-azobenzene linked asODNs with the optimized base pairs between asODN strands and inhibitory sense strands could only bind few percentage of the target RNA, while it was able to recover their binding to the target RNA and degrade it by RNase H after light irradiation. Upon optimization, it is promising to use these azobenzene linked asODNs for reversible spatial and temporal regulation of antisense activities based on both steric binding and RNA digestion by RNase H.

  1. Artificial neural network prediction of antisense oligodeoxynucleotide activity.

    PubMed

    Giddings, Michael C; Shah, Atul A; Freier, Sue; Atkins, John F; Gesteland, Raymond F; Matveeva, Olga V

    2002-10-01

    An mRNA transcript contains many potential antisense oligodeoxynucleotide target sites. Identification of the most efficacious targets remains an important and challenging problem. Building on separate work that revealed a strong correlation between the inclusion of short sequence motifs and the activity level of an oligo, we have developed a predictive artificial neural network system for mapping tetranucleotide motif content to antisense oligo activity. Trained for high-specificity prediction, the system has been cross-validated against a database of 348 oligos from the literature and a larger proprietary database of 908 oligos. In cross- validation tests the system identified effective oligos (i.e. oligos capable of reducing target mRNA expression to <25% that of the control) with 53% accuracy, in contrast to the <10% success rates commonly reported for trial-and-error oligo selection, suggesting a possible 5-fold reduction in the in vivo screening required to find an active oligo. We have implemented a web interface to a trained neural network. Given an RNA transcript as input, the system identifies the most likely oligo targets and provides estimates of the probabilities that oligos targeted against these sites will be effective. PMID:12364609

  2. Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system.

    PubMed

    Ross, Joseph A; Ellis, Michael J; Hossain, Shahan; Haniford, David B

    2013-05-01

    Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements.

  3. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

    PubMed

    Yu, Xing Xian; Watts, Lynnetta M; Manchem, Vara Prasad; Chakravarty, Kaushik; Monia, Brett P; McCaleb, Michael L; Bhanot, Sanjay

    2013-01-01

    Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

  4. Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification

    PubMed Central

    Wei, Hua; Zhao, Guojie; Hu, Tianyu; Tang, Suming; Jiang, Jiquan; Hu, Bo; Guan, Yifu

    2016-01-01

    We used a novel asymmetric cleavage analysis method based on rolling circle amplification (RCA) to determine the effects of LNA modification of substrate on the two subunits of R.BbvCI cleavage. We designed two sets of cleavage circular substrates by using two different ligation strategies and analyzed the single strand cleavage efficiency affected by different modification positions both from the cleaved strands and the uncleaved strands. Results showed that the effects of LNA on cleavage rates of modified strands and unmodified strands were both site-dependent. The Nb.BbvCI and Nt.BbvCI were affected by LNA modification in different way. Most of the modification positions showed strong inhibition of both of these two nickases cleavage. However, the modification in T3 position of bottom strand hardly affected both of the two nickases activities. The results suggested an intimated interaction between the two subunits of R.BbvCI, and the T3 position in bottom strand might be a less tight position which was hard to be disturbed. PMID:27582033

  5. Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates.

    PubMed

    Xiao, Yuliang; Li, Pingli; Cheng, Yanna; Zhang, Xinke; Sheng, Juzheng; Wang, Decai; Li, Juan; Zhang, Qian; Zhong, Chuanqing; Cao, Rui; Wang, Fengshan

    2014-04-25

    The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc.

  6. Making sense of antisense: seemingly noncoding RNAs antisense to the master regulator of Kaposi's sarcoma-associated herpesvirus lytic replication do not regulate that transcript but serve as mRNAs encoding small peptides.

    PubMed

    Xu, Yiyang; Ganem, Don

    2010-06-01

    The mammalian transcriptome is studded with putative noncoding RNAs, many of which are antisense to known open reading frames (ORFs). Roles in the regulation of their complementary mRNAs are often imputed to these antisense transcripts, but few have been experimentally examined, and such functions remain largely conjectural. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two transcripts that lack obvious ORFs and are complementary to the gene (RTA) encoding the master regulator of the latent/lytic switch. Here, we show that, contrary to expectation, these RNAs do not regulate RTA expression. Rather, they are found on polysomes, and genetic analysis indicates that translational initiation occurs at several AUG codons in the RNA, leading to the presumptive synthesis of peptides of 17 to 48 amino acids. These findings underscore the need for circumspection in the computational assessment of coding potential and raise the possibility that the mammalian proteome may contain many previously unsuspected peptides generated from seemingly noncoding RNAs, some of which could have important biological functions. Irrespective of their function, such peptides could also contribute substantially to the repertoire of T cell epitopes generated in both uninfected and infected cells. PMID:20357088

  7. Regulating malonyl-CoA metabolism via synthetic antisense RNAs for enhanced biosynthesis of natural products.

    PubMed

    Yang, Yaping; Lin, Yuheng; Li, Lingyun; Linhardt, Robert J; Yan, Yajun

    2015-05-01

    Malonyl-CoA is the building block for fatty acid biosynthesis and also a precursor to various pharmaceutically and industrially valuable molecules, such as polyketides and biopolymers. However, intracellular malonyl-CoA is usually maintained at low levels, which poses great challenges to efficient microbial production of malonyl-CoA derived molecules. Inactivation of the malonyl-CoA consumption pathway to increase its intracellular availability is not applicable, since it is usually lethal to microorganisms. In this work, we employ synthetic antisense RNAs (asRNAs) to conditionally down-regulate fatty acid biosynthesis and achieve malonyl-CoA enrichment in Escherichia coli. The optimized asRNA constructs with a loop-stem structure exhibit high interference efficiency up to 80%, leading to a 4.5-fold increase in intracellular malonyl-CoA concentration when fabD gene expression is inhibited. Strikingly, this strategy allows the improved production of natural products 4-hydroxycoumarin, resveratrol, and naringenin by 2.53-, 1.70-, and 1.53-fold in E. coli, respectively. In addition, down-regulation of other fab genes including fabH, fabB, and fabF also leads to remarkable increases in 4-hydroxycoumarin production. This study demonstrates a novel strategy to enhance intracellular malonyl-CoA and indicates the effectiveness of asRNA as a powerful tool for use in metabolic engineering.

  8. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    PubMed

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  9. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    PubMed

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  10. rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus

    PubMed Central

    Shpiz, Sergey; Kwon, Dmitry; Rozovsky, Yakov; Kalmykova, Alla

    2009-01-01

    Telomeres in Drosophila are maintained by the specialized telomeric retrotransposons HeT-A, TART and TAHRE. Sense transcripts of telomeric retroelements were shown to be the targets of a specialized RNA-interference mechanism, a repeat-associated short interfering (rasi)RNA-mediated system. Antisense rasiRNAs play a key role in this mechanism, highlighting the importance of antisense expression in retrotransposon silencing. Previously, bidirectional transcription was reported for the telomeric element TART. Here, we show that HeT-A is also bidirectionally transcribed, and HeT-A antisense transcription in ovaries is regulated by a promoter localized within its 3′ untranslated region. A remarkable feature of noncoding HeT-A antisense transcripts is the presence of multiple introns. We demonstrate that sense and antisense HeT-A-specific rasiRNAs are present in the same tissue, indicating that transcripts of both directions may be considered as natural targets of the rasiRNA pathway. We found that the expression of antisense transcripts of telomeric elements is regulated by the RNA silencing machinery, suggesting rasiRNA-mediated interplay between sense and antisense transcripts in the cell. Finally, this regulation occurs in the nucleus since disruption of the rasiRNA pathway leads to an accumulation of TART and HeT-A transcripts in germ cell nuclei. PMID:19036789

  11. Functional analysis of polyphenol oxidases by antisense/sense technology.

    PubMed

    Thipyapong, Piyada; Stout, Michael J; Attajarusit, Jutharat

    2007-01-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting

  12. Antisense oligonucleotide for tissue factor inhibits hepatic ischemic reperfusion injury.

    PubMed

    Nakamura, Kenji; Kadotani, Yayoi; Ushigome, Hidetaka; Akioka, Kiyokazu; Okamoto, Masahiko; Ohmori, Yoshihiro; Yaoi, Takeshi; Fushiki, Shinji; Yoshimura, Rikio; Yoshimura, Norio

    2002-09-27

    Tissue factor (TF) is an initiation factor for blood coagulation and its expression is induced on endothelial cells during inflammatory or immune responses. We designed an antisense oligodeoxynucleotide (AS-1/TF) for rat TF and studied its effect on hepatic ischemic reperfusion injury. AS-1/TF was delivered intravenously to Lewis rats. After 10 h, hepatic artery and portal vein were partially clamped. Livers were reperfused after 180 min and harvested. TF expression was studied using immunohistochemical staining. One of 10 rats survived in a 5-day survival rate and TF was strongly stained on endothelial cells in non-treatment group. However, by treatment with AS-1/TF, six of seven survived and TF staining was significantly reduced. Furthermore, we observed that fluorescein-labeled AS-1/TF was absorbed into endothelial cells. These results suggest that AS-1/TF can strongly suppress the expression of TF and thereby inhibit ischemic reperfusion injury to the rat liver. PMID:12270110

  13. QUANTITATIVE CONTRIBUTIONS OF DIET AND LIVER SYNTHESIS TO DOCOSAHEXAENOIC ACID HOMEOSTASIS

    PubMed Central

    Rapoport, Stanley I.; Igarashi, Miki; Gao, Fei

    2010-01-01

    Dietary requirements for maintaining brain and heart docosahexaenoic acid (DHA, 22:6n-3) homeostasis are not agreed on, in part because rates of liver DHA synthesis from circulating α-linolenic acid (α-LNA, 18:2n-3) have not been quantified. These rates can be estimated in vivo using intravenous radiotracer- or heavy isotope-labeled α-LNA infusion. In adult unanesthetized male rats, such infusion shows that liver synthesis-secretion rates of DHA from α-LNA markedly exceed brain and heart DHA synthesis rates and brain DHA consumption rate, and that liver but not heart or brain synthesis is upregulated as dietary n-3 PUFA content is reduced. These differences in rate reflect much higher expression of DHA-synthesizing enzymes in liver, and upregulation of liver but not heart or brain enzyme expression by reduced dietary n-3 PUFA content. A noninvasive intravenous [U-13C]α-LNA infusion method that produces steady-state liver tracer metabolism gives exact liver DHA synthesis-secretion rates and could be extended for human studies. PMID:20226642

  14. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    PubMed

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  15. Mutually exclusive sense–antisense transcription at FLC facilitates environmentally induced gene repression

    PubMed Central

    Rosa, Stefanie; Duncan, Susan; Dean, Caroline

    2016-01-01

    Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription. PMID:27713408

  16. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  17. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    PubMed

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  18. Natural Antisense Transcripts and Long Non-Coding RNA in Neurospora crassa

    PubMed Central

    Arthanari, Yamini; Heintzen, Christian; Griffiths-Jones, Sam; Crosthwaite, Susan K.

    2014-01-01

    The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs. PMID:24621812

  19. Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

    PubMed Central

    Lenartowicz, Elzbieta; Nogales, Aitor; Kierzek, Elzbieta; Kierzek, Ryszard; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches. PMID:27463680

  20. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3

    PubMed Central

    Toonen, Lodewijk J. A.; Schmidt, Iris; Luijsterburg, Martijn S.; van Attikum, Haico; van Roon-Mom, Willeke M. C.

    2016-01-01

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3. PMID:27731380

  1. Biophysical and biological characterization of hairpin and molecular beacon RNase H active antisense oligonucleotides.

    PubMed

    Østergaard, Michael E; Thomas, George; Koller, Erich; Southwell, Amber L; Hayden, Michael R; Seth, Punit P

    2015-05-15

    Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA in vitro, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice.

  2. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

    PubMed

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-01-01

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species. PMID:27646849

  3. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells

    PubMed Central

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-01-01

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5′ UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3′ end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species. PMID:27646849

  4. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  5. Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

    PubMed Central

    Fang, Huafeng; Yue, Xuan; Li, Xiaoxu; Taylor, John-Stephen

    2005-01-01

    We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders. PMID:16314303

  6. Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.

    PubMed

    Knutzon, D S; Thompson, G A; Radke, S E; Johnson, W B; Knauf, V C; Kridl, J C

    1992-04-01

    Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.

  7. A high-fat, high-oleic diet, but not a high-fat, saturated diet, reduces hepatic n3 fatty acid content in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While considerable research has centered upon the role of linoleic acid (LNA; 18:2n6) as a competitive inhibitor of alpha-linolenic (ALA; 18:3n3) metabolism, a growing literature indicates that the amount of fat consumed can reduce the elongation and desaturation process. However, little data exist ...

  8. Autoregulatory loop of Msx1 expression involving its antisense transcripts.

    PubMed

    Petit, Stéphane; Meary, Fleur; Pibouin, Laurence; Jeanny, Jean-Claude; Fernandes, Isabelle; Poliard, Anne; Hotton, Dominique; Berdal, Ariane; Babajko, Sylvie

    2009-08-01

    The Msx1 homeogene plays an important role in epithelial-mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis.

  9. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides

    PubMed Central

    Wang, Shiyu; Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2016-01-01

    Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release. PMID:27378781

  10. Design and Selection of Antisense Oligonucleotides Targeting Transforming Growth Factor Beta (TGF-β) Isoform mRNAs for the Treatment of Solid Tumors.

    PubMed

    Jaschinski, Frank; Korhonen, Hanna; Janicot, Michel

    2015-01-01

    Transforming growth factor beta isoforms (TGF-β1, -β2, and -β3) are cytokines associated with a wide range of biological processes in oncology including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-β signaling pathways may have a multifold therapeutic benefit for the treatment of solid tumors. Here, we describe the identification and selection processes for the development of highly potent and selective chemically modified antisense oligodeoxynucleotides (fully phosphorothioate locked nucleic acid gapmers) allowing effective and selective suppression of TGF-β isoform expression in cell-based assays and in vivo preclinical models.

  11. Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

    PubMed

    Diaz, Mara R; Jacobson, James W; Goodwin, Kelly D; Dunbar, Sherry A; Fell, Jack W

    2010-06-01

    Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

  12. Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests

    PubMed Central

    Royo, Joaquín; León, José; Vancanneyt, Guy; Albar, Juan Pablo; Rosahl, Sabine; Ortego, Félix; Castañera, Pedro; Sánchez-Serrano, José J.

    1999-01-01

    De novo jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated depletion of its mRNA. LOX-H3 depletion largely abolishes accumulation of proteinase inhibitors on wounding, indicating that this specific LOX plays an instrumental role in the regulation of wound-induced gene expression. As a consequence, weight gain of Colorado potato beetles fed on antisense plants is significantly larger than those fed on wild-type plants. The poorer performance of LOX-H3-deficient plants toward herbivory is more evident with a polyphagous insect; larvae of beet armyworm reared on the antisense lines have up to 57% higher weight than those fed on nontransformed plants. LOX-H3 thus appears to regulate gene activation in response to pest attack, and this inducible response is likely to be a major determinant for reducing performance of nonspecialized herbivores. However, the regulatory role of LOX-H3 is not caused by its involvement in the wound-induced increase of JA, as wild-type and LOX-H3 deficient plants have similar jasmonate levels after wounding. LOX-H3-deficient plants have higher tuber yields. The apparent effect of suppressing the inducible defensive response on plant vigor suggests that it may pose a penalty in plant fitness under nonstress situations. PMID:9927708

  13. The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA.

    PubMed

    Dong, Haifeng; Ding, Lin; Yan, Feng; Ji, Hanxu; Ju, Huangxian

    2011-05-01

    A simple nanocarrier of polyethylenimine-grafted graphene nanoribbon (PEI-g-GNR) was proposed as an effective gene vector. The GNR was formed by longitudinally unzipping multiwalled carbon nanotubes (MWCNTs), and treated with strong acids and sonication to obtain surface carboxylic acid groups for graft of PEI via electrostatic assembly. The PEI-g-GNR appeared to protect locked nucleic acid modified molecular beacon (LNA-m-MB) probes from nuclease digestion or single-strand binding protein interaction, thus could be used as a nanocarrier of the probes for more efficient transfection of cells than PEI or PEI-g-MWCNTs due to the large surface area of the GNR and high charge density of PEI. The cytotoxicity and apoptosis induced by the PEI-g-GNR were negligible under optimal transfection conditions. Combining with the remarkable affinity and specificity of LNA to microRNA (miRNA), a delivery system by the LNA-m-MB/PEI-g-GNR was proposed for effectively transferring LNA-m-MB into the cells to recognize the target miRNA. Using HeLa cells as model, a method for detection of miRNA in single cell was developed. These results suggested that PEI-g-GNR would be a promising nonviral vector for in situ detection of gene in cytoplasm and gene therapy in clinical application.

  14. Intracerebroventricular Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuates Salt Appetite in the Rat.

    PubMed

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

    The anterior ventral third ventricle (AV3V) region of the brain contains high concentrations of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) that are important in the maintenance of body fluid and electrolyte balance as well as other physiological processes. Daily intracerebroventricular pulse injections of MR antisense oligonucleotides significantly suppressed deoxycorticosterone acetate (DOCA) induced salt appetite in a dose-related manner. Similar administration of GR antisense or scrambled/sense oligonucleotide into the third ventricle failed to inhibit salt appetite. Salt appetite aroused after adrenalectomy was not suppressed by MR antisense oligonucleotide treatments but was suppressed by an antisense oligonucleotide directed against the angiotensin II AT1 receptor subtype. Receptor binding analysis demonstrated that MR and GR oligonucleotide treatments each reduced their respective receptor subtypes. Finally, although GR antisense oligonucleotide treatment was ineffective in suppressing DOCA-induced salt appetite, this treatment did increase stress induced corticosterone release as well as delayed the recovery of corticosterone to basal levels after stress. PMID:9787254

  15. Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

    PubMed Central

    Krieg, A M; Tonkinson, J; Matson, S; Zhao, Q; Saxon, M; Zhang, L M; Bhanja, U; Yakubov, L; Stein, C A

    1993-01-01

    Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides. Images PMID:8430072

  16. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. PMID:26553470

  17. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  18. Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

    PubMed

    Shin, Kyong-Oh; Kim, Kunpyo; Jeon, Sanghun; Seo, Cho-Hee; Lee, Yong-Moon; Cho, Yunhi

    2015-10-01

    Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1. PMID:26233818

  19. Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

    PubMed

    Shin, Kyong-Oh; Kim, Kunpyo; Jeon, Sanghun; Seo, Cho-Hee; Lee, Yong-Moon; Cho, Yunhi

    2015-10-01

    Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1.

  20. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    SciTech Connect

    Kubo, Takanori; Yanagihara, Kazuyoshi; Takei, Yoshifumi; Mihara, Keichiro; Sato, Yuichiro; Seyama, Toshio

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  1. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  2. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA.

    PubMed

    Bendahmane, M; Gronenborn, B

    1997-01-01

    One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny. PMID:9037152

  3. Antisense overlapping open reading frames in genes from bacteria to humans.

    PubMed Central

    Merino, E; Balbás, P; Puente, J L; Bolívar, F

    1994-01-01

    Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far. PMID:8208617

  4. A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

    PubMed

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  5. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  6. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast

    PubMed Central

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y.; Brem, Rachel B.

    2016-01-01

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1. Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1. Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them. PMID:27190003

  7. Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology

    PubMed Central

    Diaz, Mara R.; Jacobson, James W.; Goodwin, Kelly D.; Dunbar, Sherry A.; Fell, Jack W.

    2010-01-01

    Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid–modified oligonucleotides (LNA) and Mirus Label IT® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5′ terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions. PMID:21165155

  8. Use of an Antisense RNA Strategy To Investigate the Functional Significance of Mn-Catalase in the Extreme Thermophile Thermus thermophilus

    PubMed Central

    Moreno, Renata; Hidalgo, Aurelio; Cava, Felipe; Fernández-Lafuente, Roberto; Guisán, José Manuel; Berenguer, José

    2004-01-01

    The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a “bicistronic” transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target. PMID:15516595

  9. Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

    PubMed

    Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

    2008-06-25

    Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays. PMID:18494480

  10. Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

    PubMed

    Salvi, Sergio; D'Orso, Fabio; Morelli, Giorgio

    2008-06-25

    Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.

  11. Delivery of antisense oligonucleotide to the cornea by iontophoresis.

    PubMed

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F

    2003-04-01

    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  12. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.

  13. Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime

    PubMed Central

    Readman, John B.; Dickson, George; Coldham, Nick G.

    2016-01-01

    Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and –17 to –5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates. PMID:27047482

  14. The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.

    PubMed

    Edwards, Mary E; Choo, Tze-Siang; Dickson, Cathryn A; Scott, Catherine; Gidley, Michael J; Reid, J S Grant

    2004-03-01

    Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

  15. Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

    NASA Astrophysics Data System (ADS)

    Shen, Yuefei

    This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made. The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled

  16. Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

    PubMed

    Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

    2010-01-01

    Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

  17. Impact of DNA gyrase inhibition by antisense ribozymes on rec A in E. coli.

    PubMed

    Shilpakala, Sainath Rao; Raghunathan, Malathi

    2009-09-01

    The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluoroquinolones.

  18. Nanoparticle-Delivered Antisense MicroRNA-21 Enhances the Effects of Temozolomide on Glioblastoma Cells.

    PubMed

    Ananta, Jeyarama S; Paulmurugan, Ramasamy; Massoud, Tarik F

    2015-12-01

    Glioblastoma (GBM) generally exhibits high IC50 values for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21) is an oncomiR overexpressed in GBM, thus controlling important aspects of glioma biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 to glioblastoma cells might beneficially knock down endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating antisense miR-21 were effective in intracellular delivery and sustained silencing (p < 0.01) of miR-21 function in U87 MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly reduced the number of viable cells (p < 0.001), and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ treatment in U87 MG cells. There was overexpression of the miR-21 target genes PTEN (by 67%) and caspase-3 (by 15%) upon cotreatment. This promising PLGA nanoparticle-based platform for antisense miR-21 delivery to GBM is an effective cotherapeutic strategy in cell culture, warranting the need for further studies prior to future clinical translation. PMID:26559642

  19. An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

    PubMed Central

    Subudhi, Amit Kumar; Boopathi, P.A.; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B.; Mugasimangalam, Raja C.; Sirohi, Paramendra; Kochar, Sanjay K.; Kochar, Dhanpat K.; Das, Ashis

    2014-01-01

    Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014). PMID:26484136

  20. Drug evaluation: ISIS-301012, an antisense oligonucleotide for the treatment of hypercholesterolemia.

    PubMed

    Burnett, John R

    2006-10-01

    ISIS-301012 is an antisense oligonucleotide inhibitor of apolipoprotein B-100, which is being developed by Isis Pharmaceuticals Inc for the potential treatment of hypercholesterolemia. A subcutaneous injectable formulation is currently undergoing phase 11 clinical trials, while phase I trials are underway with an oral formulation of the drug.

  1. Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription.

    PubMed

    Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J; van Hooff, Sander; van Leenen, Dik; Dargemont, Catherine; Géli, Vincent; Holstege, Frank C P

    2012-09-01

    Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by

  2. Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein.

    PubMed

    Lee, Richard G; Fu, Wuxia; Graham, Mark J; Mullick, Adam E; Sipe, Donna; Gattis, Danielle; Bell, Thomas A; Booten, Sheri; Crooke, Rosanne M

    2013-03-01

    Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr⁻/⁻ mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile. PMID:23220583

  3. Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

    PubMed

    Shen, Z J; Kim, S K; Kwon, O S; Lee, Y S; Moon, B J

    2001-04-01

    To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.

  4. Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma

    PubMed Central

    Andreyev, H; Ross, P; Cunningham, D; Clarke, P

    2001-01-01

    BACKGROUND—Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer.
AIMS—To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction.
METHODS—Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture.
RESULTS—The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively.
CONCLUSIONS—Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA.


Keywords: Ki-ras mutation; antisense treatment; colorectal carcinoma PMID:11156646

  5. Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

    1996-01-01

    By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

  6. Linear decay of retrotransposon antisense bias across genes is contingent upon tissue specificity.

    PubMed

    Linker, Sara; Hedges, Dale

    2013-01-01

    Retrotransposons comprise approximately half of the human genome and contribute to chromatin structure, regulatory motifs, and protein-coding sequences. Since retrotransposon insertions can disrupt functional genetic elements as well as introduce new sequence motifs to a region, they have the potential to affect the function of genes that harbour insertions as well as those nearby. Partly as a result of these effects, the distribution of retrotransposons across the genome is non-uniform and there are observed imbalances in the orientation of insertions with respect to the transcriptional direction of the containing gene. Although some of the factors underlying the observed distributions are understood, much of the variability remains unexplained. Detailed characterization of retrotransposon density in genes could help inform predictions of the functional consequence of de novo as well as polymorphic insertions. In order to characterize the relationship between genes and inserted elements, we have examined the distribution of retrotransposons and their internal motifs within tissue-specific and housekeeping genes. We have identified that the previously established retrotransposon antisense bias decays at a linear rate across genes, resulting in an equal density of sense and antisense retrotransposons near the 3'-UTR. In addition, the decay of antisense bias across genes is less pronounced among tissue-specific genes. Our results provide support for the scenario in which this linear decay in antisense bias is established by natural selection shortly after retrotransposon integration, and that total antisense bias observed is above and beyond any bias introduced by the integration process itself. Finally, we provide an example of a retrotransposon acting as an eQTL on a coincident gene, highlighting one of several possible avenues through which insertions may modulate gene function.

  7. Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

    PubMed Central

    Walton, S Patrick; Stephanopoulos, Gregory N; Yarmush, Martin L; Roth, Charles M

    2002-01-01

    Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed. PMID:11751323

  8. Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes.

    PubMed

    Movassaghian, Sara; Moghimi, Hamid R; Shirazi, Farshad H; Koshkaryev, Alexander; Trivedi, Malav S; Torchilin, Vladimir P

    2013-01-30

    The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques. PMID:23262426

  9. Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria.

    PubMed

    Furukawa, Ryo; Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

    2015-07-01

    Mitochondrial genome-targeting nucleic acids are promising therapeutic candidates for treating mitochondrial diseases. To date, a number of systems for delivering genetic information to the cytosol and the nucleus have been reported, and several successful gene therapies involving gene delivery targeted to the cytosol and the nucleus have been reported. However, much less progress has been made concerning mitochondrial gene delivery systems, and mitochondrial gene therapy has never been achieved. Here, we report on the mitochondrial delivery of an antisense RNA oligonucleotide (ASO) to perform mitochondrial RNA knockdown to regulate mitochondrial function. Mitochondrial delivery of the ASO was achieved using a combination of a MITO-Porter system, which contains mitochondrial fusogenic lipid envelopes for mitochondrial delivery via membrane fusion and D-arm, a mitochondrial import signal of tRNA to the matrix. Mitochondrial delivery of the ASO induces the knockdown of the targeted mitochondria-encoded mRNA and protein, namely cytochrome c oxidase subunit II, a component of the mitochondrial respiratory chain. Furthermore, the mitochondrial membrane potential was depolarized by the down regulation of the respiratory chain as the result of the mitochondrial delivery of ASO. This finding constitutes the first report to demonstrate that the nanocarrier-mediated mitochondrial genome targeting of antisense RNA effects mitochondrial function.

  10. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    SciTech Connect

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  11. Probable presence of an ubiquitous cryptic mitochondrial gene on the antisense strand of the cytochrome oxidase I gene

    PubMed Central

    2011-01-01

    Background Mitochondria mediate most of the energy production that occurs in the majority of eukaryotic organisms. These subcellular organelles contain a genome that differs from the nuclear genome and is referred to as mitochondrial DNA (mtDNA). Despite a disparity in gene content, all mtDNAs encode at least two components of the mitochondrial electron transport chain, including cytochrome c oxidase I (Cox1). Presentation of the hypothesis A positionally conserved ORF has been found on the complementary strand of the cox1 genes of both eukaryotic mitochondria (protist, plant, fungal and animal) and alpha-proteobacteria. This putative gene has been named gau for gene antisense ubiquitous in mtDNAs. The length of the deduced protein is approximately 100 amino acids. In vertebrates, several stop codons have been found in the mt gau region, and potentially functional gau regions have been found in nuclear genomes. However, a recent bioinformatics study showed that several hypothetical overlapping mt genes could be predicted, including gau; this involves the possible import of the cytosolic AGR tRNA into the mitochondria and/or the expression of mt antisense tRNAs with anticodons recognizing AGR codons according to an alternative genetic code that is induced by the presence of suppressor tRNAs. Despite an evolutionary distance of at least 1.5 to 2.0 billion years, the deduced Gau proteins share some conserved amino acid signatures and structure, which suggests a possible conserved function. Moreover, BLAST analysis identified rare, sense-oriented ESTs with poly(A) tails that include the entire gau region. Immunohistochemical analyses using an anti-Gau monoclonal antibody revealed strict co-localization of Gau proteins and a mitochondrial marker. Testing the hypothesis This hypothesis could be tested by purifying the gau gene product and determining its sequence. Cell biological experiments are needed to determine the physiological role of this protein. Implications of

  12. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    PubMed

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  13. Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

    PubMed Central

    Datta, Santanu; Shandil, Radha Krishan; Kumar, Naveen; Robert, Nanduri; Sokhi, Upneet K.; Guptha, Supreeth; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2016-01-01

    One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. Mycobacterium tuberculosis (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro vs. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of rpoB>aroK>ppk>rpoC>ilvB. RpoB was used as the cidality control. In-vitro and in-vivo studies feature aroK (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute

  14. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligos

    PubMed Central

    Sztainberg, Yehezkel; Chen, Hong-mei; Swann, John W.; Hao, Shuang; Tang, Bin; Wu, Zhenyu; Tang, Jianrong; Wan, Ying-Wooi; Liu, Zhandong; Rigo, Frank; Zoghbi, Huda Y.

    2015-01-01

    Copy number variations have been frequently associated with developmental delay, intellectual disability, and autism spectrum disorders1. MECP2 duplication syndrome is one of the most common genomic rearrangements in males2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections, and early death3–5. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question, however, was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders6–8, including loss of MeCP2 in Rett syndrome9, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we hypothesized that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. Therefore, we first generated and characterized a conditional Mecp2-overexpressing mouse model and showed that correction of MeCP2 levels largely reversed the behavioral, molecular, and electrophysiological deficits. Next, we sought a translational strategy to reduce MeCP2 and turned to antisense oligonucleotides (ASOs). ASOs are small modified nucleic acids that can selectively hybridize with mRNA transcribed from a target gene and silence it10,11, and have been successfully used to correct deficits in different mouse models12–18. We found that ASO treatment induced a broad phenotypic rescue in adult symptomatic transgenic MECP2

  15. Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

    PubMed

    Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

    2009-06-01

    Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that

  16. Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

    PubMed Central

    Hanecak, R; Brown-Driver, V; Fox, M C; Azad, R F; Furusako, S; Nozaki, C; Ford, C; Sasmor, H; Anderson, K P

    1996-01-01

    Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and

  17. On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23.

    PubMed

    Nicole, L M; Tanguay, R M

    1987-03-01

    The specificity of action of antisense RNA for one of Drosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translation in vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45 degrees C) and low stringency (37 degrees C) conditions. This suggests that the 5' leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

  18. Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling

    PubMed Central

    2009-01-01

    Background The serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco. Results Over expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility. Conclusion We have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism. PMID:19689802

  19. LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging.

    PubMed

    Roy, Kislay; Kanwar, Rupinder K; Kanwar, Jagat R

    2015-12-01

    This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin

  20. Antisense-mediated exon skipping: taking advantage of a trick from Mother Nature to treat rare genetic diseases.

    PubMed

    Veltrop, Marcel; Aartsma-Rus, Annemieke

    2014-07-01

    Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense oligonucleotide treatment to the splicing thus has therapeutic potential for many rare diseases. In this review we will focus on the state of the art on exon skipping using antisense oligonucleotides as a potential therapy for rare genetic diseases, outlining how this versatile approach can be exploited to correct for different mutations.

  1. Strand-specific community RNA-seq reveals prevalent and dynamic antisense transcription in human gut microbiota

    PubMed Central

    Bao, Guanhui; Wang, Mingjie; Doak, Thomas G.; Ye, Yuzhen

    2015-01-01

    Metagenomics and other meta-omics approaches (including metatranscriptomics) provide insights into the composition and function of microbial communities living in different environments or animal hosts. Metatranscriptomics research provides an unprecedented opportunity to examine gene regulation for many microbial species simultaneously, and more importantly, for the majority that are unculturable microbial species, in their natural environments (or hosts). Current analyses of metatranscriptomic datasets focus on the detection of gene expression levels and the study of the relationship between changes of gene expression and changes of environment. As a demonstration of utilizing metatranscriptomics beyond these common analyses, we developed a computational and statistical procedure to analyze the antisense transcripts in strand-specific metatranscriptomic datasets. Antisense RNAs encoded on the DNA strand opposite a gene’s CDS have the potential to form extensive base-pairing interactions with the corresponding sense RNA, and can have important regulatory functions. Most studies of antisense RNAs in bacteria are rather recent, are mostly based on transcriptome analysis, and have been applied mainly to single bacterial species. Application of our approaches to human gut-associated metatranscriptomic datasets allowed us to survey antisense transcription for a large number of bacterial species associated with human beings. The ratio of protein coding genes with antisense transcription ranges from 0 to 35.8% (median = 10.0%) among 47 species. Our results show that antisense transcription is dynamic, varying between human individuals. Functional enrichment analysis revealed a preference of certain gene functions for antisense transcription, and transposase genes are among the most prominent ones (but we also observed antisense transcription in bacterial house-keeping genes). PMID:26388849

  2. PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes.

    PubMed

    Wei, N; Wang, Y; Xu, R-X; Wang, G-Q; Xiong, Y; Yu, T-Y; Yang, G-S; Pang, W-J

    2015-04-01

    Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin-like phospholipase domain containing 2 (PNPLA2)] and hormone-sensitive lipase [HSL; also known as lipase, hormone-sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through

  3. Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

    PubMed

    Dean, Zachary S; Elias, Paul; Jamilpour, Nima; Utzinger, Urs; Wong, Pak Kin

    2016-09-01

    Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

  4. Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity

    PubMed Central

    Serobyan, Vahan; Xiao, Hua; Namdeo, Suryesh; Rödelsperger, Christian; Sieriebriennikov, Bogdan; Witte, Hanh; Röseler, Waltraud; Sommer, Ralf J.

    2016-01-01

    Phenotypic plasticity has been suggested to act through developmental switches, but little is known about associated molecular mechanisms. In the nematode Pristionchus pacificus, the sulfatase eud-1 was identified as part of a developmental switch controlling mouth-form plasticity governing a predatory versus bacteriovorous mouth-form decision. Here we show that mutations in the conserved histone-acetyltransferase Ppa-lsy-12 and the methyl-binding-protein Ppa-mbd-2 mimic the eud-1 phenotype, resulting in the absence of one mouth-form. Mutations in both genes cause histone modification defects and reduced eud-1 expression. Surprisingly, Ppa-lsy-12 mutants also result in the down-regulation of an antisense-eud-1 RNA. eud-1 and antisense-eud-1 are co-expressed and further experiments suggest that antisense-eud-1 acts through eud-1 itself. Indeed, overexpression of the antisense-eud-1 RNA increases the eud-1-sensitive mouth-form and extends eud-1 expression. In contrast, this effect is absent in eud-1 mutants indicating that antisense-eud-1 positively regulates eud-1. Thus, chromatin remodelling and antisense-mediated up-regulation of eud-1 control feeding plasticity in Pristionchus. PMID:27487725

  5. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    PubMed Central

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  6. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    PubMed

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  7. An in vivo and in silico approach to study cis-antisense: a short cut to higher order response

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen; Varanasi, Usha; Chatterjee, Anushree

    2014-03-01

    Antisense interactions are present in all domains of life. Typically sense, antisense RNA pairs originate from overlapping genes with convergent face to face promoters, and are speculated to be involved in gene regulation. Recent studies indicate the role of transcriptional interference (TI) in regulating expression of genes in convergent orientation. Modeling antisense, TI gene regulation mechanisms allows us to understand how organisms control gene expression. We present a modeling and experimental framework to understand convergent transcription that combines the effects of transcriptional interference and cis-antisense regulation. Our model shows that combining transcriptional interference and antisense RNA interaction adds multiple-levels of regulation which affords a highly tunable biological output, ranging from first order response to complex higher-order response. To study this system we created a library of experimental constructs with engineered TI and antisense interaction by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences to control expression of a set of fluorescent reporter proteins. Studying this gene expression mechanism allows for an understanding of higher order behavior of gene expression networks.

  8. Composition of photosystem II antenna in light-harvesting complex II antisense tobacco plants at varying irradiances.

    PubMed Central

    Flachmann, R

    1997-01-01

    Plants with genes coding for chlorophyll a/b-binding proteins of light-harvesting complex II (LHCII) in antisense orientation (Lhcb) that are characterized by severely reduced Lhcb transcript levels (below 10% of wild type) do not show a bleached phenotype due to a specific loss of the polypeptide. To produce such a phenotype, a conceptually different antisense approach was tested with a dual-functional transcript encoding the gene for hygromycin phosphotransferase and the transit sequence of Lhcb1-2 in the antisense orientation. Using increasing concentrations of hygromycin, transformants with Lhcb steady-state levels as low as 9% of wild type were regenerated and grown in a growth chamber. Together with Lhcb antisense plants obtained in an earlier study, these antisense plants were analyzed biochemically for their photosystem II (PSII) antenna composition under varying light conditions. All antisense plants showed a characteristic low-irradiance-induced increase of their PSII antenna size as determined by higher chlorophyll concentrations, an increased content of LHCII, and a constant chlorophyll b-to-lutein ratio in comparison with control plants. One to 5% of the total Lhcb transcript amount was sufficient to allow unrestricted formation of the PSII antenna at low irradiance, suggesting that LHCH biogenesis is not controlled primarily by transcription. PMID:9085572

  9. Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene.

    PubMed

    Shiba, H; Kimura, N; Takayama, S; Hinata, K; Suzuki, A; Isogai, A

    2000-05-01

    Self-incompatible (SI) Brassica rapa (syn. B. campestris) was transformed with an antisense SLG gene by using SLG8 cDNA isolated from the B. campestris S8 homozygote. Two transformed lines were obtained and analyzed. Northern blot and Western blot analyses revealed that endogenous SLG and SRK were greatly reduced of the transcriptional and translational levels in the transformant. Pollination experiments confirmed that their SI phenotype had broken down. In addition, the progeny with the antisense SLG gene, resulting from self- or cross-pollination of the transgenic plant, also showed the self-compatible phenotype. The breakdown of SI in the tranformants was due to the change in property of the stigma and not of the pollen. These results provide strong evidence that SLG and/or SRK is implicated in the pollen-stigma recognition of SI and that they act only as stigmatic factors.

  10. Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B

    PubMed Central

    Crooke, Stanley T; Geary, Richard S

    2013-01-01

    Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50–400 mg week−1, both as a single agent and in the presence of maximal lipid lowering therapy. No drug–drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. PMID:23013161

  11. Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription

    PubMed Central

    Marquardt, Sebastian; Raitskin, Oleg; Wu, Zhe; Liu, Fuquan; Sun, Qianwen; Dean, Caroline

    2014-01-01

    Summary Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms. PMID:24725596

  12. Mechanistic principles of antisense targets for the treatment of spinal muscular atrophy.

    PubMed

    Singh, Natalia N; Lee, Brian M; DiDonato, Christine J; Singh, Ravindra N

    2015-01-01

    Spinal muscular atrophy (SMA) is a major neurodegenerative disorder of children and infants. SMA is primarily caused by low levels of SMN protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of the production of the functional SMN protein due to predominant skipping of exon 7. Several compounds, including antisense oligonucleotides (ASOs) that elevate SMN protein from SMN2 hold the promise for treatment. An ASO-based drug currently under Phase III clinical trial employs intronic splicing silencer N1 (ISS-N1) as its target. Cumulative studies on ISS-N1 reveal a wealth of information with significance to the overall therapeutic development for SMA. Here, the authors summarize the mechanistic principles behind various antisense targets currently available for SMA therapy.

  13. The role of antisense oligonucleotide therapy in patients with familial hypercholesterolemia: risks, benefits, and management recommendations.

    PubMed

    Agarwala, Anandita; Jones, Peter; Nambi, Vijay

    2015-01-01

    Antisense oligonucleotide therapy is a promising approach for the treatment of a broad variety of medical conditions. It functions at the cellular level by interfering with RNA function, often leading to degradation of specifically targeted abnormal gene products implicated in the disease process. Mipomersen is a novel antisense oligonucleotide directed at apolipoprotein (apoB)-100, the primary apolipoprotein associated with low-density lipoprotein cholesterol (LDL-C), which has recently been approved for the treatment of familial hypercholesterolemia. A number of clinical studies have demonstrated its efficacy in lowering LDL-C and apoB levels in patients with elevated LDL-C despite maximal medical therapy using conventional lipid-lowering agents. This review outlines the risks and benefits of therapy and provides recommendations on the use of mipomersen.

  14. Mechanistic principles of antisense targets for the treatment of Spinal Muscular Atrophy

    PubMed Central

    Singh, Natalia N.; Lee, Brian M.; DiDonato, Christine J.; Singh, Ravindra N.

    2015-01-01

    Spinal muscular atrophy (SMA) is a major neurodegenerative disorder of children and infants. SMA is primarily caused by low levels of SMN protein owing to deletions or mutations of the survival motor neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of the production of the functional SMN protein due to predominant skipping of exon 7. Several compounds, including antisense oligonucleotides (ASOs) that elevate SMN protein from SMN2 hold the promise for treatment. An ASO-based drug currently under phase 3 clinical trial employs intronic splicing silencer N1 (ISS-N1) as its target. Cumulative studies on the ISS-N1 reveal a wealth of information with significance to the overall therapeutic development for SMA. Here we summarize the mechanistic principles behind various antisense targets currently available for SMA therapy. PMID:26381381

  15. Kv1.1 channel antisense attenuates learning and modulation of dentate polysialylated NCAM.

    PubMed

    Gratacós, E; Ghelardini, C; Gherardini, L M; Galeotti, N; Murphy, K J; Bartolini, A; Regan, C M

    1998-08-24

    The distribution and modulation of neural cell adhesion molecule polysialylation state (NCAM PSA) and the consequence of antisense inactivation of the Kv1.1 potassium channel was investigated following avoidance learning in mice. PSA immunoreactivity was most notable on cells at the inner denate border and in cortical layer II. Task acquisition resulted in a significant 30% transient increase in the frequency of dentate polysialylated neurons at the 12 h post-training time. In contrast, animals pretreated with the Kv1.1 antisense oligonucleotide exhibited both attenuated recall avoidance latencies and polysialylated cell frequency. As Kv1.1 is enriched on the dendrites of these granule-like cells, the attenuated polysialylation response is considered secondary to NCAM-mediated events during their transient synapse production in the 6-8 h post-training period.

  16. Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.

    PubMed Central

    Maher, L J; Dolnick, B J

    1988-01-01

    Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage. Images PMID:2836793

  17. NOX2 Antisense Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis in Cardiomyocyte

    PubMed Central

    Yu, Bo; Meng, Fanbo; Yang, Yushuang; Liu, Dongna; Shi, Kaiyao

    2016-01-01

    Heart ischemia is a hypoxia related disease. NOX2 and HIF-1α proteins were increased in cardiomyocytes after acute myocardial infarction. However, the relationship of the hypoxia-induced HIF-1α. NOX2-derived oxidative stress and apoptosis in cardiomyocyte remains unclear. In the current study, we use NOX2 antisense strategy to investigate the role of NOX2 in hypoxia-induced oxidative stress and apoptosis in rat cardiomyocytes. Here, we show that transduction of ADV-NOX2-AS induces potent silencing of NOX2 in cardiomyocytes, and resulting in attenuation of hypoxia-induced oxidative stress and apoptosis. This study indicates the potential of antisense-based therapies and validates NOX2 as a potent therapeutic candidate for heart ischemia. PMID:27499697

  18. Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids.

    PubMed

    Zened, A; Troegeler-Meynadier, A; Nicot, M C; Combes, S; Cauquil, L; Farizon, Y; Enjalbert, F

    2011-11-01

    Trans isomers of fatty acids exhibit different health properties. Among them, trans-10,cis-12 conjugated linoleic acid has negative effects on milk fat production and can affect human health. A shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of dairy cows receiving high-concentrate diets, especially when the diet is supplemented with highly unsaturated fat sources. The differences of BH patterns between linoleic acid (LeA) and linolenic acid (LnA) in such ruminal conditions remain unknown; thus, the aim of this work was to investigate in vitro the effects of starch and sunflower oil in the diet of the donor cows and starch level in the incubates on the BH patterns and efficiencies of LeA and LnA. The design was a 4 × 4 Latin square design with 4 cows, 4 periods, and 4 diets with combinations of 21 or 34% starch and 0 or 5% sunflower oil. The rumen content of each cow during each period was incubated with 4 substrates, combining 2 starch levels and either LeA or LnA addition. Capillary electrophoresis single-strand conformation polymorphism of incubates showed that dietary starch decreased the diversity of the bacterial community and the high-starch plus oil diet modified its structure. High-starch diets poorly affected isomerization and first reduction of LeA and LnA, but decreased the efficiencies of trans-11,cis-15-C18:2 and trans C18:1 reduction. Dietary sunflower oil increased the efficiency of LeA isomerization but decreased the efficiency of trans C18:1 reduction. An interaction between dietary starch and dietary oil resulted in the highest trans-10 isomers production in incubates when the donor cow received the high-starch plus oil diet. The partition between trans-10 and trans-11 isomers was also affected by an interaction between starch level and the fatty acid added to the incubates, showing that the trans-10 shift only occurred with LeA, whereas LnA was mainly hydrogenated via the more usual trans-11

  19. Antisense Modulation of RNA Processing as a Therapeutic Approach in Cancer Therapy

    PubMed Central

    Spraggon, Lee

    2013-01-01

    Next-generation antisense technologies are re-emerging as viable and powerful approaches to the treatment of several genetic diseases. Similar strategies are also being applied to cancer therapy. Re-programming of the expression of endogenous oncogenic products to replace them with functional antagonists, by interfering with alternative splicing or polyadenylation, provides a promising novel approach to address acquired drug resistance and previously undruggable targets. PMID:25589899

  20. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides.

    PubMed

    Adachi, Shungo; Natsume, Tohru

    2015-01-01

    To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex.

  1. Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

    PubMed Central

    Mundodi, V; Kucknoor, AS; Alderete, JF

    2007-01-01

    Background Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. Results We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. Conclusion These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis. PMID:17608941

  2. Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex

    PubMed Central

    Kralovicova, Jana; Lages, Ana; Patel, Alpa; Dhir, Ashish; Buratti, Emanuele; Searle, Mark; Vorechovsky, Igor

    2014-01-01

    Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression. PMID:24944197

  3. Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2014-06-01

    Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in

  4. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    PubMed

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  5. Splicing of a non-coding antisense transcript controls LEF1 gene expression

    PubMed Central

    Beltran, Manuel; Aparicio-Prat, Estel; Mazzolini, Rocco; Millanes-Romero, Alba; Massó, Pere; Jenner, Richard G.; Díaz, Víctor M.; Peiró, Sandra; de Herreros, Antonio García

    2015-01-01

    In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms. PMID:25990740

  6. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

  7. Intrathecal PLC(β3) oligodeoxynucleotides antisense potentiates acute morphine efficacy and attenuates chronic morphine tolerance.

    PubMed

    Quanhong, Zhou; Ying, Xue; Moxi, Chen; Tao, Xu; Jing, Wang; Xin, Zhang; Li, Wang; Derong, Cui; Xiaoli, Zhang; Wei, Jiang

    2012-09-01

    Morphine is a mainstay for chronic pain treatment, but its efficacy has been hampered by physical tolerance. The underlying mechanism for chronic morphine induced tolerance is complicated and not well understood. PLC(β3) is regarded as an important factor in the morphine tolerance signal pathway. In this study, we determined intrathecal (i.t.) administration of an antisense oligodeoxynucleotide (ODN) of PLC(β3) could quicken the on-set antinociceptive efficacy of acute morphine treatment and prolong the maximum effect up to 4h. The antisense could also attenuate the development of morphine-induced tolerance and left shift the ED50 after 7 day of coadministration with morphine. These results probably were contributed by the PLC(β3) antisense ODN as they successfully knocked down protein expression levels and reduced activity of PLC(β3) in spinal cord in rats. The mismatch group had no such effects. The results confirmed the important involvement of PLC(β3) in both acute morphine efficacy and chronic morphine tolerance at spinal level in rats. This study may provide an idea for producing a novel adjuvant for morphine treatment.

  8. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. PMID:26534950

  9. Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

    PubMed Central

    Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2003-01-01

    Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432

  10. α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans.

    PubMed

    Lefimil, C; Jedlicki, E; Holmes, D S

    2014-03-01

    A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

  11. An antisense RNA that governs the expression kinetics of a multifunctional virulence gene

    PubMed Central

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

    Summary Genome-wide transcriptome analyses of several bacterial species have recently uncovered a hitherto unappreciated amount of antisense transcription. However, the physiological role, regulation and significance of such antisense transcripts are presently unclear. We now report the identification of a cis-encoded 1.2 kb long antisense RNA – termed AmgR – that is complementary to the mgtC portion of the mgtCBR polycistronic message from Salmonella enterica. The mgtCBR mRNA specifies the MgtC protein, which is necessary for survival within macrophages, virulence in mice and growth in low Mg2+; the Mg2+ transporter MgtB with no apparent role in virulence; and the membrane peptide MgtR mediating MgtC degradation. Expression of AmgR dimished both MgtC and MgtB protein levels in a process requiring RNase E but independent of RNase III, the RNA chaperone Hfq, and the regulatory peptide MgtR. Inactivation of the chromosomal amgR promoter increased MgtC and MgtB protein levels and enhanced Salmonella virulence. Surprisingly, AmgR transcription is governed by the regulatory protein PhoP, which also directs transcription of the sense mgtCBR mRNA. AmgR may function as a timing device that alters MgtC and MgtB levels after the onset of PhoP-inducing conditions. PMID:20398218

  12. Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

    PubMed Central

    Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J

    1994-01-01

    We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites. Images PMID:8058724

  13. Chemistry and biochemistry of 2',5'-oligoadenylate-based antisense strategy.

    PubMed

    Adah, S A; Bayly, S F; Cramer, H; Silverman, R H; Torrence, P F

    2001-08-01

    This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described. PMID:11472236

  14. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    PubMed

    Nakashima, Nobutaka; Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

    2014-01-01

    We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

  15. Inhibition of the synthesis of a cytochrome-c-oxidase subunit isoform by antisense RNA.

    PubMed

    Sandonà, D; Bisson, R

    1994-02-01

    To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity. PMID:8112318

  16. Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells. PMID:26986720

  17. A computational analysis of antisense off-targets in prokaryotic organisms.

    PubMed

    Worley-Morse, Thomas O; Gunsch, Claudia K

    2015-02-01

    The adoption of antisense gene silencing as a novel disinfectant for prokaryotic organisms is hindered by poor silencing efficiencies. Few studies have considered the effects of off-targets on silencing efficiencies, especially in prokaryotic organisms. In this computational study, a novel algorithm was developed that determined and sorted the number of off-targets as a function of alignment length in Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv. The mean number of off-targets per a single location was calculated to be 14.1 ± 13.3 and 36.1 ± 58.5 for the genomes of E. coli K-12 MG1655 and M. tuberculosis H37Rv, respectively. Furthermore, when the entire transcriptome was analyzed, it was found that there was no general gene location that could be targeted to minimize or maximize the number of off-targets. In an effort to determine the effects of off-targets on silencing efficiencies, previously published studies were used. Analyses with acpP, ino1, and marORAB revealed a statistically significant relationship between the number of short alignment length off-targets hybrids and the efficacy of the antisense gene silencing, suggesting that the minimization of off-targets may be beneficial for antisense gene silencing in prokaryotic organisms. PMID:25486012

  18. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    SciTech Connect

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

    2010-03-12

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  19. The first crystal structures of RNA–PNA duplexes and a PNA-PNA duplex containing mismatches—toward anti-sense therapy against TREDs

    PubMed Central

    Kiliszek, Agnieszka; Banaszak, Katarzyna; Dauter, Zbigniew; Rypniewski, Wojciech

    2016-01-01

    PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA–PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA–PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA–RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson–Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA. PMID:26717983

  20. Role of Omega-3 Polyunsaturated Fatty Acids in the Production of Prostaglandin E2 and Nitric Oxide during Experimental Murine Paracoccidioidomycosis

    PubMed Central

    Sargi, S. C.; Dalalio, M. M. O.; Moraes, A. G.; Visentainer, J. E. L.; Morais, D. R.; Visentainer, J. V.

    2013-01-01

    There has recently been increased interest in the potential health effects of omega-3 polyunsaturated fatty acids on the immune system. Paracoccidioidomycosis is the most important endemic mycosis in Latin America. Macrophages have a fundamental role and act as first line of organism defense. The purpose of this study was to analyze the effect of n-3 fatty acids on the production of PGE2 and NO by mice infected with Pb18 and fed a diet enriched with LNA for 8 weeks. To study the effect of omega-3 fatty acids on macrophage activity during experimental paracoccidioidomycosis, mice were infected with Pb18 and fed a diet supplemented with LNA. PGE2 in the serum of animals was analyzed and NO in the supernatants of macrophages cultured and challenged in vitro with Pb18 was measured. Omega-3 fatty acids seemed to decrease the production of PGE2 in vivo in the infected group fed an LNA-supplemented diet during the 4th and 8th weeks of the experiment. At the same time, we observed an increase in synthesis of NO by peritoneal macrophages in this group. Omega-3 fatty acids thus appear to have an immunomodulatory effect in paracoccidioidomycosis. PMID:24455741

  1. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

    PubMed

    Mitsudome, Yuya; Takahama, Mamiko; Hirose, Jun; Yoshida, Naoto

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for

  2. Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes.

    PubMed

    Lu, Xueguang; Jia, Fei; Tan, Xuyu; Wang, Dali; Cao, Xueyan; Zheng, Jiamin; Zhang, Ke

    2016-07-27

    Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics. PMID:27420413

  3. Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

    PubMed Central

    Jin, Hailing; Vacic, Vladimir; Girke, Thomas; Lonardi, Stefano; Zhu, Jian-Kang

    2008-01-01

    Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs). The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs) via the RNA interference (RNAi) pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1) inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2) generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP) and Massively Parallel Signature Sequencing (MPSS) small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64%) protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or mitochondrion

  4. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

    PubMed Central

    Nishimura, Yoshiki; Kikis, Elise A.; Zimmer, Sara L.; Komine, Yutaka; Stern, David B.

    2004-01-01

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5

  5. Oligonucleotides antisense to the interleukin 1 receptor mRNA block the effects of interleukin 1 in cultured murine and human fibroblasts and in mice.

    PubMed Central

    Burch, R M; Mahan, L C

    1991-01-01

    Phosphodiester and phosphorothioate oligodeoxynucleotides (18 mers) were constructed antisense to sequences of the recently cloned murine and human IL-1 receptors. Murine antisense oligonucleotides inhibited IL-1-stimulated PGE2 synthesis by murine fibroblasts in culture in a time (days) and concentration-dependent (3 microM-30 microM) fashion. Murine sense oligonucleotide and an oligonucleotide antisense to human IL-1 receptor were without effect. Moreover, murine antisense oligonucleotides did not affect tumor necrosis factor- or bradykinin-stimulated PGE2 synthesis by murine fibroblasts. Similarly, antisense oligonucleotides to the human, but not the murine, IL-1 receptor inhibited IL-1-stimulated PGE2 synthesis by cultured human fibroblasts. The attenuation of the cellular response to IL-1 caused by the antisense oligonucleotides correlated with a loss in cell surface receptors for IL-1, without any change in the number of bradykinin receptors on these cells. When antisense oligonucleotides were encapsulated in liposomes, they blocked completely the appearance of newly synthesized IL-1 receptors and IL-1-stimulated PGE2 synthesis. In mice, subcutaneous injection with an oligonucleotide antisense to the murine IL-1 receptor markedly inhibited the infiltration of neutrophils in response to subsequent injection of IL-1. These data suggest that antisense oligodeoxynucleotides may share a role in the design of antiinflammatory therapeutics. Images PMID:1833422

  6. Effects of antisense-mediated inhibition of 11β-hydroxysteroid dehydrogenase type 1 on hepatic lipid metabolism[S

    PubMed Central

    Li, Guoping; Hernandez-Ono, Antonio; Crooke, Rosanne M.; Graham, Mark J.; Ginsberg, Henry N.

    2011-01-01

    11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) converts inactive 11-keto derivatives to active glucocorticoids within tissues and may play a role in the metabolic syndrome (MS). We used an antisense oligonucleotide (ASO) to knock down 11β-HSD1 in livers of C57BL/6J mice consuming a Western-type diet (WTD). 11β-HSD1 ASO-treated mice consumed less food, so we compared them to ad libitum-fed mice and to food-matched mice receiving control ASO. Knockdown of 11β-HSD1 directly protected mice from WTD-induced steatosis and dyslipidemia by reducing synthesis and secretion of triglyceride (TG) and increasing hepatic fatty acid oxidation. These changes in hepatic and plasma lipids were not associated with reductions in genes involved in de novo lipogenesis. However, protein levels of both sterol regulatory element-binding protein (SREBP) 1 and fatty acid synthase were significantly reduced in mice treated with 11β-HSD1 ASO. There was no change in hepatic secretion of apolipoprotein (apo)B, indicating assembly and secretion of smaller apoB-containing lipoproteins by the liver in the 11β-HSD1-treated mice. Our results indicate that inhibition of 11β-HSD1 by ASO treatment of WTD-fed mice resulted in improved plasma and hepatic lipid levels, reduced lipogenesis by posttranslational regulation, and secretion of similar numbers of apoB-containing lipoproteins containing less TG per particle. PMID:21364201

  7. Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic

    PubMed Central

    Moreno, Pedro M. D.; Pêgo, Ana P.

    2014-01-01

    Under clinical development since the early 90's and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics has not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given toward a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field. PMID:25353019

  8. Therapeutic Antisense Oligonucleotides against Cancer: Hurdling to the Clinic

    NASA Astrophysics Data System (ADS)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

    Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  9. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

    PubMed Central

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-01-01

    Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

  10. Inhibition of the alternative complement pathway by antisense oligonucleotides targeting complement factor B improves lupus nephritis in mice.

    PubMed

    Grossman, Tamar R; Hettrick, Lisa A; Johnson, Robert B; Hung, Gene; Peralta, Raechel; Watt, Andrew; Henry, Scott P; Adamson, Peter; Monia, Brett P; McCaleb, Michael L

    2016-06-01

    Systemic lupus erythematosus is an autoimmune disease that manifests in widespread complement activation and deposition of complement fragments in the kidney. The complement pathway is believed to play a significant role in the pathogenesis and in the development of lupus nephritis. Complement factor B is an important activator of the alternative complement pathway and increasing evidence supports reducing factor B as a potential novel therapy to lupus nephritis. Here we investigated whether pharmacological reduction of factor B expression using antisense oligonucleotides could be an effective approach for the treatment of lupus nephritis. We identified potent and well tolerated factor B antisense oligonucleotides that resulted in significant reductions in hepatic and plasma factor B levels when administered to normal mice. To test the effects of factor B antisense oligonucleotides on lupus nephritis, we used two different mouse models, NZB/W F1 and MRL/lpr mice, that exhibit lupus nephritis like renal pathology. Antisense oligonucleotides mediated reductions in circulating factor B levels were associated with significant improvements in renal pathology, reduced glomerular C3 deposition and proteinuria, and improved survival. These data support the strategy of using factor B antisense oligonucleotides for treatment of lupus nephritis in humans.

  11. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

    PubMed Central

    Kuraoka, Mutsuki; Lee, Joshua J.A.; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  12. Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

    PubMed Central

    Zwicky, Roman; Müntener, Kathrin; Goldring, Mary B; Baici, Antonio

    2002-01-01

    Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies. PMID:12086583

  13. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    PubMed

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  14. Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

    PubMed

    Boopathi, P A; Subudhi, Amit Kumar; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Chand, Bipin; Mugasimangalam, Raja C; Kochar, Sanjay K; Sirohi, Parmendra; Kochar, Dhanpat K; Das, Ashis

    2013-12-01

    Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

  15. The ICAM-1 antisense oligonucleotide ISIS-3082 prevents the development of postoperative ileus in mice.

    PubMed

    The, Frans O; de Jonge, Wouter J; Bennink, Roel J; van den Wijngaard, Rene M; Boeckxstaens, Guy E

    2005-09-01

    Intestinal manipulation (IM) during abdominal surgery triggers the influx of inflammatory cells, leading to postoperative ileus. Prevention of this local muscle inflammation, using intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1-specific antibodies, has been shown to shorten postoperative ileus. However, the therapeutic use of antibodies has considerable disadvantages. The aim of the current study was to evaluate the effect of ISIS-3082, a mouse-specific ICAM-1 antisense oligonucleotide, on postoperative ileus in mice. Mice underwent a laparotomy or a laparotomy combined with IM after treatment with ICAM-1 antibodies, 0.1-10 mg kg(-1) ISIS-3082, saline or ISIS-8997 (scrambled control antisense oligonucleotides, 1 and 3 mg kg(-1)). At 24 h after surgery, gastric emptying of a 99mTC labelled semi-liquid meal was determined using scintigraphy. Intestinal inflammation was assessed by myeloperoxidase (MPO) activity in ileal muscle whole mounts. IM significantly reduced gastric emptying compared to laparotomy. Pretreatment with ISIS-3082 (0.1-1 mg kg(-1)) as well as ICAM-1 antibodies (10 mg kg(-1)), but not ISIS-8997 or saline, improved gastric emptying in a dose-dependent manner. This effect diminished with higher doses of ISIS-3082 (3-10 mg kg(-1)). Similarly, ISIS-3082 (0.1-1 mg kg(-1)) and ICAM-1 antibodies, but not ISIS-8997 or higher doses of ISIS-3082 (3-10 mg kg(-1)), reduced manipulation-induced inflammation. Immunohistochemistry showed reduction of ICAM-1 expression with ISIS-3082 only. ISIS-3082 pretreatment prevents postoperative ileus in mice by reduction of manipulation-induced local intestinal muscle inflammation. Our data suggest that targeting ICAM-1 using antisense oligonucleotides may represent a new therapeutic approach to the prevention of postoperative ileus.

  16. Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene.

    PubMed

    Sohrab, Sayed Sartaj; Kamal, Mohammad A; Ilah, Abdul; Husen, Azamal; Bhattacharya, P S; Rana, D

    2016-05-01

    Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV. PMID:27081361

  17. Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

    PubMed Central

    Sohrab, Sayed Sartaj; Kamal, Mohammad A.; Ilah, Abdul; Husen, Azamal; Bhattacharya, P.S.; Rana, D.

    2014-01-01

    Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV. PMID:27081361

  18. Antisense molecular beacon strategy for in situ visualization of snRNA and fibrillarin protein interaction in Giardia lamblia.

    PubMed

    Ganguly, Sandipan; Ghosh, Srikanta; Chattopadhyay, Dhrubajyoti; Das, Pradeep

    2004-05-01

    Use of confocal microscopy has provided many recent developments in the study of functional aspects, especially localization and distribution of proteins, DNA and RNA within the cells. In the present investigation, we have applied for the first time, antisense molecular beacon based Fluorescence Resonance Energy Transfer (FRET) and Flow Cytometric Energy Transfer (FCET) techniques to demonstrate binding and co-localization of fibrillarin protein with small nuclear RNA (snRNA) to form ribonucleoprotein particle (RNPP) complex in Giardia lamblia. It has been observed by FRET and FCET that energy transfer occurs from fluorescence tagged fibrillarin to snRNA antisense molecular beacon confirming the clear physical interaction between them during RNPP complex formation. This is the first demonstration of in situ detection of RNA-protein complex formation by antisense molecular beacon based FRET and FCET in Giardia lamblia.

  19. The effects of antisense to Gialpha2 on opioid agonist potency and Gialpha2 protein and mRNA abundance in the mouse.

    PubMed

    Shen, J; Shah, S; Hsu, H; Yoburn, B C

    1998-08-31

    In this study, mice received a single intracerebroventricular (i.c.v. ) injection of an antisense oligodeoxynucleotide (ODN) directed towards the mRNA of Gialpha2. Controls received a saline or a nonsense ODN injection. The subsequent effects on protein levels and mRNA of Gialpha2 were determined in mouse striatum, as well as, the effect on opioid ([d-Ala2, d-Leu5]-enkephalin; DADLE) inhibition of cyclic AMP (cAMP) formation in striatum and morphine analgesic potency. At 48 h after treatment, maximal inhibition (Emax) of cAMP formation was significantly reduced for the antisense group compared to controls. Antisense ODN treatment only changed the Emax and did not significantly alter the IC50s of the dose-effect curves for inhibition of cAMP formation. Antisense ODN, but not nonsense ODN, significantly reduced morphine's analgesic potency by >2-fold, 48 h following treatment. Using a quantitative immunoblotting procedure, antisense treatment was shown to decrease striatal Gialpha2 protein 48 h after antisense injection, while there were no changes in protein levels at 2, 12 and 24 h. In contrast, no changes in Gialpha2 mRNA in mouse striatum were noted at any time after antisense treatment. Taken together, these data suggest that Gialpha2 mediates opioid-induced analgesia and opioid inhibition of cAMP production in the mouse. These data also suggest that antisense reduces target protein by a mechanism independent of changes in mRNA abundance.

  20. Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development.

    PubMed

    Wang, Hua-Sen; Zhu, Zhu-Jun; Feng, Zhen; Zhang, Shi-Gang; Yu, Chao

    2012-12-01

    In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

  1. Antisense Inhibition of the 2-Oxoglutarate Dehydrogenase Complex in Tomato Demonstrates Its Importance for Plant Respiration and during Leaf Senescence and Fruit Maturation[W][OA

    PubMed Central

    Araújo, Wagner L.; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R.

    2012-01-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions. PMID:22751214

  2. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

    PubMed Central

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E.

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration. PMID:26366578

  3. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Migawa, Michael T; Vickers, Timothy A; Crooke, Stanley T

    2016-08-01

    Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression or to modify RNA splicing, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30-150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an ∼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility. PMID:27398791

  4. Identification of REST-regulated genes and pathways using a REST-targeted antisense approach.

    PubMed

    Sedaghat, Yalda; Bui, Huynh-Hoa; Mazur, Curt; Monia, Brett P

    2013-12-01

    The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is one of the first negative-acting transcriptional regulators implicated in vertebrate development thought to regulate hundreds of neuron-specific genes. However, its function in the adult system remains elusive. Here we employ second-generation antisense oligonucleotides (ASOs) to study the impact of rest-mediated suppression on gene expression. We demonstrate specific reductions in REST levels in vitro, and in vivo in mouse liver following treatment with ASOs, and we show that ASO mediated-REST suppression results in the elevation in expression of many neuronal genes including brain-derived neurotrophic factor, Synapsin1 (syn1) and β3-tubulin in BALB/c liver. Furthermore, we show the elevation of the affected proteins in plasma following ASO treatment. Finally, microarray analysis was applied to identify a broad range of genes modulated by REST suppression in mouse liver. Our findings suggest that REST may be an important target for neurodegenerative diseases like Huntington's disease, is also involved in the regulation of a broad range of additional cellular pathways, and that the antisense approach is a viable strategy for selectively modulating REST activity in vivo. PMID:24329414

  5. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

    PubMed Central

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    ABSTRACT Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation. PMID:26580233

  6. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic

    PubMed Central

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-01-01

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame −2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp. Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins—an expected outcome with overlaps in frame −2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic. PMID:27681623

  7. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  8. Antisense approaches for elucidating ranavirus gene function in an infected fish cell line.

    PubMed

    Whitley, D S; Sample, R C; Sinning, A R; Henegar, J; Chinchar, V G

    2011-09-01

    Viral virulence/immune evasion strategies and host anti-viral responses represent different sides of the continuing struggle between virus and host survival. To identify virus-encoding molecules whose function is to subvert or blunt host immune responses, we have adapted anti-sense approaches to knock down the expression of specific viral gene products. Our intention is to correlate knock down with loss of function and thus infer the role of a given viral gene. As a starting point in this process we have targeted several structural and catalytic genes using antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA). In proof of concept experiments we show the feasibility of this approach and describe recent work targeting five frog virus 3 genes. Our results indicate that both 46K and 32R, two immediate-early viral proteins, are essential for replication in vitro, and confirm earlier findings that the major capsid protein, the largest subunit of the viral homolog of RNA polymerase II, and the viral DNA methyltransferase are also essential for replication in cell culture.

  9. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  10. Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    PubMed Central

    Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M.; Fei, Jingyi; Ha, Taekjip; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2016-01-01

    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing. PMID:26826711

  11. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  12. Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense Rab11 GTPase gene.

    PubMed

    Lu, C; Zainal, Z; Tucker, G A; Lycett, G W

    2001-08-01

    A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall-modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a "mutant phenotype." Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.

  13. Targeted Intracellular Delivery of Antisense Oligonucleotides Via Conjugation With Small Molecule Ligands

    PubMed Central

    Nakagawa, Osamu; Ming, Xin; Huang, Leaf; Juliano, Rudolph L.

    2010-01-01

    Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multi-valent conjugates of oligonucleotides with small molecule ligands. Using the sigma receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid phase DNA synthesis. We generated mono- and tri-valent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The tri-valent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the mono-valent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range. PMID:20550198

  14. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    PubMed

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

  15. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  16. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

    PubMed Central

    Boiziau, C; Kurfurst, R; Cazenave, C; Roig, V; Thuong, N T; Toulmé, J J

    1991-01-01

    We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H. Images PMID:1850511

  17. Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

    SciTech Connect

    Anai, Satoshi; Brown, Bob D.; Nakamura, Kogenta; Goodison, Steve; Hirao, Yoshihiko; Rosser, Charles J. . E-mail: charles.rosser@urology.ufl.edu

    2007-07-15

    Purpose: To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. Methods and Materials: PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. Results: The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. Conclusions: These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications.

  18. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    PubMed Central

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns—multisystemic, hepatic, or in central nervous system (CNS)—and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  19. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    1999-09-28

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.

  20. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

    PubMed

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

  1. Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

    PubMed

    Glas, Julia; Dümcke, Sebastian; Zacher, Benedikt; Poron, Don; Gagneur, Julien; Tresch, Achim

    2016-03-18

    Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor. PMID:26578558

  2. Lipid-Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α.

    PubMed

    Li, Hong; Quan, Jishan; Zhang, Mengzi; Yung, Bryant C; Cheng, Xinwei; Liu, Yang; Lee, Young B; Ahn, Chang-Ho; Kim, Deog Joong; Lee, Robert J

    2016-07-01

    Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 μM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

  3. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  4. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    PubMed

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  5. Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-induced phase shift.

    PubMed

    Wakamatsu, H; Takahashi, S; Moriya, T; Inouye, S T; Okamura, H; Akiyama, M; Shibata, S

    2001-01-22

    It is well known that light induces both mPer1 and mPer2 mRNA in the suprachiasmatic nucleus. We have reported that mPer1 antisense oligonucleotides (ODNs) inhibited the light-induced phase delays of mouse locomotor rhythm. In this study, we asked whether both or either mPer1 or mPer2 expression is necessary to induce the phase shift. We examined the effects of inhibition of mRNA expression on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN, but not by mPer3 antisense or mPer1, mPer2 scrambled ODNs, whereas following simultaneous injection of both mPer1 and mPer2 antisense ODNs they disappeared. The present results suggest that acute induction of mPer1 and mPer2 gene play an additive effect on photic entrainment. PMID:11201072

  6. Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray

    PubMed Central

    Boopathi, P.A.; Subudhi, Amit Kumar; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Chand, Bipin; Mugasimangalam, Raja C.; Kochar, Sanjay K.; Sirohi, Parmendra; Kochar, Dhanpat K.; Das, Ashis

    2014-01-01

    Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013. PMID:26484095

  7. Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

    PubMed

    Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

    2013-07-01

    Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

  8. Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.

    PubMed Central

    Khillan, J S; Li, S W; Prockop, D J

    1994-01-01

    Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes. Images PMID:8022775

  9. RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts.

    PubMed

    Peters, Nick T; Rohrbach, Justin A; Zalewski, Brian A; Byrkett, Colleen M; Vaughn, Jack C

    2003-06-01

    We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

  10. Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription

    PubMed Central

    Halin, Marilène; Douceron, Estelle; Clerc, Isabelle; Journo, Chloé; Ko, Nga Ling; Landry, Sébastien; Murphy, Edward L.; Gessain, Antoine; Lemasson, Isabelle; Mesnard, Jean-Michel

    2009-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for “antisense protein of HTLV-2.” APH-2 mRNA is spliced, polyadenylated, and initiates in the 3′-long terminal repeat at different positions. This transcript was detected in all HTLV-2–infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2–infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand–encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients. PMID:19602711

  11. Targeting Long Noncoding RNA with Antisense Oligonucleotide Technology as Cancer Therapeutics.

    PubMed

    Zhou, Tianyuan; Kim, Youngsoo; MacLeod, A Robert

    2016-01-01

    Recent annotation of the human transcriptome revealed that only 2 % of the genome encodes proteins while the majority of human genome is transcribed into noncoding RNAs. Although we are just beginning to understand the diverse roles long noncoding RNAs (lncRNAs) play in molecular and cellular processes, they have potentially important roles in human development and pathophysiology. However, targeting of RNA by traditional structure-based design of small molecule inhibitors has been difficult, due to a lack of understanding of the dynamic tertiary structures most RNA molecules adopt. Antisense oligonucleotides (ASOs) are capable of targeting specific genes or transcripts directly through Watson-Crick base pairing and thus can be designed based on sequence information alone. These agents have made possible specific targeting of "non-druggable targets" including RNA molecules. Here we describe how ASOs can be applied in preclinical studies to reduce levels of lncRNAs of interest.

  12. Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

    PubMed

    Onishi, Reina; Watanabe, Ayahisa; Nakajima, Mado; Sekiguchi, Mitsuaki; Kugimiya, Akira; Kinouchi, Hiroki; Nihashi, Yoichiro; Kamimori, Hiroshi

    2015-01-01

    In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

  13. Expansion of antisense lncRNA transcriptomes since the loss of RNAi

    PubMed Central

    Alcid, Eric A.; Tsukiyama, Toshio

    2016-01-01

    Antisense long noncoding RNAs (ASlncRNAs) have been implicated in regulating gene expression in response to physiological cues. However, little is known about ASlncRNA evolutionary dynamics, and what underlies the evolution of their expression. Here, using budding yeast species Saccharomyces and Naumovozyma as models, we show that ASlncRNA repertoires have expanded since the loss of RNAi, in terms of their expression levels, their lengths, and their degree of overlap with coding genes. Furthermore, we show RNAi is inhibitory to ASlncRNA transcriptomes, and that elevation of ASlncRNAs in the presence of RNAi is deleterious to Naumovozyma castellii, a natural host of RNAi. Together, our work suggests that the loss of RNAi had a substantial impact on the genome-wide increase in expression of ASlncRNAs across budding yeast evolution. PMID:27018804

  14. Axonal trafficking of an antisense RNA transcribed from a pseudogene is regulated by classical conditioning

    PubMed Central

    Korneev, Sergei A.; Kemenes, Ildiko; Bettini, Natalia L.; Kemenes, George; Staras, Kevin; Benjamin, Paul R.; O'Shea, Michael

    2013-01-01

    Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory. We show the antiNOS-2 RNA is axonally trafficked and demonstrate that this is regulated by classical conditioning. Critically, a single conditioning trial changes the amount of antiNOS-2 RNA transported along the axon. This occurs within the critical time window when neurotransmitter NO is required for memory formation. Our data suggest a role for the antiNOS-2 RNA in establishing memories through the regulation of NO signaling at the synapse. PMID:23293742

  15. Efficient Synthesis and Biological Evaluation of 5'-GalNAc Conjugated Antisense Oligonucleotides.

    PubMed

    Østergaard, Michael E; Yu, Jinghua; Kinberger, Garth A; Wan, W Brad; Migawa, Michael T; Vasquez, Guillermo; Schmidt, Karsten; Gaus, Hans J; Murray, Heather M; Low, Audrey; Swayze, Eric E; Prakash, Thazha P; Seth, Punit P

    2015-08-19

    Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.

  16. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    PubMed Central

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  17. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    PubMed

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  18. Antisense inhibition of the photosystem I antenna protein Lhca4 in Arabidopsis thaliana.

    PubMed Central

    Zhang, H; Goodman, H M; Jansson, S

    1997-01-01

    The function of Lhca4, a gene encoding the photosystem 1 type IV chlorophyll a/b-binding protein complex in Arabidopsis, was investigated using antisense technology. Lhca4 protein was reduced in a number of mutant lines and abolished in one. The inhibition of protein was not correlated with the inhibition of mRNA. No depletion of Lhca1 was observed, but the low-temperature fluorescence emission spectrum was drastically altered in the mutants. The emission maximum was blue-shifted by 6 nm, showing that chlorophyll molecules bound to Lhca4 are responsible for most of the long-wavelength fluorescence emission. Some mutants also showed an unexplainable delay in flowering time and an increase in seed weight. PMID:9414561

  19. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    NASA Astrophysics Data System (ADS)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  20. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    PubMed

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes. PMID:26847692

  1. Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

    NASA Astrophysics Data System (ADS)

    Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

    2012-03-01

    MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve

  2. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    PubMed

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj

    2008-08-01

    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  3. Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

    PubMed

    Hotta, Carlos Takeshi; Nishiyama, Milton Yutaka; Souza, Glaucia Mendes

    2013-01-01

    Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.

  4. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    PubMed

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  5. A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

    PubMed Central

    Fortin, Marylène; D'Anjou, Hélène; Higgins, Marie-Ève; Gougeon, Jasmine; Aubé, Paméla; Moktefi, Kamel; Mouissi, Sonia; Séguin, Serge; Séguin, Rosanne; Renzi, Paolo M; Paquet, Luc; Ferrari, Nicolay

    2009-01-01

    Background Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo. Methods Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs. Results In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation

  6. Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

    PubMed Central

    Seim, Inge; Carter, Shea L; Herington, Adrian C; Chopin, Lisa K

    2008-01-01

    Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS), which spans the promoter and untranslated regions of the ghrelin gene (GHRL). Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2). Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis), as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA) genes, including 5' capping, polyadenylation, extensive splicing and short open reading frames. The gene is also

  7. Determination of essential fatty acid composition among mutant lines of Canola (Brassica napus), through high pressure liquid chromatography.

    PubMed

    Raza, Ghulam; Siddique, Aquil; Khan, Imtiaz Ahmad; Ashraf, Muhammed Yasin; Khatri, Abdullah

    2009-12-01

    The present study aimed to quantify the methyl esters of lenoleic acid (LA), gamma-lenolenic acid (LNA) and oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of B. napus cv. 'Rainbow' (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. 'Westar' (P) at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.1 to 66.3%; LA was second (15.3-41.6%) and LNA was third (18.1-28.9%). Mutant ROO-125/14 showed higher OL contents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials (NUVYT) as a new variety based on high oil quality.

  8. An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

    PubMed Central

    Pichon, Christophe; du Merle, Laurence; Caliot, Marie Elise; Trieu-Cuot, Patrick; Le Bouguénec, Chantal

    2012-01-01

    Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli. PMID:22139924

  9. Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants.

    PubMed

    Seong, Eun Soo; Yoo, Ji Hye; Lee, Jae Geun; Kim, Hee Young; Hwang, In Seong; Heo, Kweon; Kim, Jae Kwang; Lim, Jung Dae; Sacks, Erik J; Yu, Chang Yeon

    2013-02-01

    Initially, we isolated the caffeic acid O-methyltransferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, we produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidases expression, which are related to the phenylpropanoid pathway and antioxidant activity. The MsCOMT-down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT-down-regulated transgenic plants. We also observed a morphological difference, with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway.

  10. Second-generation antisense oligonucleotides against β-catenin protect mice against diet-induced hepatic steatosis and hepatic and peripheral insulin resistance.

    PubMed

    Popov, Violeta B; Jornayvaz, Francois R; Akgul, Emin O; Kanda, Shoichi; Jurczak, Michael J; Zhang, Dongyan; Abudukadier, Abulizi; Majumdar, Sachin K; Guigni, Blas; Petersen, Kitt Falk; Manchem, Vara Prasad; Bhanot, Sanjay; Shulman, Gerald I; Samuel, Varman T

    2016-03-01

    Although mutations in the Wnt/β-catenin signaling pathway are linked with the metabolic syndrome and type 2 diabetes in humans, the mechanism is unclear. High-fat-fed male C57BL/6 mice were treated for 4 wk with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) to decrease hepatic and adipose expression of β-catenin. β-Catenin mRNA decreased by ≈80% in the liver and by 70% in white adipose tissue relative to control ASO-treated mice. β-Catenin ASO improved hepatic insulin sensitivity and increased insulin-stimulated whole body glucose metabolism, as assessed during hyperinsulinemic-euglycemic clamp in awake mice. β-Catenin ASO altered hepatic lipid composition in high-fat-fed mice. There were reductions in hepatic triglyceride (44%, P < 0.05) and diacylglycerol content (60%, P < 0.01) but a 30% increase in ceramide content (P < 0.001). The altered lipid content was attributed to decreased expression of sn-1,2 diacylglycerol acyltransferase and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase and an increase in serine palmitoyl transferase. The decrease in cellular diacyglycerol was associated with a 33% decrease in PKCε activation (P < 0.05) and 64% increase in Akt2 phosphorylation (P < 0.05). In summary, Reducing β-catenin expression decreases expression of enzymes involved in hepatic fatty acid esterification, ameliorates hepatic steatosis and lipid-induced insulin resistance.

  11. Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism.

    PubMed

    Fernie, Alisdair R; Tauberger, Eva; Lytovchenko, Anna; Roessner, Ute; Willmitzer, Lothar; Trethewey, Richard N

    2002-02-01

    The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.

  12. An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains.

    PubMed

    Pichon, Christophe; du Merle, Laurence; Caliot, Marie Elise; Trieu-Cuot, Patrick; Le Bouguénec, Chantal

    2012-04-01

    Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli.

  13. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  14. An Antisense RNA-mediated Mechanism Eliminates a Meiosis-specific Copper-regulated Transcript in Mitotic Cells*

    PubMed Central

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-01-01

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1+ gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1+ sense transcript, inhibiting cum1+ expression in vegetative cells. Inhibition of cum1+ transcription was not at the level of its initiation because fusion of the cum1+ promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1+-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1+ led to the exclusive production of a shorter iss1+ mRNA isoform, thereby allowing accumulation of cum1+ sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1+ antisense mRNA could pair with cum1+ sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1+ sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells. PMID:26229103

  15. An antisense RNA-mediated mechanism eliminates a meiosis-specific copper-regulated transcript in mitotic cells.

    PubMed

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-09-11

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

  16. Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance.

    PubMed

    Iwaki, T; Iwaki, A; Tateishi, J; Goldman, J E

    1994-06-01

    The phenotypic effects of selectively altering the levels of alpha B-crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B-crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization. PMID:8207065

  17. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

    PubMed Central

    Johnson, Barbara W.; Olson, Ken E.; Allen-Miura, Tanya; Rayms-Keller, Alfredo; Carlson, Jonathan O.; Coates, Craig J.; Jasinskiene, Nijole; James, Anthony A.; Beaty, Barry J.; Higgs, Stephen

    1999-01-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  18. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

    PubMed

    Johnson, B W; Olson, K E; Allen-Miura, T; Rayms-Keller, A; Carlson, J O; Coates, C J; Jasinskiene, N; James, A A; Beaty, B J; Higgs, S

    1999-11-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  19. Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF

    PubMed Central

    Im, S-A; Kim, J-S; Gomez-Manzano, C; Fueyo, J; Liu, T-J; Cho, M-S; Seong, C-M; Lee, S N; Hong, Y-K; Yung, W K A

    2001-01-01

    Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-αVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-αVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-αVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF 165 cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-αVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336478

  20. Oral delivery of nanoparticles containing anticancer SN38 and hSET1 antisense for dual therapy of colon cancer.

    PubMed

    Dinarvand, M; Kiani, M; Mirzazadeh, F; Esmaeili, A; Mirzaie, Z; Soleimani, M; Dinarvand, R; Atyabi, F

    2015-01-01

    An oral delivery system intended for treatment of colon cancer in HT29 cancerous cells was investigated by encapsulating hSET1 antisense and SN38 anticancer in nanoparticles based on cysteine trimethyl chitosan (cysTMC) and carboxymethyl dextran (CMD). Studies have shown hSET1 as the main type of histone methyltransferase (HMT) complex, is significantly overexpressed in malignant cells. In this study, hSET1 antisense was employed to inhibit gene expression. Additionally, SN38 was incorporated into nanoparticles to enhance the efficiency of the system by inhibition of topoisomerase 1. CysTMC was synthetized and characterized by (1)H NMR and FTIR. Nanoparticles were prepared through complexation of CMD and cysTMC. Particle size and surface charge was 100-150 nm and 17-21 mV respectively with drug content of around 2.6%. Gel electrophoresis assay proved the stability of antisense in simulated gastric and intestinal fluids. Nanoparticles showed high mucoadhesion and glutathione responsive release. Cellular uptake was observed by confocal microscopy and quantified by flow cytometry. Cytotoxicity of NPs was assessed using MTT assay. Results showed hSET1/SN38 nanoparticles had significantly higher cytotoxicity against HT29 cells compared with nanoparticles containing SN38, free SN38 or naked hSET1. Therefore, present system could be considered an effective combination therapy of highly hydrophobic SN38 and hSET1.

  1. Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

    PubMed Central

    Sioud, M

    1997-01-01

    The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs. PMID:9016562

  2. Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

    PubMed Central

    Pierce, M L; Ruffner, D E

    1998-01-01

    Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

  3. Hormone-dependent expression of a steroidogenic acute regulatory protein natural antisense transcript in MA-10 mouse tumor Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3'-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5' and 3' RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.

  4. Oral delivery of nanoparticles containing anticancer SN38 and hSET1 antisense for dual therapy of colon cancer.

    PubMed

    Dinarvand, M; Kiani, M; Mirzazadeh, F; Esmaeili, A; Mirzaie, Z; Soleimani, M; Dinarvand, R; Atyabi, F

    2015-01-01

    An oral delivery system intended for treatment of colon cancer in HT29 cancerous cells was investigated by encapsulating hSET1 antisense and SN38 anticancer in nanoparticles based on cysteine trimethyl chitosan (cysTMC) and carboxymethyl dextran (CMD). Studies have shown hSET1 as the main type of histone methyltransferase (HMT) complex, is significantly overexpressed in malignant cells. In this study, hSET1 antisense was employed to inhibit gene expression. Additionally, SN38 was incorporated into nanoparticles to enhance the efficiency of the system by inhibition of topoisomerase 1. CysTMC was synthetized and characterized by (1)H NMR and FTIR. Nanoparticles were prepared through complexation of CMD and cysTMC. Particle size and surface charge was 100-150 nm and 17-21 mV respectively with drug content of around 2.6%. Gel electrophoresis assay proved the stability of antisense in simulated gastric and intestinal fluids. Nanoparticles showed high mucoadhesion and glutathione responsive release. Cellular uptake was observed by confocal microscopy and quantified by flow cytometry. Cytotoxicity of NPs was assessed using MTT assay. Results showed hSET1/SN38 nanoparticles had significantly higher cytotoxicity against HT29 cells compared with nanoparticles containing SN38, free SN38 or naked hSET1. Therefore, present system could be considered an effective combination therapy of highly hydrophobic SN38 and hSET1. PMID:25858880

  5. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    PubMed

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  6. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

    PubMed Central

    Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

    2015-01-01

    MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

  7. Small antisense oligonucleotides against G-quadruplexes: specific mRNA translational switches

    PubMed Central

    Rouleau, Samuel G.; Beaudoin, Jean-Denis; Bisaillon, Martin; Perreault, Jean-Pierre

    2015-01-01

    G-quadruplexes (G4) are intricate RNA structures found throughout the transcriptome. Because they are associated with a variety of biological cellular mechanisms, these fascinating structural motifs are seen as potential therapeutic targets against many diseases. While screening of chemical compounds specific to G4 motifs has yielded interesting results, no single compound successfully discriminates between G4 motifs based on nucleotide sequences alone. This level of specificity is best attained using antisense oligonucleotides (ASO). Indeed, oligonucleotide-based strategies are already used to modulate DNA G4 folding in vitro. Here, we report that, in human cells, the use of short ASO to promote and inhibit RNA G4 folding affects the translation of specific mRNAs, including one from the 5′UTR of the H2AFY gene, a histone variant associated with cellular differentiation and cancer. These results suggest that the relatively high specificity of ASO-based strategies holds significant potential for applications aimed at modulating G4-motif folding. PMID:25510493

  8. Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.

    PubMed

    Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke

    2016-03-01

    Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles.

  9. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models

    PubMed Central

    Osman, Erkan Y.; Miller, Madeline R.; Robbins, Kate L.; Lombardi, Abby M.; Atkinson, Arleigh K.; Brehm, Amanda J.; Lorson, Christian L.

    2014-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1MO-ASOs). A single intracerebroventricular injection in the relatively severe mouse model of SMA (SMNΔ7 mouse model) elicited a robust induction of SMN protein, and mean life span was extended from an average survival of 13 to 54 days following a single dose, consistent with large weight gains and a correction of the neuronal pathology. Additionally, E1MO-ASO treatment in an intermediate SMA mouse (SMNRT mouse model) significantly extended life span by ∼700% and weight gain was comparable with the unaffected animals. While a number of experimental therapeutics have targeted the ISS-N1 element of SMN2 pre-mRNA, the development of E1 ASOs provides a new molecular target for SMA therapeutics that dramatically extends survival in two important pre-clinical models of disease. PMID:24781211

  10. In Vitro Inhibition of Hepatitis C Virus by Antisense Oligonucleotides in PBMC Compared to Hepatoma Cells

    PubMed Central

    Youssef, Samar Samir; Fahmy, Ahmed Mohamed; Omran, Moataza Hassan; Mohamed, Amr Saad; El Desouki, Mohamed Ali; El-Awady, Mostafa K.

    2014-01-01

    Aim. To assess the efficiency of phosphorothioate antisense oligodeoxynucleotide 1 (S-ODN1) on HCV translation inhibition in PBMC compared to hepatoma cells in vitro for the first time. Materials and Methods. The study included 34 treatment naive HCV patients. IRES domain III and IV sequence variations were tested in 45 clones from 9 HCV patients. PBMC of HCV positive patients were subjected to S-ODN in vitro. Concomitantly HepG2 cells infected by the same patient's serum were also treated with S-ODN1 for 24 and 48 hours. Cellular RNA was tested for HCV plus and minus strands by reverse transcription polymerase chain reaction (RT-PCR). Results. Sequence variations were seen in HCV IRES domain III only while domain IV was conserved among all the tested patient's clones. S-ODN1 successfully inhibited HCV translation in HepG2 cells, while in PBMC inhibition was partial. Conclusion. HCV IRES domain IV is more conserved than domain IIId in genotype 4 HCV patients. S-ODN against HCV IRES domain IV was not efficient to inhibit HCV translation in PBMC under the study conditions. Further studies testing other S-ODN targeting other HCV IRES domains in PBMC should be done. PMID:24991538

  11. Expression and regulation of the Msx1 natural antisense transcript during development.

    PubMed

    Coudert, Amélie E; Pibouin, Laurence; Vi-Fane, Brigitte; Thomas, Bethan L; Macdougall, Mary; Choudhury, Anuradha; Robert, Benoît; Sharpe, Paul T; Berdal, Ariane; Lezot, Frédéric

    2005-01-01

    Bidirectional transcription, leading to the expression of an antisense (AS) RNA partially complementary to the protein coding sense (S) RNA, is an emerging subject in mammals and has been associated with various processes such as RNA interference, imprinting and transcription inhibition. Homeobox genes do not escape this bidirectional transcription, raising the possibility that such AS transcription occurs during embryonic development and may be involved in the complexity of regulation of homeobox gene expression. According to the importance of the Msx1 homeobox gene function in craniofacial development, especially in tooth development, the expression and regulation of its recently identified AS transcripts were investigated in vivo in mouse from E9.5 embryo to newborn, and compared with the S transcript and the encoded protein expression pattern and regulation. The spatial and temporal expression patterns of S, AS transcripts and protein are consistent with a role of AS RNA in the regulation of Msx1 expression in timely controlled developmental sites. Epithelial-mesenchymal interactions were shown to control the spatial organization of S and also AS RNA expression during early patterning of incisors and molars in the odontogenic mesenchyme. To conclude, this study clearly identifies the Msx1 AS RNA involvement during tooth development and evidences a new degree of complexity in craniofacial developmental biology: the implication of endogenous AS RNAs.

  12. Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration.

    PubMed

    Babajko, Sylvie; Petit, Stéphane; Fernandes, Isabelle; Méary, Fleur; LeBihan, Johanne; Pibouin, Laurence; Berdal, Ariane

    2009-01-01

    Msx homeogenes play an important role in epithelial-mesenchymal interactions leading development. Msx1 is relevant for dental and craniofacial morphogenesis, as suggested by phenotypes of Msx1 mutations in human and Msx1 KO mice. During adulthood, Msx1 is still expressed in the skeleton where its role is largely unknown. Our group showed that the Msx1 gene is submitted to bidirectional transcription generating a long noncoding antisense (AS) RNA. During tooth development, Msx1 sense (S) and AS RNAs showed specific patterns of expression. Thus, the aim of the present study was to analyze the relation between Msx1 S and AS RNAs. In vivo mapping in adult mice showed that both Msx1 RNAs were detected in tested tissues such as bone. In vitro, Msx1 AS RNA decreased endogenous Msx1 S expression and modified Msx1 protein cell distribution. Regulations of Dlx5 and Bmp4 expression involving Msx1 S and AS RNAs showed that Msx1 AS RNA could modulate Msx1 function. The study of Msx1 S and AS RNA status is interesting in the case of tooth agenesis and bone loss to see if a disturbance of this balance could be associated with a disturbance of bone homeostasis. In that sense, our current results suggest a clear involvement of Msx1 in alveolar bone.

  13. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  14. Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli.

    PubMed

    Thomason, Maureen K; Bischler, Thorsten; Eisenbart, Sara K; Förstner, Konrad U; Zhang, Aixia; Herbig, Alexander; Nieselt, Kay; Sharma, Cynthia M; Storz, Gisela

    2015-01-01

    While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

  15. Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides.

    PubMed

    Vetter, A; Martien, R; Bernkop-Schnürch, A

    2010-03-01

    The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.

  16. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

    PubMed Central

    Gramlich, Michael; Pane, Luna Simona; Zhou, Qifeng; Chen, Zhifen; Murgia, Marta; Schötterl, Sonja; Goedel, Alexander; Metzger, Katja; Brade, Thomas; Parrotta, Elvira; Schaller, Martin; Gerull, Brenda; Thierfelder, Ludwig; Aartsma-Rus, Annemieke; Labeit, Siegfried; Atherton, John J; McGaughran, Julie; Harvey, Richard P; Sinnecker, Daniel; Mann, Matthias; Laugwitz, Karl-Ludwig; Gawaz, Meinrad Paul; Moretti, Alessandra

    2015-01-01

    Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM. PMID:25759365

  17. Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

    PubMed

    Rahman, M A; Summerton, J; Foster, E; Cunningham, K; Stirchak, E; Weller, D; Schaup, H W

    1991-01-01

    Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics. PMID:1821653

  18. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-07-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

  19. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

    NASA Technical Reports Server (NTRS)

    Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

    2003-01-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

  20. Intravesical Liposome and Antisense Treatment for Detrusor Overactivity and Interstitial Cystitis/Painful Bladder Syndrome

    PubMed Central

    Kashyap, Mahendra P.; Kawamorita, Naoki; Yoshizawa, Tsuyoshi; Chancellor, Michael

    2014-01-01

    Purpose. The following review focuses on the recent advancements in intravesical drug delivery, which brings added benefit to the therapy of detrusor overactivity and interstitial cystitis/painful bladder syndrome (IC/PBS). Results. Intravesical route is a preferred route of administration for restricting the action of extremely potent drugs like DMSO for patients of interstitial cystitis/painful bladder syndrome (IC/PBS) and botulinum toxin for detrusor overactivity. Patients who are either refractory to oral treatment or need to mitigate the adverse effects encountered with conventional routes of administration also chose this route. Its usefulness in some cases can be limited by vehicle (carrier) toxicity or short duration of action. Efforts have been underway to overcome these limitations by developing liposome platform for intravesical delivery of biotechnological products including antisense oligonucleotides. Conclusions. Adoption of forward-thinking approaches can achieve advancements in drug delivery systems targeted to future improvement in pharmacotherapy of bladder diseases. Latest developments in the field of nanotechnology can bring this mode of therapy from second line of treatment for refractory cases to the forefront of disease management. PMID:24527221

  1. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    PubMed

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  2. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    PubMed Central

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  3. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

    PubMed Central

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

    2016-01-01

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

  4. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana.

    PubMed

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-06-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications. PMID:25922535

  5. Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages

    PubMed Central

    Wan, W. Brad; Migawa, Michael T.; Vasquez, Guillermo; Murray, Heather M.; Nichols, Josh G.; Gaus, Hans; Berdeja, Andres; Lee, Sam; Hart, Christopher E.; Lima, Walt F.; Swayze, Eric E.; Seth, Punit P.

    2014-01-01

    Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability. PMID:25398895

  6. Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes.

    PubMed Central

    Crooke, S T; Lemonidis, K M; Neilson, L; Griffey, R; Lesnik, E A; Monia, B P

    1995-01-01

    1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8526876

  7. Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica

    PubMed Central

    Morf, Laura; Pearson, Richard J.; Wang, Angelia S.; Singh, Upinder

    2013-01-01

    RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects. PMID:23935116

  8. Effect of Antisense Oligodeoxynucleotides Glucose Transporter-1 on Enhancement of Radiosensitivity of Laryngeal Carcinoma

    PubMed Central

    Yan, Sen-Xiang; Luo, Xing-Mei; Zhou, Shui-Hong; Bao, Yang-Yang; Fan, Jun; Lu, Zhong-Jie; Liao, Xin-Biao; Huang, Ya-Ping; Wu, Ting-Ting; Wang, Qin-Ying

    2013-01-01

    Purpose: Laryngeal carcinomas always resist to radiotherapy. Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is considered to be a possible intrinsic marker of hypoxia in malignant tumors. We speculated that the inhibition of GLUT-1 expression might improve the radiosensitivity of laryngeal carcinoma. Methods: We assessed the effect of GLUT-1 expression on radioresistance of laryngeal carcinoma and the effect of GLUT-1 expressions by antisense oligodeoxynucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma in vitro and in vivo. Results: After transfection of GLUT-1 AS-ODNs: MTS assay showed the survival rates of radiation groups were reduced with the prolongation of culture time (p<0.05); Cell survival rates were significantly reduced along with the increasing of radiation dose (p<0.05). There was significant difference in the expression of GLUT-1mRNA and protein in the same X-ray dose between before and after X-ray radiation (p<0.05). In vivo, the expressions of GLUT-1 mRNA and protein after 8Gy radiation plus transfection of GLUT-1 AS-ODNs were significant decreased compared to 8Gy radiation alone (p<0.001). Conclusion: Radioresistance of laryngeal carcinoma may be associated with increased expression of GLUT-1 mRNA and protein. GLUT-1 AS-ODNs may enhance the radiosensitivity of laryngeal carcinoma mainly by inhibiting the expression of GLUT-1. PMID:23983599

  9. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

    PubMed

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  10. Stress-induced glucocorticoids suppress the antisense molecular regulation of FGF-2 expression.

    PubMed

    Frank, Matthew G; Der-Avakian, Andre; Bland, Sondra T; Watkins, Linda R; Maier, Steven F

    2007-05-01

    Psychological stress can upregulate basic fibroblast growth factor (FGF-2) expression. Because glucocorticoids can also upregulate FGF-2 expression, the present studies investigated whether stress-induced glucocorticoids mediate the effects of stress on FGF-2. FGF-2 is regulated by an FGF-2 antisense (AS) molecular mechanism and so the present experiments also, for the first time, assessed the effects of stress on FGF-2-AS mRNA, as well as the mediating role of glucocorticoids. The effects of either escapable shock (ES) or yoked-inescapable tail shock (IS) on FGF-2 and FGF-2-AS were determined. To test whether glucocorticoids mediate the effect of stress on FGF-2 and FGF-2-AS, animals were pretreated with temporary corticosterone (CORT) synthesis inhibitors and exposed to IS. To test whether glucocorticoids are sufficient to modulate FGF-2 and FGF-2-AS mRNA, animals were injected with CORT and mRNA measured. ES and IS similarly downregulated FGF-2-AS mRNA at 0 h post-stress and upregulated FGF-2 mRNA 2 h post-stress. Inhibition of CORT synthesis abrogated the effect of IS on both FGF-2-AS and FGF-2 mRNA. Exogenous CORT mimicked the effects of ES and IS on FGF-2, but not FGF-2-AS mRNA. The present study demonstrates that glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS.

  11. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana.

    PubMed

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-06-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications.

  12. Gene expression analysis by a competitive and differential PCR with antisense competitors.

    PubMed

    de Kant, E; Rochlitz, C F; Herrmann, R

    1994-11-01

    We report a sensitive method for the reproducible and accurate measurement of gene expression from small samples of RNA. This method is based on a combination of two PCR techniques: First, an endogenous reporter gene and the gene of interest are simultaneously amplified in one tube after random-primed reverse transcription (RT) of RNA (differential RT-PCR). Second, exogenous homologous fragments of both genes with artificially introduced mutations are added and coamplified in the same reaction (competitive PCR). The first-strand cDNA, and the mutated antisense homologues of the reporter as well as the target gene compete for their respective primers and are therefore amplified with equal efficiencies. After PCR, restriction enzyme digestion allows visualization of the quantitative differences between the four resulting reaction products. The ratios of products that competed during PCR provide the quantitative information. The initial amount of a specific cDNA can be calculated from any competitor/cDNA ratio of reliably measurable PCR product amounts. Extensive competitor titration to experimentally approach the equilibrium is therefore unnecessary. The differential counterpart of competitive and differential RT-PCR (CD-RT-PCR) allows expression of the levels in reference to a reporter gene. MDR1 expression was determined in tumor cells by CD-RT-PCR.

  13. Proteome changes in tomato lines transformed with phytoene synthase-1 in the sense and antisense orientations.

    PubMed

    Robertson, Francesca P; Koistinen, P Kaisa; Gerrish, Christopher; Halket, John M; Patel, Raj K P; Fraser, Paul D; Bramley, Peter M

    2012-10-01

    The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.

  14. Rational genomics I: antisense open reading frames and codon bias in short-chain oxido reductase enzymes and the evolution of the genetic code.

    PubMed

    Duax, William L; Huether, Robert; Pletnev, Vladimir Z; Langs, David; Addlagatta, Anthony; Connare, Sonjay; Habegger, Lukas; Gill, Jay

    2005-12-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6000 members, extending from bacteria and archaea to humans. Nucleic acid sequence analysis reveals that significant numbers of these genes are remarkably free of stopcodons in reading frames other than the coding frame, including those on the antisense strand. The genes from this subset also use almost entirely the GC-rich half of the 64 codons. Analysis of a million hypothetical genes having random nucleotide composition shows that the percentage of SCOR genes having multiple open reading frames exceeds random by a factor of as much as 1 x 10(6). Nevertheless, screening the content of the SWISS-PROT TrEMBL database reveals that 15% of all genes contain multiple open reading frames. The SCOR genes having multiple open reading frames and a GC-rich coding bias exhibit a similar GC bias in the nucleotide triple composition of their DNA. This bias is not correlated with the GC content of the species in which the SCOR genes are found. One possible explanation for the conservation of multiple open reading frames and extreme bias in nucleic acid composition in the family of Rossman folds is that the primordial member of this family was encoded early using only very stable GC-rich DNA and that evolution proceeded with extremely limited introduction of any codons having two or more adenine or thymine nucleotides. These and other data suggest that the SCOR family of enzymes may even have diverged from a common ancestor before most of the AT-rich half of the genetic code was fully defined.

  15. Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.

    PubMed Central

    Wagner, J A; McDonald, T V; Nghiem, P T; Lowe, A W; Schulman, H; Gruenert, D C; Stryer, L; Gardner, P

    1992-01-01

    Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway. Images PMID:1379720

  16. Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

    PubMed

    Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

    2016-09-01

    Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

  17. Antisense against Amyloid-β Protein Precursor Reverses Memory Deficits and Alters Gene Expression in Neurotropic and Insulin-Signaling Pathways in SAMP8 Mice.

    PubMed

    Armbrecht, Harvey J; Siddiqui, Akbar M; Green, Michael; Farr, Susan A; Kumar, Vijaya B; Banks, William A; Patrick, Ping; Shah, Gul N; Morley, John E

    2015-01-01

    The senescence-accelerated mouse (SAMP8) strain exhibits an age-related decrease in memory accompanied by an increase in hippocampal amyloid-β protein precursor (AβPP) and amyloid-β peptide (Aβ). We have shown that administration of an antisense oligonucleotide against the Aβ region of AβPP (AβPP antisense) reverses the memory deficits. The purpose of this study was to determine the effect of peripheral (IV) administration of AβPP antisense on hippocampal gene expression. The AβPP antisense reversed the memory deficits and altered expression of 944 hippocampal genes. Pathway analysis showed significant gene expression changes in nine pathways. These include the MAPK signaling pathway (p = 0.0078) and the phosphatidylinositol signaling pathway (p = 0.043), which we have previously shown to be altered in SAMP8 mice. The changes in these pathways contributed to significant changes in the neurotropin (p = 0.0083) and insulin signaling (p = 0.015) pathways, which are known to be important in learning and memory. Changes in these pathways were accompanied by phosphorylation changes in the downstream target proteins p70S6K, GSK3β, ERK, and CREB. These changes in hippocampal gene expression and protein phosphorylation may suggest specific new targets for antisense therapy aimed at improving memory.

  18. Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA

    PubMed Central

    Player, Mark R.; Barnard, Dale L.; Torrence, Paul F.

    1998-01-01

    The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication. PMID:9671772

  19. Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

    PubMed Central

    Sullenger, B A; Lee, T C; Smith, C A; Ungers, G E; Gilboa, E

    1990-01-01

    NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies. Images PMID:2247070

  20. Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys

    PubMed Central

    Shemesh, Colby S; Yu, Rosie Z; Gaus, Hans J; Greenlee, Sarah; Post, Noah; Schmidt, Karsten; Migawa, Michael T; Seth, Punit P; Zanardi, Thomas A; Prakash, Thazha P; Swayze, Eric E; Henry, Scott P; Wang, Yanfeng

    2016-01-01

    Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of 3H-radiolabeled (3H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5′-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs. PMID:27164023

  1. The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

    PubMed Central

    Heinrich, J; Riedel, H D; Rückert, B; Lurz, R; Schuster, H

    1995-01-01

    The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon. Images PMID:7784198

  2. Sense-antisense gene-pairs in breast cancer and associated pathological pathways

    PubMed Central

    Grinchuk, Oleg V.; Motakis, Efthymios; Yenamandra, Surya Pavan; Ow, Ghim Siong; Jenjaroenpun, Piroon; Tang, Zhiqun; Yarmishyn, Aliaksandr A.; Ivshina, Anna V.; Kuznetsov, Vladimir A.

    2015-01-01

    More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers. PMID:26517092

  3. Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene1

    PubMed Central

    Jiménez-Bermúdez, Silvia; Redondo-Nevado, José; Muñoz-Blanco, Juan; Caballero, José L.; López-Aranda, José M.; Valpuesta, Victoriano; Pliego-Alfaro, Fernando; Quesada, Miguel A.; Mercado, José A.

    2002-01-01

    Strawberry (Fragaria × ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry. PMID:11842178

  4. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    SciTech Connect

    Feng Xian Lu; Aiyar, N.; Chaiken, I. )

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  5. Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease.

    PubMed

    Ripple, Michael J; You, Dahui; Honnegowda, Srinivasa; Giaimo, Joseph D; Sewell, Andrew B; Becnel, David M; Cormier, Stephania A

    2010-10-15

    Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Severe RSV infections in infants cause bronchiolitis, wheeze, and/or cough and significantly increase the risk for developing asthma. RSV pathogenesis is thought to be due to a Th2-type immune response initiated in response to RSV infection, specifically in the infant. Using a neonatal mouse system as an appropriate model for human infants, we sought to determine whether local inhibition of IL-4Rα expression during primary RSV infection in the neonate would prevent Th2-skewed responses to secondary RSV infection and improve long-term pulmonary function. To reduce IL-4Rα expression, antisense oligonucleotides (ASOs) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at 1 wk of age and were reinfected at 6 wk of age. Administration of IL-4Rα ASOs during primary RSV infection in neonatal mice abolished the pulmonary dysfunction normally observed following reinfection in the adult. This ablation of pulmonary dysfunction correlated with a persistent rebalancing of the Th cell compartment with decreased Th2 responses (i.e., reduced goblet cell hyperplasia, Th2 cells, and cytokine secretion) and increased Th1 responses (i.e., elevated Th1 cell numbers and type I Abs and cytokines). Our data support our hypothesis that a reduction in the Th2 immune response during primary infection in neonates prevents Th2-mediated pulmonary pathology initially and upon reinfection and further suggest that vaccine strategies incorporating IL-4Rα ASOs may be of significant benefit to infants.

  6. Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models

    SciTech Connect

    Roedel, Franz; Capalbo, Gianni; Weiss, Christian; Roedel, Claus

    2008-05-01

    Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

  7. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    PubMed

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-01

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

  8. Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys.

    PubMed

    Shemesh, Colby S; Yu, Rosie Z; Gaus, Hans J; Greenlee, Sarah; Post, Noah; Schmidt, Karsten; Migawa, Michael T; Seth, Punit P; Zanardi, Thomas A; Prakash, Thazha P; Swayze, Eric E; Henry, Scott P; Wang, Yanfeng

    2016-01-01

    Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs. PMID:27164023

  9. A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse

    PubMed Central

    Porensky, Paul N.; Mitrpant, Chalermchai; McGovern, Vicki L.; Bevan, Adam K.; Foust, Kevin D.; Kaspar, Brain K.; Wilton, Stephen D.; Burghes, Arthur H.M.

    2012-01-01

    Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(−10,−29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn−/−, SMN2+/+, SMN▵7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients. PMID:22186025

  10. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

    PubMed Central

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies. PMID:27124114

  11. alpha-Linolenic acid- and docosahexaenoic acid-enriched eggs from hens fed flaxseed: influence on blood lipids and platelet phospholipid fatty acids in humans.

    PubMed

    Ferrier, L K; Caston, L J; Leeson, S; Squires, J; Weaver, B J; Holub, B J

    1995-07-01

    This study was undertaken to examine the effects that consumption of eggs from hens fed diets containing flaxseed would have on plasma and platelet lipids of male volunteers. Feeding diets containing 0%, 10%, and 20% ground flaxseed to Leghorn pullets provided a marked progressive increase in n-3 fatty acid content as alpha-linolenic acid (alpha-LNA) (28, 261, and 527 mg/egg) and docosahexaenoic acid (DHA) (51, 81, and 87 mg/egg) but no alteration in the cholesterol concentration of the egg yolk. Twenty-eight male volunteers, divided into three groups, were fed four eggs per day for 2 wk according to a cyclic Latin-square design. No statistically significant changes were observed in total cholesterol, high-density-lipoprotein cholesterol, or plasma triglyceride concentrations. Significant increases in total n-3 fatty acids and in DHA content (which rose from 1.5 to 2.0% by wt or 33% overall), and a significant decrease in ratio of n-6 to n-3 fatty acids were found in platelet phospholipids of subjects consuming eggs from flaxseed-fed hens. Health and Welfare Canada in 1990 set recommended intakes for dietary n-3 fatty acids and for the ratio of n-6 to n-3 fatty acids, which are not being met currently by the overall population. Eggs modified by the inclusion of flaxseed in the laying hens' diet could provide an important nutritional source of n-3 fatty acid. PMID:7598070

  12. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  13. Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

    PubMed Central

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

    2013-01-01

    The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  14. Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

    PubMed Central

    Tortora, Giampaolo; Caputo, Rosa; Damiano, Vincenzo; Bianco, Roberto; Pepe, Stefano; Bianco, A. Raffaele; Jiang, Zhiwei; Agrawal, Sudhir; Ciardiello, Fortunato

    1997-01-01

    Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs. PMID:9356493

  15. Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

    PubMed Central

    Zabaleta, E; Mouras, A; Hernould, M; Suharsono; Araya, A

    1996-01-01

    We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855343

  16. Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

    PubMed

    Xu, Houguo; Dong, Xiaojing; Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

  17. Regulation of Tissue LC-PUFA Contents, Δ6 Fatty Acyl Desaturase (FADS2) Gene Expression and the Methylation of the Putative FADS2 Gene Promoter by Different Dietary Fatty Acid Profiles in Japanese Seabass (Lateolabrax japonicus)

    PubMed Central

    Ai, Qinghui; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Zuo, Rantao

    2014-01-01

    The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53±0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter. PMID:24498178

  18. Coinhibition of overexpressed genes in acute myeloid leukemia subtype M2 by gold nanoparticles functionalized with five antisense oligonucleotides and one anti-CD33(+)/CD34(+) aptamer.

    PubMed

    Zaimy, M A; Jebali, A; Bazrafshan, B; Mehrtashfar, S; Shabani, S; Tavakoli, A; Hekmatimoghaddam, S H; Sarli, A; Azizi, H; Izadi, P; Kazemi, B; Shojaei, A; Abdalaian, A; Tavakkoly-Bazzaz, J

    2016-09-01

    The aim of this study was to evaluate an engineered nanostructure to silence five important oncogenes, including BAG1, MDM2, Bcl-2, BIRC5 (survivin) and XIAP, in acute myeloid leukemia subtype 2 (AML-M2). The smart nanostructures were functionalized gold nanoparticles (FGNs) containing five antisense oligonucleotides (AOs) and one anti-CD33(+)/CD34(+) aptamer. First, the best AO for each gene was selected with the OligoWalk online software, and then different arrangements of AOs were evaluated with the RNAstructure software. Thereafter, naked gold nanoparticles (NGNs) were synthesized by the reaction of 1000 mm HAuCl4 with 10 μg ml(-1) ascorbic acid. Next, five AOs and one anti-CD33(+)/CD34(+) aptamer were attached to NGNs through serial reactions. Later, 5 ml of heparinized blood samples from five AML-M2 patients were prepared, cancerous cells were isolated and then incubated with three concentrations (75, 150 and 300 μg ml(-1)) each of FGNs, NGNs, gold nanoparticles functionalized with scrambled oligonucleotides (GNFSONs) and doxorubicin. Finally, cell death percentage and gene expressions were measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and real-time PCR, respectively. This study showed that FGNs and doxorubicin led to more cell death compared with NGNs and GNFSONs (P<0.05). Interestingly, all concentrations of FGNs led to a decrease in gene expression. As an important finding, although all concentrations of doxorubicin could also inhibit the expression of genes, FGNs had more effect (P<0.05). Moreover, both NGNs and GNFSONs could silence all genes only at a concentration of 300 μg ml(-1). For BCL2 and XIAP, a dose-dependent pattern was observed, but there was no similar pattern for others. PMID:27514505

  19. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening

    PubMed Central

    Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A.; Waldron, Keith W.; Quesada, Miguel A.; Muñoz-Blanco, Juan; Mercado, José A.

    2016-01-01

    Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

  20. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

    PubMed

    Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A; Waldron, Keith W; Quesada, Miguel A; Muñoz-Blanco, Juan; Mercado, José A

    2016-02-01

    Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

  1. Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

    PubMed Central

    1990-01-01

    The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation. PMID:2202734

  2. Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments.

    PubMed Central

    Kandimalla, E R; Manning, A; Zhao, Q; Shaw, D R; Byrn, R A; Sasisekharan, V; Agrawal, S

    1997-01-01

    We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2'-5'linkages at each end) with the complementary 3'-5'-DNA and -RNA target strands suggest that 2'-5'-ribonucleoside incorporation into 3'-5'-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3'-5'-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2'-5'linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2'-5'modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3'-5'-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3'-5'linkages. The current results suggest that a limited number of 2'-5'linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents. PMID:9016567

  3. Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II.

    PubMed Central

    Siebke, K.; Von Caemmerer, S.; Badger, M.; Furbank, R. T.

    1997-01-01

    It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production. PMID:12223865

  4. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

    PubMed

    Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A; Waldron, Keith W; Quesada, Miguel A; Muñoz-Blanco, Juan; Mercado, José A

    2016-02-01

    Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

  5. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    PubMed

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.

  6. Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158.

    PubMed

    López-Aguilar, Celeste; del Solar, Gloria

    2013-07-01

    Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3'-terminus. On the other hand, two different target transcripts, mRNA₆₀ and mRNA₈₀, were produced, characterized and tested for efficient binding to the antisense product. The mRNA₆₀ and mRNA₈₀ run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3'-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3'-terminal hairpin wherein the extra nucleotides base-paired to the expected 3'-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3'-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5'-terminus of RNAII in a stable 3'-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA.

  7. Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen.

    PubMed

    Maia, Margarida R G; Chaudhary, Lal C; Figueres, Lauren; Wallace, R John

    2007-05-01

    Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12-18:2) substantially. The most common product was vaccenic acid (trans-11-18:1), produced by species related to Butyrivibrio fibrisolvens. alpha-Linolenic acid (LNA; cis-9,cis-12,cis-15-18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n - 3)) and docosahexaenoic acid (DHA; 22:6(n - 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 microg ml(-1), nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11-18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.

  8. Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (codons 2-7) oligodeoxynucleotide: potential relationship to antisense c-myb inhibition.

    PubMed

    Vilenchik, M; Benimetsky, L; Kolbanovsky, A; Miller, P; Stein, C A

    2001-04-01

    We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements. For the last, the 18-mer phosphorothioate oligonucleotide was synthesized with a 5'-fluorescein group. Similar to the molecular beacon model, its fluorescence was quenched when combined in solution with tetraplex-forming oligomers that contained a 3'-Dabcyl moiety. 7-Deazaguanosine inhibits the formation of tetraplexes by eliminated Hoogsteen base pair interactions. The wild-type and 7-deazaguanosine-substituted antisense c-myb oligomers differentially downregulated the expression of the c-myb proto-oncogene in K562 and HL60 cells, with the wild-type oligomer being the least active. The 18-mer c-myb molecule can, therefore, form highly complex structures, whose analysis in solution cannot be limited to examination of slab gel electrophoresis results alone.

  9. 2'-O-[2-[2-(N,N-Dimethylamino)ethoxy]ethyl] Modified Antisense Oligonucleotides: Symbiosis of Charge Interaction Factors and Stereoelectronic Effects

    SciTech Connect

    Prhavc, M.; Prakash, T.P.; Minasov, G.; Egli, M.; Manoharan, M.

    2010-03-08

    Oligonucleotides with a novel, 2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] (2'-O-DMAEOE) modification have been synthesized. This modification, a cationic analogue of the 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, exhibits high binding affinity to target RNA (but not to DNA) and exceptional resistance to nuclease degradation. Analysis of the crystal structure of a self-complementary oligonucleotide containing a single 2'-O-DMAEOE modification explains the importance of charge factors and gauche effects on the observed antisense properties. 2'-O-DMAEOE modified oligonucleotides are ideal candidates for antisense drugs.

  10. Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

    PubMed Central

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

    2014-01-01

    Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression. PMID:25255382

  11. Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion

    PubMed Central

    Tang, Nan-Hong; Chen, Yan-Ling; Wang, Xiao-Qian; Li, Xiu-Jin; Yin, Feng-Zhi; Wang, Xiao-Zhong

    2004-01-01

    AIM: To explore the cooperative effects of antisense oligonucleotide (ASON) of cell adhesion molecules and cimetidine on the expression of E-selectin and ICAM-1 in endothelial cells and their adhesion to tumor cells. METHODS: After treatment of endothelial cells with ASON and/or cimetidine and induction with TNF-α, the protein and mRNA changes of E-selectin and ICAM-1 in endothelial cells were examined by flow cytometry and RT-PCR, respectively. The adhesion rates of endothelial cells to tumor cells were measured by cell adhesion experiment. RESULTS: In comparison with TNF-α inducing group, lipo-ASON and lipo-ASON/cimetidine could significantly decrease the protein and mRNA levels of E-selectin and ICAM-1 in endothelial cells, and lipo-ASON/cimetidine had most significant inhibitory effect on E-selectin expression (from 36.37 ± 1.56% to 14.23 ± 1.07%, P < 0.001). Meanwhile, cimetidine alone could inhibit the expression of E-selectin (36.37 ± 1.56% vs 27.2 ± 1.31%, P < 0.001), but not ICAM-1 (69.34 ± 2.50% vs 68.07 ± 2.10%, P > 0.05)and the two kinds of mRNA, either. Compared with TNF-α inducing group, the rate of adhesion was markedly decreased in lipo-E-selectin ASON and lipo-E-selectin ASON/cimetidine treated groups(P < 0.05), and lipo-E-selectin ASON/cimetidine worked better than lipo-E-selectin ASON alone except for HepG2/ECV304 group (P < 0.05). However, the decrease of adhesion was not significant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/cimetidine treated groups except for HepG2/ECV304 group (P > 0.05). CONCLUSION: These data demonstrate that ASON in combination with cimetidine in vitro can significantly reduce the adhesion between endothelial cells and hepatic or colorectal cancer cells, which is stronger than ASON or cimetidine alone. This study provides some useful proofs for gene therapy of antiadhesion. PMID:14695770

  12. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

    PubMed

    Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

    2015-10-15

    Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants

  13. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

    PubMed Central

    Villamizar, Olga; Chambers, Christopher B.; Mo, Yin-Yuan; Torry, Donald S.; Hofstrand, Reese; Riberdy, Janice M.; Persons, Derek A.; Wilber, Andrew

    2016-01-01

    This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5′ untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf. PMID:27141526

  14. Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

    PubMed

    Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

    2001-02-01

    Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

  15. Pathogenic C9ORF72 Antisense Repeat RNA Forms a Double Helix with Tandem C:C Mismatches.

    PubMed

    Dodd, David W; Tomchick, Diana R; Corey, David R; Gagnon, Keith T

    2016-03-01

    Expansion of a GGGGCC/CCCCGG repeat sequence in the first intron of the C9ORF72 gene is a leading cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this combined disorder, called c9FTD/ALS, the expansion is bidirectionally transcribed into sense and antisense repeat RNA associated with disease. To better understand the role of C9ORF72 repeat RNA in molecular disease pathology, we determined crystal structures of a [(CCCCGG)3(CCCC)] model antisense repeat RNA to 1.47 Å resolution. The RNA structure was an A-form-like double helix composed of repeating and regularly spaced tandem C:C mismatch pairs that perturbed helical geometry and surface charge. Solution studies revealed a preference for A-form-like helical conformations as the repeat number increased. Results provide a structural starting point for rationalizing the contribution of repeat RNA to c9FTD/ALS molecular disease mechanisms and for developing molecules to target C9ORF72 repeat RNA as potential therapeutics.

  16. Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

    PubMed

    Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

    2001-02-01

    Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives. PMID:11262007

  17. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

    PubMed

    Donnelly, Christopher J; Zhang, Ping-Wu; Pham, Jacqueline T; Haeusler, Aaron R; Heusler, Aaron R; Mistry, Nipun A; Vidensky, Svetlana; Daley, Elizabeth L; Poth, Erin M; Hoover, Benjamin; Fines, Daniel M; Maragakis, Nicholas; Tienari, Pentti J; Petrucelli, Leonard; Traynor, Bryan J; Wang, Jiou; Rigo, Frank; Bennett, C Frank; Blackshaw, Seth; Sattler, Rita; Rothstein, Jeffrey D

    2013-10-16

    A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

  18. Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo

    SciTech Connect

    Stoyanova, Radka; Hachem, Paul; Hensley, Harvey; Khor, L.-Y.; Mu Zhaomei; Hammond, M. Elizabeth H.; Agrawal, Sudhir; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2007-07-15

    Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease.

  19. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  20. Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation

    SciTech Connect

    Bariola, P.A.; MacIntosh, G.C.; Green, P.J.

    1999-01-01

    The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T{sub 2} ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In this study the authors examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for NS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

  1. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides

    PubMed Central

    Lennox, Kim A.; Behlke, Mark A.

    2016-01-01

    Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. PMID:26578588

  2. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    PubMed

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

  3. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    PubMed Central

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in an