Science.gov

Sample records for acid lna antisense

  1. C5-amino acid functionalized LNA: positively poised for antisense applications.

    PubMed

    Guenther, Dale C; Kumar, Pawan; Anderson, Brooke A; Hrdlicka, Patrick J

    2014-08-18

    Incorporation of positively charged C5-amino acid functionalized LNA uridines into oligodeoxyribonucleotides (ONs) results in extraordinary RNA affinity, binding specificity and stability towards 3'-exonucleases.

  2. How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

    PubMed

    Plashkevych, Oleksandr; Li, Qing; Chattopadhyaya, Jyoti

    2017-03-29

    A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg(2+) ion availability at the cleavage site

  3. Locked nucleic acid (LNA): Based single-stranded oligonucleotides are not genotoxic.

    PubMed

    Guérard, Melanie; Andreas, Zeller; Erich, Koller; Christine, Marchand; Martina, Müller B; Christian, Weile; Franz, Schuler; Thomas, Singer; Yann, Tessier

    2017-04-01

    Over the last decade, single stranded oligonucleotides (ON) have gained increased attention as a new drug modality. Because the assessment of genotoxicity risk during early development of pharmaceuticals is essential, we evaluated the potential of locked nucleic acids (LNA)-ONs to induce DNA damage in L5178Y tk(+/-) cells both with the mouse lymphoma assay (MLA) and the micronucleus test (MNT). Further, the MLA was performed to assess gene and chromosome mutation over 3 and 24h (± metabolic activation). In addition, the MNT was performed to assess, in addition, a potential aneugenic liability. None of the experiments demonstrated a genotoxic effect for the five tested LNA-ONs. We further show data from four proprietary LNA-ONs tested in standard genotoxicity assays in vitro and partially in vivo, which were all negative. In addition, cellular and nuclear uptake of LNA-ONs in L5178Y tk(+/-) cells was demonstrated. Based on the results presented here as well as in the literature about other representatives of this class, we consider LNA-ONs as generally not DNA reactive and question whether genotoxicity testing of this class of ONs should be required. This is in line with recent recommendation made by the OSWG that extensively assessed the genotoxicity of oligonucleotides. Environ. Mol. Mutagen. 58:112-121, 2017. © 2017 Wiley Periodicals, Inc.

  4. 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

  5. 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

  6. 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

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

    2016-09-29

    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.

  7. Antisense 2'-Deoxy, 2'-Fluoroarabino Nucleic Acid (2'F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids.

    PubMed

    Souleimanian, Naira; Deleavey, Glen F; Soifer, Harris; Wang, Sijian; Tiemann, Katrin; Damha, Masad J; Stein, Cy A

    2012-01-01

    Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2'-deoxy-2'fluoroarabino nucleic acid (2'F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2'F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2'F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded oligos in vitro.

  8. 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

  9. 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.

  10. Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes.

    PubMed

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

    2013-01-01

    The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.

  11. Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling.

    PubMed

    Moisan, Annie; Gubler, Marcel; Zhang, Jitao David; Tessier, Yann; Dumong Erichsen, Kamille; Sewing, Sabine; Gérard, Régine; Avignon, Blandine; Huber, Sylwia; Benmansour, Fethallah; Chen, Xing; Villaseñor, Roberto; Braendli-Baiocco, Annamaria; Festag, Matthias; Maunz, Andreas; Singer, Thomas; Schuler, Franz; Roth, Adrian B

    2017-03-17

    Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

  12. Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation.

    PubMed

    Lundin, Karin E; Hasan, Maroof; Moreno, Pedro M; Törnquist, Elisabeth; Oprea, Iulian; Svahn, Mathias G; Simonson, E Oscar; Smith, C I Edvard

    2005-12-01

    Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.

  13. Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

    PubMed Central

    Shimo, Takenori; Tachibana, Keisuke; Saito, Kiwamu; Yoshida, Tokuyuki; Tomita, Erisa; Waki, Reiko; Yamamoto, Tsuyoshi; Doi, Takefumi; Inoue, Takao; Kawakami, Junji; Obika, Satoshi

    2014-01-01

    Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing. PMID:24935206

  14. Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

    DTIC Science & Technology

    2012-01-10

    Friedrich, U. & Lenke, J. Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real...messenger RNA using locked nucleic acid probes. Anal. Biochem. 390, 109-114 (2009). 13. Waters, L. & Storz, G. Regulatory RNAs in bacteria . Cell. 136, 615...Video Article Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection Kelly

  15. Amplification and re-generation of LNA-modified libraries.

    PubMed

    Doessing, Holger; Hansen, Lykke H; Veedu, Rakesh N; Wengel, Jesper; Vester, Birte

    2012-11-05

    Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts) and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together, these results suggest that dispersed LNA monomers are tolerated in our in vitro selection protocol, and that LNA-modified libraries can be sustained for up to at least seven selection rounds, albeit at reduced levels. This enables the discovery of native LNA aptamers and similar oligonucleotide structures.

  16. 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 Central

    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. PMID:25775001

  17. 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.

  18. Kinetics of membrane damage to high (HNA) and low (LNA) nucleic acid bacterial clusters in drinking water by ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate.

    PubMed

    Ramseier, Maaike K; von Gunten, Urs; Freihofer, Pietro; Hammes, Frederik

    2011-01-01

    Drinking water was treated with ozone, chlorine, chlorine dioxide, monochloramine, ferrate(VI), and permanganate to investigate the kinetics of membrane damage of native drinking water bacterial cells. Membrane damage was measured by flow cytometry using a combination of SYBR Green I and propidium iodide (SGI+PI) staining as indicator for cells with permeabilized membranes and SGI alone to measure total cell concentration. SGI+PI staining revealed that the cells were permeabilized upon relatively low oxidant exposures of all tested oxidants without a detectable lag phase. However, only ozonation resulted in a decrease of the total cell concentrations for the investigated reaction times. Rate constants for the membrane damage reaction varied over seven orders of magnitude in the following order: ozone > chlorine > chlorine dioxide ≈ ferrate > permanganate > chloramine. The rate constants were compared to literature data and were in general smaller than previously measured rate constants. This confirmed that membrane integrity is a conservative and therefore safe parameter for disinfection control. Interestingly, the cell membranes of high nucleic acid (HNA) content bacteria were damaged much faster than those of low nucleic acid (LNA) content bacteria during treatment with chlorine dioxide and permanganate. However, only small differences were observed during treatment with chlorine and chloramine, and no difference was observed for ferrate treatment. Based on the different reactivity of these oxidants it was suggested that HNA and LNA bacterial cell membranes have a different chemical constitution.

  19. In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts.

    PubMed

    Mook, Orf; Vreijling, Jeroen; Wengel, Suzy L; Wengel, Jesper; Zhou, Chuanzheng; Chattopadhyaya, Jyoti; Baas, Frank; Fluiter, Kees

    2010-07-01

    The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.

  20. 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

  1. Cationic derivatives of biocompatible hyaluronic acids for delivery of siRNA and antisense oligonucleotides.

    PubMed

    Han, Su-Eun; Kang, Hyungu; Shim, Ga Yong; Kim, Sun Jae; Choi, Han-Gon; Kim, Jiseok; Hahn, Sei Kwang; Oh, Yu-Kyoung

    2009-02-01

    In this study, we tested the use of cationic polymer derivatives of biocompatible hyaluronic acid (HA) as a delivery system of siRNA and antisense oligonucleotides. HA was modified with cationic polymer polyethylenimine (PEI). When compared with PEI alone, cationic PEI derivatives of HA (HA-PEI) provided increased cellular delivery of Small interfering RNA (siRNA) in B16F1, A549, HeLa, and Hep3B tumor cells. Indeed, more than 95% of the cells were positive for siRNA following its delivery with HA-PEI. A survivin-specific siRNA that was delivered using HA-PEI potently reduced the mRNA expression levels of the target gene in all of the cell lines. By contrast, survivin-specific siRNA delivered by PEI alone did not induce a significant reduction in mRNA levels. In green fluorescent protein (GFP)-expressing 293 T cells, a loss of GFP expression was evident in the cells that had been treated with GFP-specific siRNA and HA-PEI complex. The inhibition of target gene expression by antisense oligonucleotide G3139 was also enhanced after delivery with HA-PEI. Moreover, HA-PEI displayed lower cytotoxicity than PEI alone. These results suggest that HA-PEI could be further developed as biocompatible delivery systems of siRNA and antisense oligonucleotides for enhanced cellular uptake and inhibition of target gene expression.

  2. Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers.

    PubMed

    Kaura, Mamta; Hrdlicka, Patrick J

    2015-07-14

    LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides. We hypothesized that the LNA monomers would alter the sugar rings of the modified DNA monomers toward more RNA-like North-type conformations for maximal DNA/RNA affinity and specificity. Indeed, the incorporation of LNA monomers almost invariably results in increased target affinity and specificity relative to the corresponding LNA-free ONs, but the magnitude of the stabilization varies greatly. Introduction of LNA nucleotides as direct neighbors into C5-pyrene-functionalized pyrimidine DNA monomers yields oligonucleotide probes with more desirable photophysical properties as compared to the corresponding LNA-free probes, including more intense fluorescence emission upon target binding and improved discrimination of single nucleotide polymorphisms (SNPs). These hybrid oligonucleotides are therefore promising probes for diagnostic applications.

  3. 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

  4. Antisense oligodeoxynucleotide-conjugated hyaluronic acid/protamine nanocomplexes for intracellular gene inhibition.

    PubMed

    Mok, Hyejung; Park, Ji Won; Park, Tae Gwan

    2007-01-01

    Green fluorescent protein (GFP) antisense oligodeoxynucleotide (ODN) was covalently conjugated to hyaluronic acid (HA) via a reducible disulfide linkage, and the HA-ODN conjugate was complexed with protamine to increase the extent of cellular uptake and enhance the gene inhibition efficiency of GFP expression. The HA-ODN conjugate formed more stable polyelectrolyte complexes with protamine as compared to naked ODN, probably because of its increased charge density. The higher cellular uptake of protamine/HA-ODN complexes than that of protamine/naked ODN complexes was attributed to the formation of more compact nanosized complexes (approximately 200 nm in diameter) in aqueous solution. Protamine/HA-ODN complexes also showed a comparable level of GFP gene inhibition to that of cytotoxic polyethylenimine (PEI)/ODN complexes. Since both HA and protamine are naturally occurring biocompatible materials, the current formulation based on a cleavable conjugation strategy of ODN to HA could be potentially applied as safe and effective nonviral carriers for ODN and siRNA nucleic acid therapeutics.

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

    PubMed

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

    2015-03-31

    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 (CUG(exp)) 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 CUG(exp)/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 CUG(exp) foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUG(exp)-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.

  6. Why Carba-LNA-modified oligonucleotides show considerably improved 3'-exonuclease stability compared to that of the LNA modified or the native counterparts: A Michaelis-Menten kinetic analysis.

    PubMed

    Zhou, Chuanzheng; Chattopadhyaya, Jyoti

    2010-04-02

    In this study, 12 different native or LNA, carba-LNA-modified dinucleoside phosphates were designed as simple chemical models to study how carba-LNA modifications improve the 3'-exonuclease (SVPDE in this study) resistance of internucleotidic phosphate compared to those exhibited by LNA-modified and the native counterparts. Michaelis-Menten kinetic studies for dimers 3 - 7, in which the LNA or carba-LNA modifications are located at the 5'-end, showed that (i) increased 3'-exonuclease resistance of (5')[LNA-T](p)T (3) compared to the native (5')T(p)T (1) was mainly attributed to steric hindrance imposed by the LNA modification that retards the nuclease binding (K(M)) and (ii) digestion of (5')[carba-LNA-dT](p)T (4) and (5')[LNA-T](p)T (3), however, exhibit similar K(M)s, whereas the former shows a 100x decrease in K(cat) and is hence more stable than the latter. By studying the correlation between log k(cat) and pK(a) of the departing 3'(or 6')-OHs for 3-7, we found the pK(a) of 3'-OH of carba-LNA-T was 1.4 pK(a) units higher than that of LNA-T, and this relatively less acidic character of the 3'-OH in the former leads to the 100x decrease in the catalytic efficiency for the digestion of (5')[carba-LNA-T](p)T (4). In contrast, Michaelis-Menten kinetic studies for dimers 9-12, with the LNA or carba-LNA modifications at the 3'-end, showed that the digestion of (5')T(p)[LNA-T] (9) exhibited similar K(M) but k(cat) decreased around 40 times compared to that of the native (5')T(p)T (1). Similar k(cat) values have been observed for digestion of (5')T(p)[carba-LNA-T] (10) and (5')T(p)[LNA-T] (9). The higher stability of carba-LNA modified dimer 10 compared with LNA modified dimer 9 comes solely from the increased K(M).

  7. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-05

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs.

  8. Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Oyama, Takuya; Akagi, Isao; Sakai, Masao

    2015-01-01

    Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

  9. C5-Alkynyl-Functionalized α-L-LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability

    PubMed Central

    2015-01-01

    Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers. PMID:24797769

  10. 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

  11. Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

    PubMed

    Gifford, Lida K; Opalinska, Joanna B; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C; Do, Bao T; Lu, Ponzy; Gewirtz, Alan M

    2005-02-17

    We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

  12. An Exocyclic Methylene Group Acts As A Bio-isostere of the 2’-Oxygen Atom in LNA

    PubMed Central

    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.

    2010-01-01

    We show for the first time that it is possible to obtain LNA (Locked Nucleic Acid 1) like binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2’-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’-position and a propynyl group at C-4’ 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’-deoxyribose residue of the 5’-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’-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA ASOs showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moeities in nucleic acid structure and emphasizes greater use of this bio-isostere to improve the properties of nucleic acids for therapeutic and diagnostic applications. PMID:20886816

  13. Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

    PubMed

    Lou, Chenguang; Samuelsen, Simone V; Christensen, Niels Johan; Vester, Birte; Wengel, Jesper

    2017-04-05

    Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.

  14. 2′-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping

    PubMed Central

    Lee, Tomoko; Awano, Hiroyuki; Yagi, Mariko; Matsumoto, Masaaki; Watanabe, Nobuaki; Goda, Ryoya; Koizumi, Makoto; Takeshima, Yasuhiro; Matsuo, Masafumi

    2017-01-01

    Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO)-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51), which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA), which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2′-O-methyl RNA(2′-OMeRNA)/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2′-OMeRNA/ENA chimera (AO85) had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2′-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD. PMID:28208626

  15. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes

    PubMed Central

    Jackson, Alexis; Jani, Saumya; Sala, Carol Davies; Zorreguieta, Angeles; Tolmasky, Marcelo E.

    2016-01-01

    EGSs (external guide sequences) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNA), modified ribonucleotides that contain a bridge at the 2′,4′-position of the ribose. LNA and the second generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6′)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5′- and 3′-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell penetrating peptide (CPP), the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and

  16. Continuous administration of antisense oligonucleotides to c-fos reduced the development of seizure susceptibility after ethacrynic acid-induced seizure in mice.

    PubMed

    Suzukawa, Junko; Omori, Kyoko; Yang, Li; Inagaki, Chiyoko

    2003-09-25

    We previously demonstrated that seizure susceptibility developed by the 14th day post-ethacrynic acid (EA)-induced seizure in mice, with a prolonged increase in the expression of c-fos mRNA in the brain during days 10-14. To examine whether such c-fos increase contributes to the development of seizure susceptibility, we administered antisense oligodeoxynucleotide to c-fos by continuous infusion into the lateral ventricle of mice that had shown a moderate stage of EA seizure, and evaluated the seizure susceptibility to kainic acid (10 mg/kg) on the 14th day. Antisense-infused mice displayed significant reduction of the c-Fos level in the hippocampus and cerebral cortex on the 7th and 14th days, and a significant decrease in seizure severity. These findings suggest that the prolonged increase in c-fos expression after EA seizure may lead to the development of seizure susceptibility.

  17. Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse.

    PubMed

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew J A

    2010-04-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy.

  18. 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.

  19. Current Status and Perspectives Regarding LNA-anti-miR Oligonucleotides and microRNA miR-21 Inhibitors as a Potential Therapeutic Option in Treatment of Colorectal Cancer.

    PubMed

    Nedaeinia, Reza; Avan, Amir; Ahmadian, Mehdi; Nedaee Nia, Sasan; Ranjbar, Maryam; Sharifi, Mohammadreza; Goli, Mohammad; Piroozmand, Ahmad; Nourmohammadi, Esmail; Manian, Mostafa; Ferns, Gordon A; Ghayour-Mobarhan, Majid; Salehi, Rasoul

    2017-04-12

    Colorectal cancer (CRC) is among the leading causes of cancer-related death, principally due to its metastatic spread and multifactorial chemoresistance. The therapeutic failure can also be explained by inter- or intra-tumor genetic heterogeneity and tumor stromal content. Thus, the identification of novel prognostic biomarkers and therapeutic options are warranted in the management of CRC patients. There are data showing that microRNA-21 is elevated in different types of cancer, particularly colon adenocarcinoma and that this is association with a poor prognosis. This suggests that microRNA-21 may be of value as a potential therapeutic target. Furthermore, locked nucleic acid (LNA)-modified oligonucleotides have recently emerged as a therapeutic option for targeting dysregulated miRNAs in cancer therapy, through antisense-based gene silencing. Further work is required to identify innovative anticancer drugs that improve the current therapy either through novel combinatorial approaches or with better efficacy than conventional drugs. We aimed to provide an overview of the preclinical and clinical studies targeting key dysregulated signaling pathways in CRC as well as the therapeutic application of LNA-modified oligonucleotides and miR inhibitors in the treatment of CRC patients. This article is protected by copyright. All rights reserved.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

    PubMed Central

    Cordier, Céline; Boutimah, Fatima; Bourdeloux, Mathilde; Dupuy, Florian; Met, Elisabeth; Alberti, Patrizia; Loll, François; Chassaing, Gérard; Burlina, Fabienne; Saison-Behmoaras, Tula Ester

    2014-01-01

    Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization. PMID:25127364

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

    PubMed Central

    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

  7. Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture.

    PubMed

    Araújo, Wagner L; Nunes-Nesi, Adriano; Osorio, Sonia; Usadel, Björn; Fuentes, Daniela; Nagy, Réka; Balbo, Ilse; Lehmann, Martin; Studart-Witkowski, Claudia; Tohge, Takayuki; Martinoia, Enrico; Jordana, Xavier; Damatta, Fábio M; Fernie, Alisdair R

    2011-02-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell-specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.

  8. Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA, and 2'-Amino-LNA Monomers.

    PubMed

    Ries, Annika; Kumar, Rajesh; Lou, Chenguang; Kosbar, Tamer; Vengut-Climent, Empar; Jørgensen, Per T; Morales, Juan C; Wengel, Jesper

    2016-11-18

    Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.

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

    PubMed Central

    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.

    2011-01-01

    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 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 inter-nucleotide C-F…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 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)…H-C(Pu) pseudo hydrogen bonds in nucleic acid structures. PMID:21919455

  10. 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.

  11. Evaluation of LNA, MGB and non-modified DNA probes to improve the detection limit of TaqMan real-time PCR assay for Pantoea stewartii subsp. stewartii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this study was to compare the sensitivity and amplification efficiency of the TaqMan assay using locked nucleic acid (LNA), minor groove binder (MGB) ligands and non-modified DNA probes. In monoplex or single target TaqMan assays for P. stewartii subsp. stewartii, LNA and MGB probes impr...

  12. 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)

  13. Different HIV-1 env frames: gp120 and ASP (antisense protein) biosynthesis, and theirs co-variation tropic amino acid signatures in X4- and R5-viruses.

    PubMed

    Dimonte, Salvatore

    2017-01-01

    Antisense protein (ASP) is the new actor of viral life of Human Immunodeficiency Virus type 1 (HIV-1) although proposed above 20 years ago. The asp ORF is into complementary strand of the gp120/gp41 junction of env gene. The ASP biological role remains little known. Knowing the Env markers of viral tropism, a dataset of sequences (660 strains) was used to analyze the hypothetical ASP involvement in CCR5 (R5) and/or CXCR4 (X4) co-receptor interaction. Preliminarily, prevalence of ASP and gp120V3 mutations was performed; following association among mutations were elaborate. The classical V3 tropic-signatures were confirmed, and 36 R5- and 22 X4-tropic ASP mutations were found. Moreover, by analyzing the ASP sequences, 36 out of 179 amino acid positions significantly associated with different co-receptor usage were found. Several statistically significant associations between gp120V3 and ASP mutations were observed. The dendrogram showed the existence of a cluster associated with R5-usage and a large cluster associated with X4-usage. These results show that gp120V3 and specific amino acid changes in ASP are associated together with CXCR4 and/or CCR5-usage. These findings implement previous observations on unclear ASP functions. J. Med. Virol. 89:112-122, 2017. © 2016 Wiley Periodicals, Inc.

  14. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

    modifications, chimeric oligonucleotides, or peptide nucleic acids. Continued progress in this arena will require that many of the preclinical challenges confronting antisense development are satisfactory resolved.

  15. A novel noise optimization technique for inductively degenerated CMOS LNA

    NASA Astrophysics Data System (ADS)

    Zhiqing, Geng; Haiyong, Wang; Nanjian, Wu

    2009-10-01

    This paper proposes a novel noise optimization technique. The technique gives analytical formulae for the noise performance of inductively degenerated CMOS low noise amplifier (LNA) circuits with an ideal gate inductor for a fixed bias voltage and nonideal gate inductor for a fixed power dissipation, respectively, by mathematical analysis and reasonable approximation methods. LNA circuits with required noise figure can be designed effectively and rapidly just by using hand calculations of the proposed formulae. We design a 1.8 GHz LNA in a TSMC 0.25 μm CMOS process. The measured results show a noise figure of 1.6 dB with a forward gain of 14.4 dB at a power consumption of 5 mW, demonstrating that the designed LNA circuits can achieve low noise figure levels at low power dissipation.

  16. 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

  17. Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters.

    PubMed Central

    Faiella, A; Zappavigna, V; Mavilio, F; Boncinelli, E

    1994-01-01

    Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated first, whereas upstream Hox genes are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity. Images PMID:7911240

  18. HOX antisense lincRNA HOXA-AS2 is an apoptosis repressor in all trans retinoic acid treated NB4 promyelocytic leukemia cells.

    PubMed

    Zhao, Hang; Zhang, Xueqing; Frazão, Josias Brito; Condino-Neto, Antonio; Newburger, Peter E

    2013-10-01

    HOXA cluster antisense RNA 2 (HOXA-AS2) is a long non-coding RNA located between the HOXA3 and HOXA4 genes in the HOXA cluster. Its transcript is expressed in NB4 promyelocytic leukemia cells and human peripheral blood neutrophils, and expression is increased in NB4 cells treated with all trans retinoic acid (ATRA). Knockdown of HOXA-AS2 expression by transduced shRNA decreases the number of viable cells and increases the proportion of apoptotic cells, measured by annexin V binding and by activity and cleavage of caspases-3, -8, and -9. The increase in death of HOXA-AS2 knockdown cells was accompanied by an elevated TNF-related apoptosis-inducing ligand (TRAIL) levels, but ATRA-induced NB4 cells treated with TRAIL did show an increase in HOXA-AS2 expression. These results demonstrate that ATRA induction of HOXA-AS2 suppresses ATRA-induced apoptosis, possibly through a TRAIL-mediated pathway. HOXA-AS2-mediated negative regulation thus contributes to the fine-tuning of apoptosis during ATRA-induced myeloid differentiation in NB4 cells.

  19. Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy

    PubMed Central

    Govan, Jeane M.; Uprety, Rajendra; Thomas, Meryl; Lusic, Hrvoje; Lively, Mark O.; Deiters, Alexander

    2013-01-01

    Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity. PMID:23915424

  20. 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

  1. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    PubMed

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation.

  2. Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter.

    PubMed

    Kuwano, Mio; Mimura, Tetsuro; Takaiwa, Fumio; Yoshida, Kaoru T

    2009-01-01

    Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

  3. [Effects of antisense acid invertase gene on reducing sugar and starch contents of potato (Solanum tuberosum L.) tuber stored at low temperature].

    PubMed

    Wang, Qing; Huang, Hui-Ying; Zhang, Jin-Wen; Li, Xue-Cai

    2005-10-01

    Two transgenic potato lines with antisense AcInv gene and the non-transgenic varieties were used to test the reducing sugar and starch contents. The results indicated that the reducing sugar content increased and total starch content decreased in all samples after tubers were stored at 4 degrees C for 40 d. The reducing sugar content in tubers of transgenic line "Anti-AcInv Atlantic" and "Anti-AcInv Gannongshu No.2" were lower about 23% and 18% than those of Atlantic and Gannongshu No.2 (Table 1). The total starch and the amylopectin were also decreased by 1% and 1.3% in "Anti-AcInv Atlantic" and 1.4% and 1.7% in "Anti-AcInv Gannongshu No.2" respectively (Table 2). The proportions of amylose and amylopectin were lower in the transgenic lines than the non-transgenic varieties (Figs. 1 and 2). It was only 0.29 for "Anti-AcInv Atlantic" and 0.38 for "Anti-AcInv Gannongshu No.2". Meanwhile, there are fewer dark blue starch particles in transgenic tuber, which is less than in tuber of the non-transgenic varieties by paraffin-cut section method (Fig. 3). After the tubers were transferred to the storage temperature 15-17 degrees C for 20 d, the reducing sugar contents in tubers of the two transgenic lines were significantly lower than that of non-transgenic varieties. It was 0.23% for "Anti-AcInv Atlantic" and 0.24% for "Anti-AcInv Gannongshu No.2" (Table 1). It is suggested that the trans-antisense AcInv gene in the transgenic potato down-regulates the AcInv gene expression after the tubers were stored under low temperature.

  4. 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

  5. Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    PubMed Central

    Bergquist, Helen; Rocha, Cristina S. J.; Álvarez-Asencio, Rubén; Nguyen, Chi-Hung; Rutland, Mark. W.; Smith, C. I. Edvard; Good, Liam; Nielsen, Peter E.; Zain, Rula

    2016-01-01

    Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression. PMID:27846236

  6. 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

  7. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

    SciTech Connect

    Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; Singh, Anup K.

    2014-08-20

    Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155 and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.

  8. miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

    DOE PAGES

    Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

    2014-08-20

    Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less

  9. Antisense Treatments for Biothreat Agents

    DTIC Science & Technology

    2006-08-01

    oligomers (ASOs) represent a promising technology to treat viral and bacterial infections, and have already been shown to be successful against a...viral and bacterial agents have a history of state- sponsored ’weaponization’, including Marburg, Ebola, Junin, Machupo, yellow fever viruses and...14. ABSTRACT Antisense oligomers (ASOs) represent a promising technology to treat viral and bacterial infections, and have already been shown to be

  10. Neighboring gene regulation by antisense long non-coding RNAs.

    PubMed

    Villegas, Victoria E; Zaphiropoulos, Peter G

    2015-02-03

    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.

  11. Threshold changes in rat brain docosahexaenoic acid incorporation and concentration following graded reductions in dietary alpha-linolenic acid

    PubMed Central

    Taha, Ameer Y.; Chang, Lisa; Chen, Mei

    2016-01-01

    Background This study tested the dietary level of alpha-linolenic acid (α-LNA, 18:3n-3) sufficient to maintain brain 14C-Docosahexaenoic acid (DHA, 22:6n-3) metabolism and concentration following graded α-LNA reduction. Methods 18–21 day male Fischer-344 (CDF) rats were randomized to the AIN-93G diet containing as a % of total fatty acids, 4.6% (“n-3 adequate”), 3.6%, 2.7%, 0.9% or 0.2% (“n-3 deficient”) α-LNA for 15 weeks. Rats were intravenously infused with 14C-DHA to steady state for 5 minutes, serial blood samples collected to obtain plasma and brains excised following microwave fixation. Labeled and unlabeled DHA concentrations were measured in plasma and brain to calculate the incorporation coefficient, k*, and incorporation rate, Jin. Results Compared to 4.6% α-LNA controls, k* was significantly increased in ethanolamine glycerophospholipids in the 0.2% α-LNA group. Circulating unesterified DHA and brain incorporation rates (Jin) were significantly reduced at 0.2% α-LNA. Brain total lipid and phospholipid DHA concentrations were reduced at or below 0.9% α-LNA. Conclusion Threshold changes for brain DHA metabolism and concentration were maintained at or below 0.9% dietary α-LNA, suggesting the presence of homeostatic mechanisms to maintain brain DHA metabolism when dietary α-LNA intake is low. PMID:26869088

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

    PubMed

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

    2016-04-12

    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.

  13. 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

  14. 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.

  15. Stability and free energy calculation of LNA modified quadruplex: a molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Chaubey, Amit Kumar; Dubey, Kshatresh Dutta; Ojha, Rajendra Prasad

    2012-03-01

    Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, which are the fundamental in protecting the cell from recombination and degradation. Telomeric DNA sequences can form four stranded quadruplex structures, which are involved in the structure of telomere ends. The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadrulex as an attractive target for cancer therapeutic intervention. Molecular dynamic simulation offers the prospects of detailed description of the dynamical structure with ion and water at molecular level. In this work we have taken a oligomeric part of human telomeric DNA, d(TAGGGT) to form different monomeric quadruplex structures d(TAGGGT)4. Here we report the relative stabilities of these structures under K+ ion conditions and binding interaction between the strands, as determined by molecular dynamic simulations followed by energy calculation. We have taken locked nucleic acid (LNA) in this study. The free energy molecular mechanics Poission Boltzman surface area calculations are performed for the determination of most stable complex structure between all modified structures. We calculated binding free energy for the combination of different strands as the ligand and receptor for all structures. The energetic study shows that, a mixed hybrid type quadruplex conformation in which two parallel strands are bind with other two antiparallel strands, are more stable than other conformations. The possible mechanism for the inhibition of the cancerous growth has been discussed. Such studies may be helpful for the rational drug designing.

  16. An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system.

    PubMed

    Montagner, Giulia; Bezzerri, Valentino; Cabrini, Giulio; Fabbri, Enrica; Borgatti, Monica; Lampronti, Ilaria; Finotti, Alessia; Nielsen, Peter E; Gambari, Roberto

    2017-02-03

    Discovery of novel antimicrobial agents against Pseudomonas aeruginosa able to inhibit bacterial growth as well as the resulting inflammatory response is a key goal in cystic fibrosis research. We report in this paper that a peptide nucleic acid (PNA3969) targeting the translation initiation region of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits PAO1 induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably, no effect on PAO1 induction of VEGF, GM-CSF and IL-17 was observed. Analogous experiments using a two base mis-match control PNA did not show such inhibition. Furthermore, no significant effects of the PNAs were seen on cell growth, apoptosis or secretome profile in uninfected IB3-1 cells (with the exception of a PNA-mediated up-regulation of PDGF, IL-17 and GM-CSF). Thus, we conclude that in cell culture an antimicrobial PNA against Pseudomonas can inhibit the expression of pro-inflammatory cytokines otherwise induced by the infection. In particular, the effects of PNA-3969 on IL-8 gene expression are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection.

  17. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

    PubMed

    Olson, Xiaoping; Kotani, Shohei; Yurke, Bernard; Graugnard, Elton; Hughes, William L

    2017-03-30

    Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

  18. Functionalization of an Antisense Small RNA.

    PubMed

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

    2016-02-27

    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.

  19. 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

  20. Design and optimization of CMOS LNA with ESD protection for 2.4 GHz WSN application

    NASA Astrophysics Data System (ADS)

    Zhiqun, Li; Liang, Chen; Hao, Zhang

    2011-10-01

    A new optimization method of a source inductive degenerated low noise amplifier (LNA) with electrostatic discharge protection is proposed. It can achieve power-constrained simultaneous noise and input matching. An analysis of the input impedance and the noise parameters is also given. Based on the developed method, a 2.4 GHz LNA for wireless sensor network application is designed and optimized using 0.18-μm RF CMOS technology. The measured results show that the LNA achieves a noise figure of 1.59 dB, a power gain of 14.12 dB, an input 1 dB compression point of -8 dBm and an input third-order intercept point of 1 dBm. The DC current is 4 mA under a supply of 1.8 V.

  1. The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot.

    PubMed Central

    Gogarten, J P; Fichmann, J; Braun, Y; Morgan, L; Styles, P; Taiz, S L; DeLapp, K; Taiz, L

    1992-01-01

    Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35S promoter. Bafilomycin-sensitive ATPase, H(+)-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. Two-dimensional protein gel blots of total microsomes of wild-type and control transformant cell lines revealed two major immunoreactive polypeptides in the acidic pI range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carrot. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct. PMID:1392599

  2. Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

    PubMed

    Jastrzębska, Katarzyna; Maciaszek, Anna; Dolot, Rafał; Bujacz, Grzegorz; Guga, Piotr

    2015-10-21

    3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.

  3. Development of Antisense Drugs for Dyslipidemia

    PubMed Central

    Wada, Fumito; Harada-Shiba, Mariko

    2016-01-01

    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

  4. Development of a 2′,4′-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo

    PubMed Central

    Wada, Shunsuke; Obika, Satoshi; Shibata, Masa-Aki; Yamamoto, Tsuyoshi; Nakatani, Moeka; Yamaoka, Tetsuji; Torigoe, Hidetaka; Harada-Shiba, Mariko

    2012-01-01

    Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2′,4′-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2′,4′-BNA/LNA in its sense strand and with 2′-methoxy (2′-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2′,4′-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5′-UG-3′ in the sense strand would ameliorate the negative immune response. PMID:23344237

  5. Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

    PubMed

    Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

    2014-07-03

    Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

  6. Lysine metabolism in antisense C-hordein barley grains.

    PubMed

    Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A; Medici, Leonardo O; Vincze, Eva; Kozak, Marcin; Lea, Peter J; Azevedo, Ricardo A

    2015-02-01

    The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise) and five antisense C-hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-hordein suppression did not only alter the metabolism of at least one aspartate-derived amino acid (threonine), but major changes were also detected in the metabolism of lysine and methionine. Modifications in the activities and regulation of aspartate kinase, dihydrodipicolinate synthase and homoserine dehydrogenase were observed in most transgenic lines. Furthermore the activities of lysine α-ketoglutarate reductase and saccharopine dehydrogenase were also altered, although the extent varied among the transgenic lines.

  7. Plasmid Replication Control by Antisense RNAs.

    PubMed

    Brantl, Sabine

    2014-08-01

    Plasmids are selfish genetic elements that normally constitute a burden for the bacterial host cell. This burden is expected to favor plasmid loss. Therefore, plasmids have evolved mechanisms to control their replication and ensure their stable maintenance. Replication control can be either mediated by iterons or by antisense RNAs. Antisense RNAs work through a negative control circuit. They are constitutively synthesized and metabolically unstable. They act both as a measuring device and a regulator, and regulation occurs by inhibition. Increased plasmid copy numbers lead to increasing antisense-RNA concentrations, which, in turn, result in the inhibition of a function essential for replication. On the other hand, decreased plasmid copy numbers entail decreasing concentrations of the inhibiting antisense RNA, thereby increasing the replication frequency. Inhibition is achieved by a variety of mechanisms, which are discussed in detail. The most trivial case is the inhibition of translation of an essential replication initiator protein (Rep) by blockage of the rep-ribosome binding site. Alternatively, ribosome binding to a leader peptide mRNA whose translation is required for efficient Rep translation can be prevented by antisense-RNA binding. In 2004, translational attenuation was discovered. Antisense-RNA-mediated transcriptional attenuation is another mechanism that has, so far, only been detected in plasmids of Gram-positive bacteria. ColE1, a plasmid that does not need a plasmid-encoded replication initiator protein, uses the inhibition of primer formation. In other cases, antisense RNAs inhibit the formation of an activator pseudoknot that is required for efficient Rep translation.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Electronic coupling between base pair dimers of LNA:DNA oligomers.

    PubMed

    Ivanova, Anela; Jezierski, Grzegorz; Rösch, Notker

    2008-01-21

    We calculated ab initio electronic coupling elements between neighboring base-pair dimers in a set of LNA:DNA oligomers with different numbers of locked nucleotides and compared them by averaging the values over ensembles of snapshots from molecular dynamics trajectories. Averaging was based on coupling elements for various ensembles comprising of 33,000 structures. The known pronounced variations of coupling elements on the nanosecond timescale due to thermal fluctuations of the DNA structure were confirmed. We found significant differences in electronic coupling at the dimer level between a non-modified DNA:DNA duplex and the corresponding duplex containing one fully LNA-substituted strand. We rationalized these differences by very dissimilar overlap in the pi-stack as a consequence of the LNA-modified system approximating an A-DNA-type helix. The calculated coupling elements for the non-modified reference duplex were similar to those of standard B-DNA and those for the fully modified oligomer resembled the matrix elements estimated for standard A-DNA.

  14. Selection of LNA-containing DNA aptamers against recombinant human CD73.

    PubMed

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G; Larsen, Niels; Nielsen, Ronni; Derbyshire, Nicola; Mandrup, Susanne; Ditzel, Henrik J; Wengel, Jesper

    2015-05-01

    LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

  15. 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

  16. Antisense Oligodeoxynucleotide Inhibition of HIV Gene Expression

    DTIC Science & Technology

    1989-03-20

    of HtL -60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examined and compared with Me2 SO. Differentiation of HL-60...reticulocyte eIF4B binds specifically to AUG (36). Both eIF4F, which includes eIF4A, eIF4E, and a 220 kD subunit, and eIF4B may be crosslinked to the

  17. 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

    2015-03-03

    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.

  18. Overlapping Antisense Transcription in the Human Genome

    PubMed Central

    Fahey, M. E.; Moore, T. F.

    2002-01-01

    Accumulating evidence indicates an important role for non-coding RNA molecules in eukaryotic cell regulation. A small number of coding and non-coding overlapping antisense transcripts (OATs) in eukaryotes have been reported, some of which regulate expression of the corresponding sense transcript. The prevalence of this phenomenon is unknown, but there may be an enrichment of such transcripts at imprinted gene loci. Taking a bioinformatics approach, we systematically searched a human mRNA database (RefSeq) for complementary regions that might facilitate pairing with other transcripts. We report 56 pairs of overlapping transcripts, in which each member of the pair is transcribed from the same locus. This allows us to make an estimate of 1000 for the minimum number of such transcript pairs in the entire human genome. This is a surprisingly large number of overlapping gene pairs and, clearly, some of the overlaps may not be functionally significant. Nonetheless, this may indicate an important general role for overlapping antisense control in gene regulation. EST databases were also investigated in order to address the prevalence of cases of imprinted genes with associated non-coding overlapping, antisense transcripts. However, EST databases were found to be completely inappropriate for this purpose. PMID:18628857

  19. Polymorphism of linoleic acid (cis-9, cis-12-octadecadienoic acid) and alpha-linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid).

    PubMed

    Ueno, S; Miyazaki, A; Yano, J; Furukawa, Y; Suzuki, M; Sato, K

    2000-10-01

    Crystallization and polymorphic properties of linoleic acid (cis-9, cis-12-Octadecadienoic acid) (LA) and alpha-linolenic acid (cis-9, cis-12, cis-15-Octadecatrienoic acid) (alpha-LNA) have been studied by optical microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The DSC analyses presented three polymorphs in LA, and two polymorphs in alpha-LNA. The XRD patterns of the higher- and lower-temperature forms in LA and alpha-LNA showed orthorhombic O'(//)+O-like and O'(//) subcell, which were similar to those of alpha- and gamma-forms of mono-unsaturated fatty acids, respectively. From the solvent crystallization of LA and alpha-LNA in acetonitrile, single crystals of the higher temperature polymorphs have been obtained. The crystal habits of truncated rhombic shape were also similar to those of alpha-forms of the mono-unsaturated fatty acids. The enthalpy and entropy values of fusion and dissolution of the alpha-forms of LA, alpha-LNA and oleic acid showed that the two values decreased with increasing number of the cis-double bond.

  20. 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.

  1. A good antisense molecule is hard to find.

    PubMed

    Branch, A D

    1998-02-01

    Antisense molecules and ribozymes capture the imagination with their promise of rational drug design and exquisite specificity. However, they are far more difficult to produce than was originally anticipated, and their ability to eliminate the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light. Although some of these side effects will almost certainly have clinical value, they make it hard to produce drugs that act primarily through true antisense mechanisms and complicate the use of antisense compounds as research reagents. To minimize unwanted non-antisense effects, investigators are searching for antisense compounds and ribozymes whose target sites are particularly vulnerable to attack. This is a challenging quest.

  2. 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

  3. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles.

    PubMed

    Ita, Kevin

    2017-03-01

    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted.

  4. Antisense antibiotics: a brief review of novel target discovery and delivery.

    PubMed

    Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing

    2010-06-01

    The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

  5. Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

    PubMed Central

    2015-01-01

    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. PMID:25198246

  6. 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.

  7. Natural antisense and noncoding RNA transcripts as potential drug targets.

    PubMed

    Wahlestedt, Claes

    2006-06-01

    Information on the complexity of mammalian RNA transcription has increased greatly in the past few years. Notably, thousands of sense transcripts (conventional protein-coding genes) have antisense transcript partners, most of which are noncoding. Interestingly, a number of antisense transcripts regulate the expression of their sense partners, either in a discordant (antisense knockdown results in sense-transcript elevation) or concordant (antisense knockdown results in concomitant sense-transcript reduction) manner. Two new pharmacological strategies based on the knockdown of antisense RNA transcripts by siRNA (or another RNA targeting principle) are proposed in this review. In the case of discordant regulation, knockdown of antisense transcript elevates the expression of the conventional (sense) gene, thereby conceivably mimicking agonist-activator action. In the case of concordant regulation, knockdown of antisense transcript, or concomitant knockdown of antisense and sense transcripts, results in an additive or even synergistic reduction of the conventional gene expression. Although both strategies have been demonstrated to be valid in cell culture, it remains to be seen whether they provide advantages in other contexts.

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26151747

  10. Adsorption and surface dynamics of short DNA and LNA oligonucleotides on single-crystal Au(1 1 1) electrode surfaces

    NASA Astrophysics Data System (ADS)

    Wackerbarth, Hainer; Grubb, Mikala; Wengel, Jesper; Chorkendorff, Ib; Ulstrup, Jens

    2006-05-01

    We have studied the surface dynamics of a double-strand decanucleotide (HS-10AT L) with 10 adenine-thymine base pairs linked to a Au(1 1 1)-electrode surface via a hexamethylene thiol linker. The study is based on a combination of voltammetry, interfacial capacitance data, electrochemical in situ scanning tunnelling microscopy, and X-ray photoelectron spectroscopy. The thymine bases of the oligonucleotide are connected to furanoses locked in a C3'- endo configuration called LNA (locked nucleic acid). Hybridization in solution is effected prior to linking to the Au(1 1 1)-surface. The melting point of the linker-free locked decanucleotide, 10AT L is >63 °C. However, voltammetric reductive desorption of the adsorbed thiol-modified double-strand decanucleotide, HS-10AT L, gives almost the same charge as single-strand HS-10A, 29 ± 3 and 27 ± 5 μC cm -2, respectively. In situ STM after HS-10AT L-immobilization also gives images showing highly ordered domains, virtually indistinguishable from those of immobilized HS-10A. X-ray photoelectron spectroscopy gives an N/P ration of 5.0 for HS-10AT L in line with the expected value for single-strand HS-10A (5.0). All three sets of data suggest that HS-10AT L hybridized in solution is significantly dissociated on binding to the Au(1 1 1)-electrode surface. This points to an adsorption mechanism in which a stable high density of Au-S bonds is achieved but at the expense of significant unzipping of the more voluminous duplex form.

  11. Splice-switching antisense oligonucleotides as therapeutic drugs

    PubMed Central

    Havens, Mallory A.; Hastings, Michelle L.

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  12. Gene Isoform Specificity through Enhancer-Associated Antisense Transcription

    PubMed Central

    Onodera, Courtney S.; Underwood, Jason G.; Katzman, Sol; Jacobs, Frank; Greenberg, David; Salama, Sofie R.; Haussler, David

    2012-01-01

    Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates. PMID:22937057

  13. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    PubMed

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  14. On primordial sense-antisense coding.

    PubMed

    Rodin, Andrei S; Rodin, Sergei N; Carter, Charles W

    2009-11-01

    The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These 20 enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sense-antisense (SAS) coding (Rodin and Ohno, Orig Life Evol Biosph 25:565-589, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achlya klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in Williams et al. (Mol Biol Evol 26:445-450, 2009), this could indeed be a "Rosetta stone" gene (Carter and Duax, Mol Cell 10:705-708, 2002) (eroded somewhat, though) for the SAS origin of the two aaRS classes.

  15. 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.

  16. Synthesis of 6'-branched locked nucleic acid by a radical TEMPO-scavanged stereoselective mercury cyclization.

    PubMed

    Enderlin, Gerald; Nielsen, Poul

    2008-09-05

    A 6'(R)-hydroxymethyl derivative of the locked nucleic acid (LNA)-thymidine monomer has been synthesized by a stereoselective mercury cyclization and subsequent use of TEMPO as a radical scavenger. This compound was converted to an azide derivative, which in a Huisgen-type [3 + 2] cycloaddition afforded a double-headed nucleoside with a triazole linking an additional thymine to the 6'-position of the LNA-nucleoside monomer.

  17. Natural antisense transcripts of Alzheimer's disease associated genes.

    PubMed

    Guo, Jin-Hu; Cheng, Hai-Peng; Yu, Long; Zhao, Shouyuan

    2006-04-01

    Natural antisense transcripts (NATs), also named endogenous antisense transcripts, are a class of genes whose role in controlling gene expression is becoming more and more relevant. NATs might play important roles in gene expression and translation regulation. Present work investigated the presence of NATs of Alzheimer's disease associated genes including PRESENILIN1, PRESENILIN2, BACE1, BACE2, APP, APOE, TAU (MAPT), PRION, alpha-SYNUCLEIN (SNCA), NICASTRIN, PEN2, APH1A, APH1B as well as CD147 (BASIGIN), and the results revealed that APP, BACE2, APH1A, TAU, CD147 and alpha-SYNUCLEIN contain natural antisense transcripts. These NATs were characterized according to the sense-antisense overlapping information and potential functional mechanisms were proposed. Present findings provide preliminary but important information about transcription regulation of AD associated genes, which would further our understanding of the gene expression regulation of AD, and also suggest a novel potential strategy for the therapy of AD.

  18. 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.

  19. 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.

  20. 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.

  1. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1998-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinary important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time because much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regard to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of chronic myelogenous leukemia and other neoplastic disorders.

  2. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinarily important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time since much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regards to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of CML and other neoplastic disorders.

  3. 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.

  4. 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.

  5. Bacterial type B RNase P: functional characterization of the L5.1-L15.1 tertiary contact and antisense inhibition.

    PubMed

    Walczyk, Dennis; Willkomm, Dagmar K; Hartmann, Roland K

    2016-11-01

    Ribonuclease P is the ubiquitous endonuclease that generates the mature 5'-ends of precursor tRNAs. In bacteria, the enzyme is composed of a catalytic RNA (∼400 nucleotides) and a small essential protein subunit (∼13 kDa). Most bacterial RNase P RNAs (P RNAs) belong to the architectural type A; type B RNase P RNA is confined to the low-G+C Gram-positive bacteria. Here we demonstrate that the L5.1-L15.1 intradomain contact in the catalytic domain of the prototypic type B RNase P RNA of Bacillus subtilis is crucial for adopting a compact functional conformation: Disruption of the L5.1-L15.1 contact by antisense oligonucleotides or mutation reduced P RNA-alone and holoenzyme activity by one to two orders of magnitude in vitro, largely retarded gel mobility of the RNA and further affected the structure of regions P7/P8/P10.1, P15 and L15.2, and abolished the ability of B. subtilis P RNA to complement a P RNA-deficient Escherichia coli strain. We also provide mutational evidence that an L9-P1 tertiary contact, as found in some Mycoplasma type B RNAs, is not formed in canonical type B RNAs as represented by B. subtilis P RNA. We finally explored the P5.1 and P15 stem-loop structures as targets for LNA-modified antisense oligonucleotides. Oligonucleotides targeting P15, but not those directed against P5.1, were found to efficiently anneal to P RNA and to inhibit activity (IC50 of ∼2 nM) when incubated with preassembled B. subtilis RNase P holoenzymes.

  6. Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides.

    PubMed Central

    Goodchild, J; Agrawal, S; Civeira, M P; Sarin, P S; Sun, D; Zamecnik, P C

    1988-01-01

    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. PMID:3041414

  7. 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.

  8. 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.

  9. 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

  10. 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...

  11. Expression of antisense or sense RNA of an ankyrin repeat-containing gene blocks chloroplast differentiation in arabidopsis.

    PubMed Central

    Zhang, H; Scheirer, D C; Fowle, W H; Goodman, H M

    1992-01-01

    The Arabidopsis AKR gene that encodes a protein with four ankyrin repeats (a 33-amino acid motif that appears in the 89K domain of the human protein ankyrin) was isolated and characterized. A short sequence outside the ankyrin repeats is similar to that of the protein of the Drosophila muscle segment homeobox (msh) gene. The expression of the AKR gene is light dependent, and transgenic Arabidopsis plants with two or more copies of an antisense or sense AKR construct became chlorotic in a developmentally regulated manner. The chlorotic phenotype was genetically transmitted to the next generation, although most chlorotic plants produced much less seed. Reduced presence of thylakoid membranes and loss of grana are found in the plastids of chlorotic leaves, indicating that antisense or sense AKR has blocked chloroplast differentiation. This study indicates the importance of ankyrin repeat-containing proteins, not only in yeast and animals, but in plants as well. PMID:1281700

  12. Effect of 2′-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells

    PubMed Central

    Matsui, Masayuki; Threlfall, Richard N; Caruthers, Marvin H; Corey, David R

    2014-01-01

    ABSTRACT Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2′-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions. PMID:26865404

  13. 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...

  14. Reduction of polygalacturonase activity in tomato fruit by antisense RNA.

    PubMed

    Sheehy, R E; Kramer, M; Hiatt, W R

    1988-12-01

    Polygalacturonase [PG; poly(1,4-alpha-D-galacturonide) glycanhydrolase; EC 3.2.1.15] is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by Agrobacterium-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity in ripening fruit. The steady-state levels of PG antisense RNA in green fruit of transgenic plants were lower than the levels of PG mRNA normally attained during ripening. However, analysis of transcription in isolated nuclei demonstrated that the antisense RNA construct was transcribed at a higher rate than the tomato PG gene(s). Analysis of fruit from transgenic plants demonstrated a reduction in PG mRNA and enzymatic activity of 70-90%. The reduction in PG activity did not prevent the accumulation of the red pigment lycopene.

  15. Vertical and Seasonal Variations of Bacterioplankton Subgroups with Different Nucleic Acid Contents: Possible Regulation by Phosphorus†

    PubMed Central

    Nishimura, Yoko; Kim, Chulgoo; Nagata, Toshi

    2005-01-01

    We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups: bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop. PMID:16204494

  16. Antisense-Based Progerin Downregulation in HGPS-Like Patients' Cells.

    PubMed

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

    2016-07-11

    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.

  17. No rosetta stone for a sense-antisense origin of aminoacyl tRNA synthetase classes.

    PubMed

    Williams, Tom A; Wolfe, Kenneth H; Fares, Mario A

    2009-02-01

    Aminoacyl tRNA synthetases (aaRS) are crucial enzymes that join amino acids to their cognate tRNAs, thereby implementing the genetic code. These enzymes fall into two unrelated structural classes whose evolution has not been explained. The leading hypothesis, proposed by Rodin and Ohno, is that the two classes originated as a pair of sense-antisense genes encoded on opposite strands of a single DNA molecule. This unusual idea obtained its main support from reports of a "Rosetta stone": a locus where genes for heat shock protein 70 (HSP70) and an Nicotinamide adenine dinulecotide-specific glutamate dehydrogenase (NAD-GDH), which are structurally homologous to the two classes of aaRS, overlap extensively on complementary DNA strands. This remarkable locus was first characterized in the oomycete Achlya klebsiana and has since been reported in many other species. Here we present evidence that the open reading frames on the antisense strand of HSP70 genes are spurious, and we identify a more probable candidate for the gene encoding the oomycete NAD-GDH enzyme. These results cast extensive doubt on the Rosetta Stone argument.

  18. Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants1

    PubMed Central

    Zeh, Michaela; Casazza, Anna Paola; Kreft, Oliver; Roessner, Ute; Bieberich, Katrin; Willmitzer, Lothar; Hoefgen, Rainer; Hesse, Holger

    2001-01-01

    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

  19. Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy.

    PubMed

    Falzarano, Maria Sofia; Passarelli, Chiara; Ferlini, Alessandra

    2014-02-01

    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.

  20. Dietary linoleic acid elevates the endocannabinoids 2-AG and anandamide and promotes weight gain in mice fed a low fat diet.

    PubMed

    Alvheim, Anita Røyneberg; Torstensen, Bente E; Lin, Yu Hong; Lillefosse, Haldis Haukås; Lock, Erik-Jan; Madsen, Lise; Frøyland, Livar; Hibbeln, Joseph R; Malde, Marian Kjellevold

    2014-01-01

    Dietary intake of linoleic acid (LNA, 18:2n-6) has increased dramatically during the 20th century and is associated with greater prevalence of obesity. The endocannabinoid system is involved in regulation of energy balance and a sustained hyperactivity of the endocannabinoid system may contribute to obesity. Arachidonic acid (ARA, 20:4n-6) is the precursor for 2-AG and anandamide (AEA), and we sought to determine if low fat diets (LFD) could be made obesogenic by increasing the endocannabinoid precursor pool of ARA, causing excessive endocannabinoid signaling leading to weight gain and a metabolic profile associated with obesity. Mice (C57BL/6j, 6 weeks of age) were fed 1 en% LNA and 8 en% LNA in low fat (12.5 en%) and medium fat diets (MFD, 35 en%) for 16 weeks. We found that increasing dietary LNA from 1 to 8 en% in LFD and MFD significantly increased ARA in phospholipids (ARA-PL), elevated 2-AG and AEA in liver, elevated plasma leptin, and resulted in larger adipocytes and more macrophage infiltration in adipose tissue. In LFD, dietary LNA of 8 en% increased feed efficiency and caused greater weight gain than in an isocaloric reduction to 1 en% LNA. Increasing dietary LNA from 1 to 8 en% elevates liver endocannabinoid levels and increases the risk of developing obesity. Thus a high dietary content of LNA (8 en%) increases the adipogenic properties of a low fat diet.

  1. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

  2. 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

  3. Induction of Radiosensitization by Antisense Oligonucleotide Gene Therapy

    DTIC Science & Technology

    2002-07-01

    Miraglia L and Strobl JS: Sensitization of breast cancer cells to ionizing radiation by protein kinase C inhibition. Proc. of the 9 0 ,h American Assoc...sensitizes human tumor cells to ionizing radiation . Radiat Res 129:345-350. O’Brian C, Vogel VG, Singletary SE and Ward NE (1989) Elevated protein...Antisense Oligonucleotides, Ionizing Radiation , Breast Cancer, Abbreviations: IR, ionizing radiation ; PKC, protein kinase C; MCF-7, Michigan Cancer

  4. Intravesical NGF Antisense Therapy using Lipid Nanoparticle for Interstitial Cystitis

    DTIC Science & Technology

    2013-10-01

    disease of the urinary bladder . The goal of this project is to advance key preclinical experiments towards the development of a new drug. Specific...factor (NGF) bladder drug delivery system targeting Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS), IC/PBS is a chronic, severely debilitating...interstitial cystitis, painful bladder syndrome, liposome, nerve growth factor, afferent hyper-excitability, antisense 16. SECURITY CLASSIFICATION OF

  5. Intravesical NGF Antisense Therapy Using Lipid Nanoparticle for Interstitial Cystitis

    DTIC Science & Technology

    2014-10-01

    cystitis” evaluates the feasibility of an anti-nerve growth factor (NGF) bladder drug delivery as a treatment for Interstitial Cystitis/Painful Bladder...the project include early stage development, initial manufacture, and animal testing of an experimental liposome NGF-antisense formulations for...a novel diseased animal model. 15. SUBJECT TERMS Interstitial cystitis/painful bladder syndrome, Liposome, Peer Reviewed Medical Research Program

  6. Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study.

    PubMed

    Kiani, Melika; Mirzazadeh Tekie, Farnaz Sadat; Dinarvand, Meshkat; Soleimani, Masoud; Dinarvand, Rassoul; Atyabi, Fatemeh

    2016-05-01

    Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~115nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense.

  7. Viral Vector-Mediated Antisense Therapy for Genetic Diseases

    PubMed Central

    Imbert, Marine; Dias-Florencio, Gabriella; Goyenvalle, Aurélie

    2017-01-01

    RNA plays complex roles in normal health and disease and is becoming an important target for therapeutic intervention; accordingly, therapeutic strategies that modulate RNA function have gained great interest over the past decade. Antisense oligonucleotides (AOs) are perhaps the most promising strategy to modulate RNA expression through a variety of post binding events such as gene silencing through degradative or non-degradative mechanisms, or splicing modulation which has recently demonstrated promising results. However, AO technology still faces issues like poor cellular-uptake, low efficacy in target tissues and relatively rapid clearance from the circulation which means repeated injections are essential to complete therapeutic efficacy. To overcome these limitations, viral vectors encoding small nuclear RNAs have been engineered to shuttle antisense sequences into cells, allowing appropriate subcellular localization with pre-mRNAs and permanent correction. In this review, we outline the different strategies for antisense therapy mediated by viral vectors and provide examples of each approach. We also address the advantages and limitations of viral vector use, with an emphasis on their clinical application. PMID:28134780

  8. Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides.

    PubMed

    El Andaloussi, Samir; Said Hassane, Fatouma; Boisguerin, Prisca; Sillard, Rannar; Langel, Ulo; Lebleu, Bernard

    2011-01-01

    Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.

  9. Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.

    PubMed

    Mook, Olaf R; Baas, Frank; de Wissel, Marit B; Fluiter, Kees

    2007-03-01

    RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered siRNAs entered the first clinical trials, but strategies for successful systemic delivery of siRNA are still under development. Challenges still exist about the stability, delivery, and therapeutic efficacy of siRNA. In the present study, we compare the efficacy of two methods of systemic siRNA delivery and the effects of siRNA modifications using locked nucleic acids (LNA) in a xenograft cancer model. Low volume tail vein bolus injections and continuous s.c. delivery using osmotic minipumps yielded similar uptake levels of unmodified siRNA by tumor xenografts. Both routes of administration mediated sequence-specific inhibition of two unrelated targets inside tumor xenografts. Previous studies have shown that LNA can be incorporated into the sense strand of siRNA while the efficacy is retained. Modification of siRNA targeting green fluorescent protein with LNA results in a significant increase in serum stability and thus may be beneficial for clinical applications. We show that minimal 3' end LNA modifications of siRNA are effective in stabilization of siRNA. Multiple LNA modifications in the accompanying strand further increase the stability but negate the efficacy in vitro and in vivo. In vivo, LNA-modified siRNA reduced off-target gene regulation compared with nonmodified siRNA. End-modified siRNA targeting green fluorescent protein provides a good trade-off between stability and efficacy in vivo using the two methods of systemic delivery in the nude mouse model. Therefore, LNA-modified siRNA should be preferred over unmodified siRNA.

  10. 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

  11. 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

  12. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

    PubMed

    Yui, Rika; Iketani, Satoru; Mikami, Tetsuo; Kubo, Tomohiko

    2003-04-01

    We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.

  13. Application of locked nucleic acids to improve aptamer in vivo stability and targeting function

    PubMed Central

    Schmidt, Kathrin S.; Borkowski, Sandra; Kurreck, Jens; Stephens, Andrew W.; Bald, Rolf; Hecht, Maren; Friebe, Matthias; Dinkelborg, Ludger; Erdmann, Volker A.

    2004-01-01

    Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure–activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degradation. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2′-O-methyl RNA (2′-OMe), 2′-Fluoro RNA (2′-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex. TTA1 derivatives with LNA and 2′-OMe modifications within the non-binding stem have subsequently been synthesized. Especially, the LNA-modified TTA1 derivative exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addition, higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties. PMID:15509871

  14. Design, assembly, and activity of antisense DNA nanostructures.

    PubMed

    Keum, Jung-Won; Ahn, Jin-Ho; Bermudez, Harry

    2011-12-16

    Discrete DNA nanostructures allow simultaneous features not possible with traditional DNA forms: encapsulation of cargo, display of multiple ligands, and resistance to enzymatic digestion. These properties suggested using DNA nanostructures as a delivery platform. Here, DNA pyramids displaying antisense motifs are shown to be able to specifically degrade mRNA and inhibit protein expression in vitro, and they show improved cell uptake and gene silencing when compared to linear DNA. Furthermore, the activity of these pyramids can be regulated by the introduction of an appropriate complementary strand. These results highlight the versatility of DNA nanostructures as functional devices.

  15. 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…

  16. Effect of pH on conjugated linoleic acid (CLA) formation of linolenic acid biohydrogenation by ruminal microorganisms.

    PubMed

    Lee, Yongjae

    2013-08-01

    Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of (13)C-LNA with different pH conditions (5.5 and 6.5). The (13)C enrichment was calculated as a (13)C/(12)C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from (13)C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.

  17. Design of a LNA in the frequency band 1.8-2.2GHz in 0.13μm CMOS Technology

    NASA Astrophysics Data System (ADS)

    di Gioia, E.; Hermann, C.; Klar, H.

    2005-05-01

    The subject of this work is a low noise amplifier (LNA), operating in the frequency range 1.8-2.1GHz. The CMOS 0.13μm technology is used in respect to the low cost of the final device. Among the specifications, a variable gain and an adjustable working frequency are required. In particular, four different working modes are provided: 1.8, 1.9 and 2.1GHz high gain and 2.1GHz low gain. The amplifier is designed to be used as first stage of a receiver for mobile telephony. For this reason low power consumption is taken into consideration (low supply voltage and low drain currents). A simple digital circuit, integrated on-chip, is used to select the operating mode of the LNA by means of two input pins. A Noise figure of 1dB is obtained with a supply voltage of 0.8V.

  18. 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

  19. Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

    PubMed Central

    Förster, Charlotte; Eichert, André; Oberthür, Dominik; Betzel, Christian; Geßner, Reinhard; Nitsche, Andreas; Fürste, Jens P.

    2012-01-01

    “Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery. PMID:22666550

  20. Fate of orally administered radioactive fatty acids in the late-pregnant rat.

    PubMed

    López-Luna, Pilar; Ortega-Senovilla, Henar; López-Soldado, Iliana; Herrera, Emilio

    2016-03-01

    To investigate the biodisponibility of placental transfer of fatty acids, rats pregnant for 20 days were given tracer amounts of [(14)C]palmitic (PA), oleic (OA), linoleic (LA), α-linolenic (LNA), or docosahexaenoic acid (DHA) orally and euthanized at 0.5, 1.0, 2.0, or 8.0 h thereafter. Maternal plasma radioactivity in lipids initially increased only to decline at later times. Most of the label appeared first as triacylglycerols (TAG); later, the proportion in phospholipids (PhL) increased. The percentage of label in placental lipids was also always highest shortly after administration and declined later; again, PhL increased with time. Fetal plasma radioactivity increased with time, with its highest value at 8.0 h after DHA or LNA administration. DHA initially appeared primarily in the nonesterified fatty acids (NEFA) and PA, OA, LA, and LNA as TAG followed by NEFA; in all cases, there was an increase in PhL at later times. Measurement of fatty acid concentrations allowed calculation of specific (radio)activities, and the ratio (fetal/maternal) of these in the plasmas gave an index of placental transfer activity, which was LNA > LA > DHA = OA > PA. It is proposed that a considerable proportion of most fatty acids transferred through the placenta are released into the fetal circulation in the form of TAG.

  1. 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.

  2. Antisense-mediated exon skipping to reframe transcripts.

    PubMed

    Turczynski, Sandrina; Titeux, Matthias; Pironon, Nathalie; Hovnanian, Alain

    2012-01-01

    Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.

  3. Skipping multiple exons of dystrophin transcripts using cocktail antisense oligonucleotides.

    PubMed

    Echigoya, Yusuke; Yokota, Toshifumi

    2014-02-01

    Duchenne muscular dystrophy (DMD) is one of the most common and lethal genetic disorders, with 20,000 children per year born with DMD globally. DMD is caused by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is a promising therapeutic approach that uses short DNA-like molecules called antisense oligonucleotides (AOs) to skip over/splice out the mutated part of the gene to produce a shortened but functional dystrophin protein. One major challenge has been its limited applicability. Multiple exon skipping has recently emerged as a potential solution. Indeed, many DMD patients need exon skipping of multiple exons in order to restore the reading frame, depending on how many base pairs the mutated exon(s) and adjacent exons have. Theoretically, multiple exon skipping could be used to treat approximately 90%, 80%, and 98% of DMD patients with deletion, duplication, and nonsense mutations, respectively. In addition, multiple exon skipping could be used to select deletions that optimize the functionality of the truncated dystrophin protein. The proof of concept of systemic multiple exon skipping using a cocktail of AOs has been demonstrated in dystrophic dog and mouse models. Remaining challenges include the insufficient efficacy of systemic treatment, especially for therapies that target the heart, and limited long-term safety data. Here we review recent preclinical developments in AO-mediated multiple exon skipping and discuss the remaining challenges.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

  9. 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.

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

    PubMed

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

    2007-07-27

    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

  11. Anticubilin antisense RNA ameliorates adriamycin-induced tubulointerstitial injury in experimental rats.

    PubMed

    Liu, Jun; Li, Kailong; He, Yani; Zhang, Jianguo; Wang, Huiming; Yang, Jurong; Zhan, Jun; Liang, Haijun

    2011-12-01

    This study was designed to determine the effects of in vivo anticubilin antisense RNA on the uptake of albumin in tubules and on the tubulointerstitial injury in adriamycin-induced proteinuric rats. Adriamycin-treated rats were subjected to intrarenal delivery of adenoviral vectors encoding empty plasmid, cubilin sense RNA expression vector pAd-CUB or anticubilin antisense RNA expression vector pAd-ACUB on day 3. On days 14 and 28, half of the rats in each group were randomly selected to be killed, and blood samples, kidney tissues and 24-hour urine were collected. The diseased rats treated with pAdEasy-ACUB showed a 60% decrease in serum creatinine and glomerular filtration rate. Interestingly, the anticubilin antisense treatment led to a marked increase in albuminuria. Antisense treatment attenuated the histologic changes on both day 14 and day 28. The antisense treatment induced more than 60% recovery of adriamycin-induced injury, accompanied with 85% knockdown in the expression of cubilin protein and markedly decreased albumin deposition. Adriamycin induced an increase in the expression of monocyte chemoattractant protein-1, transforming growth factor-β and regulated on activation in normal T-cell expressed and secreted and the number of infiltrating cells, which was reversed by the antisense treatment. Anticubilin antisense RNA delivered by an adenoviral vector ameliorates albuminuria-induced glomerulosclerosis and tubulointerstitial damage in adriamycin nephrotic rats, indicating that cubilin could be a potential therapeutic target in proteinuric nephropathy.

  12. 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

  13. Protection of shrimp Penaeus monodon from WSSV infection using antisense constructs.

    PubMed

    Ahanger, Sajad; Sandaka, Supriyanka; Ananad, Deepika; Mani, Madhu K; Kondadhasula, Ravinder; Reddy, Chandra Sekhar; Marappan, Makesh; Valappil, Rajendran K; Majumdar, Kshitish C; Mishra, Rakesh K

    2014-02-01

    White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

  14. Identification of differentially expressed sense and antisense transcript pairs in breast epithelial tissues

    PubMed Central

    Grigoriadis, Anita; Oliver, Gavin R; Tanney, Austin; Kendrick, Howard; Smalley, Matt J; Jat, Parmjit; Neville, A Munro

    2009-01-01

    Background More than 20% of human transcripts have naturally occurring antisense products (or natural antisense transcripts – NATs), some of which may play a key role in a range of human diseases. To date, several databases of in silico defined human sense-antisense (SAS) pairs have appeared, however no study has focused on differential expression of SAS pairs in breast tissue. We therefore investigated the expression levels of sense and antisense transcripts in normal and malignant human breast epithelia using the Affymetrix HG-U133 Plus 2.0 and Almac Diagnostics Breast Cancer DSA microarray technologies as well as massively parallel signature sequencing (MPSS) data. Results The expression of more than 2500 antisense transcripts were detected in normal breast duct luminal cells and in primary breast tumors substantially enriched for their epithelial cell content by DSA microarray. Expression of 431 NATs were confirmed by either of the other two technologies. A corresponding sense transcript could be identified on DSA for 257 antisense transcripts. Of these SAS pairs, 163 have not been previously reported. A positive correlation of differential expression between normal and malignant breast samples was observed for most SAS pairs. Orientation specific RT-QPCR of selected SAS pairs validated their expression in several breast cancer cell lines and solid breast tumours. Conclusion Disease-focused and antisense enriched microarray platforms (such as Breast Cancer DSA) confirm the assumption that antisense transcription in the human breast is more prevalent than previously anticipated. Expression of a proportion of these NATs has already been confirmed by other technologies while the true existence of the remaining ones has to be validated. Nevertheless, future studies will reveal whether the relative abundances of antisense and sense transcripts have regulatory influences on the translation of these mRNAs. PMID:19615061

  15. 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.

  16. Safety of antisense oligonucleotide and siRNA-based therapeutics.

    PubMed

    Chi, Xuan; Gatti, Philip; Papoian, Thomas

    2017-01-31

    Oligonucleotide-based therapy is an active area of drug development designed to treat a variety of gene-specific diseases. Two of the more promising platforms are the antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs), both of which are often directed against similar targets. In light of recent reports on clinical trials of severe thrombocytopenia with two different ASO drugs and increased peripheral neuropathy with an siRNA drug, we compared and contrasted the specific safety characteristics of these two classes of oligonucleotide therapeutic. The objectives were to assess factors that could contribute to the specific toxicities observed with these two classes of promising drugs, and get a better understanding of the potential mechanism(s) responsible for these rare, but serious, adverse events.

  17. Orlistat and antisense-miRNA-loaded PLGA-PEG nanoparticles for enhanced triple negative breast cancer therapy

    PubMed Central

    Bhargava-Shah, Aarohi; Foygel, Kira; Devulapally, Rammohan; Paulmurugan, Ramasamy

    2016-01-01

    Background: This study explores the use of hydrophilic poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NPs) as delivery system to improve the antitumor effect of antiobesity drug orlistat for triple-negative breast cancer (TNBC) therapy by improving its bioavailability. Materials & methods: PLGA-PEG-NPs were synthesized by emulsion-diffusion-evaporation method, and the experiments were conducted in vitro in MDA-MB-231 and SKBr3 TNBC and normal breast fibroblast cells. Results: Delivery of orlistat via PLGA-PEG-NPs reduced its IC50 compared with free orlistat. Combined treatment of orlistat-loaded NPs and doxorubicin or antisense-miR-21-loaded NPs significantly enhanced apoptotic effect compared with independent doxorubicin, anti-miR-21-loaded NPs, orlistat-loaded NPs or free orlistat treatments. Conclusion: We demonstrate that orlistat in combination with antisense-miR-21 or current chemotherapy holds great promise as a novel and versatile treatment agent for TNBC. PMID:26787319

  18. HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing.

    PubMed

    Cho, Cheong Weon; Poo, Haryoung; Cho, Young Sik; Cho, Min Chul; Lee, Kyung Ae; Lee, Shin Je; Park, Sue Nie; Kim, In Ki; Jung, Yong Keun; Choe, Yong Kyung; Yeom, Young I I; Choe, In Seong; Yoon, Do Young

    2002-05-31

    Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.

  19. Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

    PubMed

    Oustric, Vincent; Manceau, Hana; Ducamp, Sarah; Soaid, Rima; Karim, Zoubida; Schmitt, Caroline; Mirmiran, Arienne; Peoc'h, Katell; Grandchamp, Bernard; Beaumont, Carole; Lyoumi, Said; Moreau-Gaudry, François; Guyonnet-Dupérat, Véronique; de Verneuil, Hubert; Marie, Joëlle; Puy, Herve; Deybach, Jean-Charles; Gouya, Laurent

    2014-04-03

    In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

  20. Antisense Oligonucleotide-Based Therapy in Human Erythropoietic Protoporphyria

    PubMed Central

    Oustric, Vincent; Manceau, Hana; Ducamp, Sarah; Soaid, Rima; Karim, Zoubida; Schmitt, Caroline; Mirmiran, Arienne; Peoc’h, Katell; Grandchamp, Bernard; Beaumont, Carole; Lyoumi, Said; Moreau-Gaudry, François; Guyonnet-Dupérat, Véronique; de Verneuil, Hubert; Marie, Joëlle; Puy, Herve; Deybach, Jean-Charles; Gouya, Laurent

    2014-01-01

    In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315−48T>C) localized in intron 3. The 315−48C allele increases usage of the 3′ cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals. PMID:24680888

  1. Effects of metal ions on lipid peroxidation in cultured rat hepatocytes loaded with alpha-linolenic acid.

    PubMed

    Furono, K; Suetsuga, T; Sugihara, N

    1996-06-07

    We investigated the ability of various redox-active metal ions to induce lipid peroxidation in normal and alpha-linolenic acid-loaded (LNA-loaded) cultured rat hepatocytes. Lipid peroxidation was estimated by the accumulation of malondialdehyde (MDA) in the culture medium. At low concentrations induction was highest with ferrous ions (Fe), whereas at high concentrations, vanadium (V) and copper ions (Cu) had the greatest effect on both groups of hepatocytes. With any one of the three metal ions, the extent of lipid peroxidation in LNA-loaded hepatocytes was several times greater compared to normal cells. In addition, upon the addition of Fe or V, LNA-loaded hepatocytes were injured whereas normal cells were not. The addition of Cu caused substantial cell injury in normal hepatocytes, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant like N,N'-diphenyl-p-phenylene-diamine (DPPD) almost completely prevented Fe- and V-induced cell injury, and reduced Cu-induced cell injury. alpha-Tocopherol behaved in a way similar to but less effective than DPPD. .OH radical scavengers such as mannitol and dimethyl sulfoxide (DMSO) had no effect on lipid peroxidation induced by any metal ions in LNA-loaded hepatocytes. Addition of cadmium ions (Cd), which required the lowest concentration to cause cell injury, induced a slight increase in lipid peroxidation in normal hepatocytes, but did not induce lipid peroxidation to the same extent as seen in LNA-loaded cells treated with any of the three metal ions already mentioned. The inhibition of lipid peroxidation by DPPD scarcely protected LNA-loaded hepatocytes from Cd-induced cell injury. None of the other metal ions including aluminum (Al), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and tin (Sn) ions, effectively induced lipid peroxidation in either group of hepatocytes, except cobalt ions (Co), which had a peroxidative effect in LNA

  2. Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells.

    PubMed

    Thanasak, J; Müller, K E; Dieleman, S J; Hoek, A; Noordhuizen, J P T M; Rutten, V P M G

    2005-04-08

    The present study aimed at analysis of the effects of polyunsaturated fatty acid (PUFA), linoleic acid (LA, C18:2n - 6) and linolenic acid (LNA, C18:3n - 3) on bovine peripheral blood mononuclear cells (PBMC) in vitro. Both mitogen (ConA)-induced proliferative lymphocyte responsiveness during 4 days of culture and eicosanoid (prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4))) production during 36 h were determined in relation to the absence or presence of various concentrations of LA and LNA (0, 1, 5, 25, 125 and 250 microM). Mitogen-driven proliferative responses of lymphocytes tended to be uninfluenced in the presence of lower concentrations of LA, whereas significant inhibition was observed at the higher concentrations of LA (125 and 250 microM). However, increasing amounts of LNA did not affect the proliferation. ConA stimulation induced a clear PGE(2) response, which significantly decreased in the presence of 250 microM of LA. In addition, increasing amounts of LNA, but not LA, led to a significant decrease in LTB(4) levels. However, The production of LTB(4) did not alter due to mitogenic stimulation. In conclusion, the present study shows that bovine mononuclear cells may functionally be influenced by the presence of PUFA in their environment. Further studies need to be conducted to clarify in vivo consequences of these findings in a situation of PUFA enriched rations in ruminants.

  3. 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

  4. Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1.

    PubMed Central

    Rittner, K; Sczakiel, G

    1991-01-01

    Antisense RNA, transcribed intracellularly from constitutive expression cassettes, inhibits the replication of the human immunodeficiency virus type 1 (HIV-1) as demonstrated by a quantitative microinjection assay in human SW480 cells. Infectious proviral HIV-1 DNA was co-microinjected together with a fivefold molar excess of plasmids expressing antisense RNA complementary to a set of ten different HIV-1 target regions. The most inhibitory antisense RNA expression plasmids were targeted against a 1 kb region within the gag open reading frame and against a 562 base region containing the coding sequences for the regulatory viral proteins tat and rev. Experimental evidence is presented that the antisense principle is the inhibitory mechanism in this assay system. PMID:2027749

  5. 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.

  6. Crucial role of antisense transcription across the Xist promoter in Tsix-mediated Xist chromatin modification.

    PubMed

    Ohhata, Tatsuya; Hoki, Yuko; Sasaki, Hiroyuki; Sado, Takashi

    2008-01-01

    Expression of Xist, which triggers X inactivation, is negatively regulated in cis by an antisense gene, Tsix, transcribed along the entire Xist gene. We recently demonstrated that Tsix silences Xist through modification of the chromatin structure in the Xist promoter region. This finding prompted us to investigate the role of antisense transcription across the Xist promoter in Tsix-mediated silencing. Here, we prematurely terminated Tsix transcription before the Xist promoter and addressed its effect on Xist silencing in mouse embryos. We found that although 93% of the region encoding Tsix was transcribed, truncation of Tsix abolished the antisense regulation of Xist. This resulted in a failure to establish the repressive chromatin configuration at the Xist promoter on the mutated X, including DNA methylation and repressive histone modifications, especially in extraembryonic tissues. These results suggest a crucial role for antisense transcription across the Xist promoter in Xist silencing.

  7. Robust Inhibitory Effects of Conjugated Linolenic Acids on a Cyclooxygenase-related Linoleate 10S-Dioxygenase: Comparison with COX-1 and COX-2

    PubMed Central

    Mashhadi, Zahra; Boeglin, William E.; Brash, Alan R.

    2015-01-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49 nM and ~125 nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1 μM, respectively. By contrast, COX-2 was ≤ 30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data. PMID:26209563

  8. Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

    PubMed

    Mashhadi, Zahra; Boeglin, William E; Brash, Alan R

    2015-10-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49nM and ~125nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1μM, respectively. By contrast, COX-2 was ≤30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2016-10-12

    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.

  13. 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

  14. 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.

  15. Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe

    PubMed Central

    Bitton, Danny A; Grallert, Agnes; Scutt, Paul J; Yates, Tim; Li, Yaoyong; Bradford, James R; Hey, Yvonne; Pepper, Stuart D; Hagan, Iain M; Miller, Crispin J

    2011-01-01

    Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3′ termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent ‘horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply ‘genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe. PMID:22186733

  16. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients.

  17. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy.

    PubMed

    Kendall, Genevieve C; Mokhonova, Ekaterina I; Moran, Miriana; Sejbuk, Natalia E; Wang, Derek W; Silva, Oscar; Wang, Richard T; Martinez, Leonel; Lu, Qi L; Damoiseaux, Robert; Spencer, Melissa J; Nelson, Stanley F; Miceli, M Carrie

    2012-12-12

    Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

  18. Shock waves: a novel method for cytoplasmic delivery of antisense oligonucleotides.

    PubMed

    Tschoep, K; Hartmann, G; Jox, R; Thompson, S; Eigler, A; Krug, A; Erhardt, S; Adams, G; Endres, S; Delius, M

    2001-06-01

    Intracytoplasmic delivery of oligonucleotides (ODN) can improve ODN-based strategies such as the antisense approach and the use of immunostimulatory CpG dinucleotide containing ODN. Shock waves are established for the treatment of nephrolithiasis and other diseases. Here we describe the use of shock waves as a new physical method for the direct transport of antisense ODN into the cytoplasm and the nucleus of cells. Human peripheral blood mononuclear cells together with antisense ODN were exposed to shock waves generated by an electrohydraulic lithotripter. ODN uptake was examined by flow cytometry and fluorescence microscopy. By optimization of physical parameters we achieved the transfer of high amounts of ODN which were detected within less than 5 min after shock wave exposure, with viability of cells higher than 95%. Transfection of human peripheral blood mononuclear cells with an antisense ODN directed against tumor necrosis factor (TNF) alpha resulted in a reduction in lipopolysaccharide-induced TNF production by 62% (n=5, P=0.006). Specificity of TNF suppression was confirmed with a four-mismatch oligonucleotide. Positive atmospheric pressure abolished antisense-mediated inhibition of TNF synthesis by blocking shock wave-induced cavitation and formation of oscillating air bubbles. Electroporation was less effective. The use of shock waves is thus an efficient physical tool for ODN delivery to cells. Shock waves may allow the evaluation of target proteins in cell types difficult to transfect with other methods and thus may improve the antisense technique for the analysis of unknown genes.

  19. In situ hybridization for Coccidioides immitis 5.8S ribosomal RNA sequences in formalin-fixed, paraffin-embedded pulmonary specimens using a locked nucleic acid probe: a rapid means for identification in tissue sections.

    PubMed

    Montone, Kathleen T; Litzky, Leslie A; Feldman, Michael D; Peterman, Heather; Mathis, Benjamin; Baliff, Jeffrey; Kaiser, Larry R; Kucharczuk, John; Nachamkin, Irving

    2010-06-01

    Coccidioides immitis/Coccidioides posadasii are common causes of pulmonary infection in certain geographic areas, and are highly infectious when working with culture isolates in the laboratory. Rapid techniques to accurately identify this pathogen in tissues may be of benefit for diagnosis and in limiting the exposure of laboratory personnel to this agent. Locked nucleic acids (LNA) are modified nucleotides in which a ribonucleoside is linked between the 2'-oxygen and the 4'-carbon atoms with a methylene unit. LNA oligonucleotides exhibit increased thermal stability and make excellent probes for in situ hybridization (ISH). In this study, ISH utilizing a biotin-labeled LNA probe targeting Coccidioides sp. ribosomal RNA sequences in 6 formalin-fixed, paraffin-embedded pulmonary tissue specimens from 6 patients with culture positive or histologic findings suggestive of Coccidioides sp. infection is described. The cultures of the pulmonary specimens confirmed C. immitis in 3 of 6 patients. The ISH procedure with the LNA probe was positive in all 6 cases, although the number of organisms that were highlighted varied from rare to numerous. ISH with a biotin-labeled DNA probe of the same sequence was positive in 4 of the 6 cases and the signal intensity and number of organisms was much less than that observed with the LNA probe. Negative control tissues containing a variety of different fungal pathogens including Aspergillus sp., Fusarium sp., Blastomyces dermatitidis, Candida sp, Histoplasma capsulatum, and Zygomyces did not hybridize with the LNA and DNA probes. ISH with an LNA oligonucleotide probe targeting Coccidioides sp. ribosomal RNA is useful for rapid ISH. ISH could be rapidly performed when fungal pathogens are observed in tissue but cultures are negative or have not been performed.

  20. Periostin antisense oligonucleotide prevents adhesion formation after surgery in mice.

    PubMed

    Takai, Shinji; Yoshino, Masafumi; Takao, Kazumasa; Yoshikawa, Kazunori; Jin, Denan

    2017-02-09

    To study the role of periostin in adhesion formation, the effect of periostin antisense oligonucleotide (PAO) on adhesion formation was evaluated in mice. Under anesthesia, the serous membrane of the cecum was abraded, and the adhesion score and mRNA levels of periostin and its related factors were determined after surgery. Saline, 40 mg/kg of negative sense oligonucleotide (NSO), or 40 mg/kg of PAO were injected into the abdomen after surgery, and the adhesion score and mRNA levels were evaluated 14 days later. Filmy adhesion formation was observed 1 day after surgery, and the adhesion score increased gradually to 14 days. The mRNA levels of periostin, transforming growth factor (TGF)-β, and collagen I increased gradually from 3 days to 14 days. The adhesion score of PAO was significantly lower than of saline or NSO 14 days after surgery. The mRNA levels of periostin, TGF-β, and collagen I were also significantly attenuated by treatment with PAO compared with saline or NSO. Thus, these results demonstrated that the periostin mRNA level increased in the abraded cecum, and PAO prevented adhesion formation along with attenuation of the periostin mRNA level.

  1. Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions.

    PubMed Central

    van Duin, M; van Den Tol, J; Hoeijmakers, J H; Bootsma, D; Rupp, I P; Reynolds, P; Prakash, L; Prakash, S

    1989-01-01

    We report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3' regions. Since naturally occurring antisense transcription is rare in S. cerevisiae and humans (this is the first example in human cells), our findings indicate that antisense transcription in the ERCC-1-RAD10 gene regions represents an evolutionarily conserved feature. Images PMID:2471070

  2. Incorporation of n-3 fatty acids into plasma and liver lipids of rats: importance of background dietary fat.

    PubMed

    MacDonald-Wicks, Lesley K; Garg, Manohar L

    2004-06-01

    The health benefits of long-chain n-3 PUFA (20:5n-3 and 22:6n-3) depend on the extent of incorporation of these FA into plasma and tissue lipids. This study aimed to investigate the effect of the background dietary fat (saturated, monounsaturated, or n-6 polyunsaturated) on the quantitative incorporation of dietary 18:3n-3 and its elongated and desaturated products into the plasma and the liver lipids of rats. Female weanling Wistar rats (n = 54) were randomly assigned to six diet groups (n = 9). The fat added to the semipurified diets was tallow (SFA), tallow plus linseed oil (SFA-LNA), sunola oil (MUFA), sunola oil plus linseed oil (MUFA-LNA), sunflower oil (PUFA), or sunflower oil plus linseed oil (PUFA-LNA). At the completion of the 4-wk feeding period, quantitative FA analysis of the liver and plasma was undertaken by GC. The inclusion of linseed oil in the rat diets increased the level of 18:3n-3, 20:5n-3, and, to a smaller degree, 22:6n-3 in plasma and liver lipids regardless of the background dietary fat. The extent of incorporation of 18:3n-3, 20:5n-3, and 22:5n-3 followed the order SFA-LNA > MUFA-LNA > PUFA-LNA. Levels of 22:6n-3 were increased to a similar extent regardless of the type of major fat in the rat diets. This indicates that the background diet affects the incorporation in liver and plasma FA pools of the n-3 PUFA with the exception of 22:6n-3 and therefore the background diet has the potential to influence the already established health benefits of long-chain n-3 fatty acids.

  3. Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus.

    PubMed

    Colliver, S P; Morris, P; Robbins, M P

    1997-11-01

    Three clonal genotypes of Lotus corniculatus L. (bird's foot trefoil) were transformed with an antisense chalcone synthase (CHS) gene construct made using a stress induced CHS17 cDNA from Phaseolus vulgaris under the control of the constitutive CaMV 35S promoter and Nos terminator via Agrobacterium rhizogenes. After initial screening, ten antisense and five control co-transformation events from each recipient clonal genotype were analysed. After elicitation with glutathione, the level of tannin accumulation was found to be increased in a number of antisense root cultures derived from the low (S33) and moderate (S50) tannin recipient genotypes. Six antisense and four control transformed lines from genotype S50 were selected for more detailed study. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from 1 to 5 and antisense orientation was confirmed by PCR. In transformed root cultures, increased CHS transcript levels were noted in a number of antisense lines. Biochemical analyses of glutathione-elicited-root cultures indicated a significant increase in tannin accumulation in antisense CHS lines and mean vestitol levels were reduced. These results show that the introduction of a heterologous antisense chalcone synthase construct into L. corniculatus resulted in an unpredicted molecular and biochemical phenotype. Such findings are discussed in relation to manipulation of this complex multigene family.

  4. A High-Fat, High-Oleic Diet, But Not a High-Fat, Saturated Diet, Reduces Hepatic α-Linolenic Acid and Eicosapentaenoic Acid Content in Mice.

    PubMed

    Picklo, Matthew J; Murphy, Eric J

    2016-05-01

    Considerable research has focused upon the role of linoleic acid (LNA; 18:2n-6) as a competitive inhibitor of α-linolenic (ALA; 18:3n-3) 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 hypothesis that a high SFA diet, compared to a high MUFA (oleic acid 18:1n-9) diet, reduces ALA conversion to long chain n-3 fatty acids. Mice were fed for 12 weeks on three diets: (1) a control, 16 % fat energy diet consisting of similar levels of SFA and MUFA (2) a 50 % fat energy high MUFA energy diet (35 % MUFA and 7 % SFA) or (3) a 50 % fat energy, high SFA energy diet (34 % SFA, 8 % MUFA). ALA and LNA content remained constant. Analysis of hepatic lipids demonstrated a selective reduction (40 %) in ALA but not LNA and a 35 % reduction in eicosapentaenoic acid (EPA; 20:5n-3) in the high MUFA mice compared to the other groups. Lower content of ALA was reflected in the neutral lipid fraction, while smaller levels of phospholipid esterified EPA and docosapentaenoic acid (DPA; 22:5n-3) were evident. Docosahexaenoic acid (DHA; 22:6n-3) content was elevated by the high SFA diet. Expression of Fads1 (Δ5 desaturase) and Fads2 (Δ6 desaturase) was elevated by the high MUFA and reduced by the high SFA diet. These data indicate that a high MUFA diet, but not a high SFA diet, reduces ALA metabolism and point to selective hepatic disposition of ALA versus LNA.

  5. 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 ...

  6. Stage of harvest and polyunsaturated essential fatty acid concentrations in purslane (Portulaca oleraceae) leaves.

    PubMed

    Palaniswamy, U R; McAvoy, R J; Bible, B B

    2001-07-01

    Purslane is a nutritious vegetable crop rich in the polyunsaturated essential fatty acids (PUEFA) alpha-linolenic acid (LNA) and linoleic acid (LA), which are essential for normal human growth, health promotion, and disease prevention. Total lipids and fatty acid concentrations at three stages of harvest (6-, 10-, and 14-true-leaf stages) were examined in a cultivated variety of purslane (Portulaca oleraceae L. var. sativa). The 14-true-leaf stage of growth was found to be ideal for harvest because at this stage the leaf area, shoot fresh weight, shoot dry weight, and PUEFA concentrations per gram of leaf fresh weight were higher (P < or = 0.05) than at the 6- and 10-true-leaf stages of growth. The LNA to LA ratio was also highest at the 14-true-leaf stage.

  7. Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.

    PubMed

    Koty, Patrick P; Tyurina, Yulia Y; Tyurin, Vladimir A; Li, Shang-Xi; Kagan, Valerian E

    2002-01-01

    Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance.

  8. Administration of antisense DNA for hepatocyte growth factor causes an depressive and anxiogenic response in rats.

    PubMed

    Wakatsuki, Masatoshi; Akiyoshi, Jotaro; Ichioka, Shugo; Tanaka, Yoshihiro; Tsuru, Jusen; Matsushita, Hirotaka; Hanada, Hiroaki; Isogawa, Koichi

    2007-12-01

    Hepatocyte growth factor (HGF) is induced in neurons during ischemia and is neuroprotective against post-ischemic delayed neuronal death in the hippocampus. HGF might play an important role in the maturation and functioning of these neurons in the hippocampus. Our aim was to determine what effect HGF antisense has on depression and anxiety in rats. HGF antisense was infused at a constant rate into cerebral lateral ventricles and its effect on anxiety in rats was monitored. In forced swimming test, rats that received antisense DNA increased the length of time that they were immobile in the water. In the elevated plus maze test, the black and white box test and conditioned fear test, HGF antisense administration caused all indicators of anxiety to increase. Number of HGF-positive cells in C1 of hippocampus was significantly decreased in the HGF antisense-infused group compared to the vehicle- and scrambled oligonucleotide-treated group. No significant effect on general locomotor activity was seen. These results indicate that inhibition of HGF induces an increase in depression and anxiety-related behaviors suggesting a depressive and anxiogenic-like effect.

  9. Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.

    PubMed Central

    Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.

    1997-01-01

    On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential. PMID:12223765

  10. The conditional inhibition of gene expression in cultured Drosophila cells by antisense RNA.

    PubMed Central

    Bunch, T A; Goldstein, L S

    1989-01-01

    Genes producing antisense RNA are becoming important tools for the selective inhibition of gene expression. Experiments in different biological systems, targeting different mRNAs have yielded diverse results with respect to the success of the technique and its mechanism of action. We have examined the potential of three antisense genes, whose transcription is driven by a Drosophila metallothionein promoter, to inhibit the expression of alcohol dehydrogenase (ADH) or a microtubule associated protein (205K MAP) in cultured Drosophila cells. Expression of ADH was significantly reduced upon induction of the anti-ADH genes. The ADH mRNA does not appear to be destabilized by the presence of antisense RNA but rather exists at similar levels in hybrid form. Hybrids are detected with both spliced and unspliced ADH RNA. In contrast to these results, antisense genes producing antisense RNA in great excess to 205K MAP mRNA, which is itself far less abundant than the ADH mRNA, failed to show any inhibition of 205K MAP expression. Images PMID:2481266

  11. Genome-wide antisense transcription drives mRNA processing in bacteria

    PubMed Central

    Lasa, Iñigo; Toledo-Arana, Alejandro; Dobin, Alexander; Villanueva, Maite; de los Mozos, Igor Ruiz; Vergara-Irigaray, Marta; Segura, Víctor; Fagegaltier, Delphine; Penadés, José R.; Valle, Jaione; Solano, Cristina; Gingeras, Thomas R.

    2011-01-01

    RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels. PMID:22123973

  12. The production of an inducible antisense alternative oxidase (Aox1a) plant.

    PubMed

    Potter, F J; Wiskich, J T; Dry, I B

    2001-01-01

    Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system.

  13. Systemic administration of antisense p75(NTR) oligodeoxynucleotides rescues axotomised spinal motor neurons.

    PubMed

    Lowry, K S; Murray, S S; Coulson, E J; Epa, R; Bartlett, P F; Barrett, G; Cheema, S S

    2001-04-01

    The 75 kD low-affinity neurotrophin receptor (p75(NTR)) is expressed in developing and axotomised spinal motor neurons. There is now convincing evidence that p75(NTR) can, under some circumstances, become cytotoxic and promote neuronal cell death. We report here that a single application of antisense p75(NTR) oligodeoxynucleotides to the proximal nerve stumps of neonatal rats significantly reduces the loss of axotomised motor neurons compared to controls treated with nonsense oligodeoxynucleotides or phosphate-buffered saline. Our investigations also show that daily systemic intraperitoneal injections of antisense p75(NTR) oligodeoxynucleotides for 14 days significantly reduce the loss of axotomised motor neurons compared to controls. Furthermore, we found that systemic delivery over a similar period continues to be effective following axotomy when intraperitoneal injections were 1) administered after a delay of 24 hr, 2) limited to the first 7 days, or 3) administered every third day. In addition, p75(NTR) protein levels were reduced in spinal motor neurons following treatment with antisense p75(NTR) oligodeoxynucleotides. There were also no obvious side effects associated with antisense p75(NTR) oligodeoxynucleotide treatments as determined by behavioural observations and postnatal weight gain. Our findings indicate that antisense-based strategies could be a novel approach for the prevention of motor neuron degeneration associated with injuries or disease.

  14. Expression of XIST sense and antisense in bovine fetal organs and cell cultures.

    PubMed

    Farazmand, Ali; Basrur, Parvathi K; Stranzinger, Gerald; Graphodatskaya, Daria; Reyes, Ed R; King, W Allan

    2004-01-01

    Untranslated RNAs transcribed from sense and antisense strands of a gene referred to as X-inactive specific transcript (XIST) play crucial roles in the genetic inactivation and condensation of one of the two X chromosomes in the somatic cells of female mammals. X inactivation is also thought to occur in mammalian male germ cells mainly based on the formation of a condensed structure referred to as a sex body or XY-body, during spermatogenesis. Molecular identity of the sex body, the roles of sense and antisense XIST RNAs in its formation, and the relevance of the sex body to spermatogenesis are not known. Here we report the results of our strand-specific RT-PCR approach to identify the amplicon detected in fetal bovine testes previously referred to as XIST and to test for sense/antisense expression in male and female organs and cell cultures of different sex chromosome constitution. Our results showed that the transcript detected consistently in male gonads and variably in somatic organs represents XIST antisense RNA and that XIST sense and antisense RNAs are co-expressed in female somatic tissues and cultured cells including cells of sex chromosome aneuploids (XXY and XXX). Our results, which differ from those of other investigators in this area, are discussed in the light of the recently reported differences in the expression pattern of murine Xist/Tsix loci and their structural and functional differences in different mammalian species.

  15. SupraMolecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression.

    PubMed Central

    Allal, C.; Sixou, S.; Kravtzoff, R.; Soulet, N.; Soula, G.; Favre, G.

    1998-01-01

    New therapeutic strategies are now being developed against adenocarcinoma associated with erbB-2 amplification, particularly by inhibiting p185erbB-2 expression. Antisense oligodeoxynucleotides seem promising for this purpose as long as they are efficiently protected against degradation and targeted into the cells. We present antisense oligonucleotide carriers, the supramolecular biovectors (SMBVs), for which we have already demonstrated the ability to improve both cellular uptake and protection of oligodeoxynucleotide. The present work demonstrates that SMBVs elicit a specific and non-toxic action of antisense compounds in a cell model, irrespective of their sensitivity to nucleases. This is a major point, considering the specificity problems associated with the use of nuclease-resistant phosphorothioate oligodeoxynucleotide. SMBVs improve antisense efficiency of oligodeoxynucleotide designed against p185erbB-2, with a complete growth arrest of SK-Br-3, human adenocarcinoma mammary cells that overexpress p185erbB-2 and no effect on MCF-7 cells that normally express p185erbB-2. The comparison of SMBVs with DOTAP reveals the statistically higher efficiency of SMBVs, which allows the antisense inhibition of p185erbB-2 expression in 65-75% of SK-Br-3 cells (P < 0.05). The efficiency and controlled synthesis of SMBVs underline their potentialities as oligodeoxynucleotide carriers for in vivo experiments. PMID:9652760

  16. Antisense oligonucleotides targeted to the p53 gene modulate liver regeneration in vivo.

    PubMed

    Arora, V; Iversen, P L

    2000-02-01

    The rapidly proliferating cells of the regenerating liver after partial hepatectomy (PH) present a reproducible in vivo model to study the functional role of the tumor suppressor gene p53. The present study uses the rat 70% PH model along with systemic administration of three different structural types of antisense oligonucleotides (ODNs) designed to suppress p53 expression. We tested the hypothesis that antisense ODNs can inhibit the expression of p53, resulting in the loss of the G(1)-S cell cycle checkpoint and an altered pattern of liver regeneration. Intraperitoneal administration of 5 mg/kg/day antisense phosphorothioate ODN after 70% PH resulted in reduced expression of the p53 protein in the regenerating liver. There were concomitant increases in weight gain of remnant-regenerating liver and expression of proliferating cell nuclear antigen and p21(waf-1) compared with either saline or 5 mg/kg/day mispaired phosphorothioate ODN treatment. Flow cytometric analysis of DNA content of isolated hepatocytes revealed a reduction in the G(0)/G(1) cell population and accumulation of cells with more than 4n DNA in antisense-treated rats. The regenerating livers had significantly diminished cytochrome P-450 (CYP) enzyme activities. Rats treated with p53 antisense ODNs, but not saline or mispair ODN controls, had significantly elevated CYP activities. These observations functionally link the expression of p53 with diminished expression of several CYP isoforms in the liver regeneration model.

  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. Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.

    PubMed

    Halitschke, Rayko; Baldwin, Ian T

    2003-12-01

    Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.

  19. Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells.

    PubMed Central

    Giles, R V; Ruddell, C J; Spiller, D G; Green, J A; Tidd, D M

    1995-01-01

    We have previously demonstrated, in vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methylphosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherichia coli and human RNases H in vitro. Intact human leukaemia cells 'biochemically microinjected' with antisense effectors demonstrated efficient suppression of target mRNA expression. It was noted that the chimeric methylphosphonodiester/phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity. Images PMID:7731809

  20. Delivery is key: lessons learnt from developing splice-switching antisense therapies.

    PubMed

    Godfrey, Caroline; Desviat, Lourdes R; Smedsrød, Bård; Piétri-Rouxel, France; Denti, Michela A; Disterer, Petra; Lorain, Stéphanie; Nogales-Gadea, Gisela; Sardone, Valentina; Anwar, Rayan; El Andaloussi, Samir; Lehto, Taavi; Khoo, Bernard; Brolin, Camilla; van Roon-Mom, Willeke Mc; Goyenvalle, Aurélie; Aartsma-Rus, Annemieke; Arechavala-Gomeza, Virginia

    2017-03-13

    The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre-clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides.

  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. 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.

  3. 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.

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

    PubMed

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

    2016-06-27

    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.

  5. 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

  6. 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.

  7. 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

  8. The zebrafish progranulin gene family and antisense transcripts

    PubMed Central

    Cadieux, Benoît; Chitramuthu, Babykumari P; Baranowski, David; Bennett, Hugh PJ

    2005-01-01

    Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs

  9. 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

  10. 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.

  11. LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

    PubMed

    Maciaszek, Anna; Krakowiak, Agnieszka; Janicka, Magdalena; Tomaszewska-Antczak, Agnieszka; Sobczak, Milena; Mikołajczyk, Barbara; Guga, Piotr

    2015-02-28

    Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

  12. Bacterial production of conjugated linoleic and linolenic Acid in foods: a technological challenge.

    PubMed

    Gorissen, Lara; Leroy, Frédéric; De Vuyst, Luc; De Smet, Stefaan; Raes, Katleen

    2015-01-01

    Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers are present in foods derived from ruminants as a result of the respective linoleic acid (LA) and α-linolenic acid (LNA) metabolism by ruminal microorganisms and in animals' tissues. CLA and CLNA have isomer-specific, health-promoting properties, including anticarcinogenic, antiatherogenic, anti-inflammatory, and antidiabetic activity, as well as the ability to reduce body fat. Besides ruminal microorganisms, such as Butyrivibrio fibrisolvens, many food-grade bacteria, such as bifidobacteria, lactic acid bacteria (LAB), and propionibacteria, are able to convert LA and LNA to CLA and CLNA, respectively. Linoleate isomerase activity, responsible for this conversion, is strain-dependent and probably related to the ability of the producer strain to tolerate the toxic effects of LA and LNA. Since natural concentrations of CLA and CLNA in ruminal food products are relatively low to exert their health benefits, food-grade bacteria with linoleate isomerase activity could be used as starter or adjunct cultures to develop functional fermented dairy and meat products with increased levels of CLA and CLNA or included in fermented products as probiotic cultures. However, results obtained so far are below expectations due to technological bottlenecks. More research is needed to assess if bacterial production kinetics can be increased and can match food processing requirements.

  13. 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

  14. 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.

  15. Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas

    PubMed Central

    Nehyba, Jiri; Malhotra, Sanandan; Winans, Shelby; O'Hare, Thomas H.; Justice, James

    2016-01-01

    ABSTRACT Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT. Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. IMPORTANCE The data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas. PMID:27512065

  16. Antisense Inhibition of the Photosynthetic Antenna Proteins CP29 and CP26

    PubMed Central

    Andersson, Jenny; Walters, Robin G.; Horton, Peter; Jansson, Stefan

    2001-01-01

    The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching. PMID:11340191

  17. 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.

  18. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    PubMed

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  19. In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

    PubMed

    Maksimenko, Andrei; Polard, Valerie; Villemeur, Marie; Elhamess, Hind; Couvreur, Patrick; Bertrand, Jean-Remi; Aboubakar, Malam; Gottikh, Marina; Malvy, Claude

    2005-11-01

    The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

  20. The association between H3K4me3 and antisense transcription.

    PubMed

    Cui, Peng; Liu, Wanfei; Zhao, Yuhui; Lin, Qiang; Ding, Feng; Xin, Chengqi; Geng, Jianing; Song, Shuhui; Sun, Fanglin; Hu, Songnian; Yu, Jun

    2012-04-01

    Histone H3 lysine 4 trimethylation (H3K4me3) is well known to occur in the promoter region of genes for transcription activation. However, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3' end of actively transcribed (sense) genes, named as 3'-H3K4me3. 3'-H3K4me3 is associated with ~15% of protein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II (RNAPII) binding sites and 5'-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3'-H3K4me3 is associated with the initiation of antisense transcription. Furthermore, 3'-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3'-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3' end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3'-H3K4me3 modification. More importantly, we observed the 3'-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3'-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in vertebrate. Therefore, we speculate that these 3'-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3'-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

  1. Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

    PubMed Central

    Nakashima, Nobutaka; Tamura, Tomohiro

    2009-01-01

    In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications. PMID:19515932

  2. Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

    PubMed Central

    Kitamoto, Noriyuki; Yoshino, Shoko; Ohmiya, Kunio; Tsukagoshi, Norihiro

    1999-01-01

    β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain. PMID:9872754

  3. Antisense suppression of 4-coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition.

    PubMed Central

    Lee, D; Meyer, K; Chapple, C; Douglas, C J

    1997-01-01

    The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) is considered necessary to activate the hydroxycinnamic acids for the biosynthesis of the coniferyl and sinapyl alcohols subsequently polymerized into lignin. To clarify the role played by 4CL in the biosynthesis of the guaiacyl (G) and syringyl (S) units characteristic of angiosperm lignin, we generated 4CL antisense Arabidopsis lines having as low as 8% residual 4CL activity. The plants had decreases in thioglycolic acid-extractable lignin correlating with decreases in 4CL activity. Nitrobenzene oxidation of cell walls from bolting stems revealed a significant decrease in G units in 4CL-suppressed plants; however, levels of S lignin units were unchanged in even the most severely 4CL-suppressed plants. These effects led to a large decrease in the G/S ratio in these plants. Our results suggest that an uncharacterized metabolic route to sinapyl alcohol, which is independent of 4CL, may exist in Arabidopsis. They also demonstrate that repression of 4CL activity may provide an avenue to manipulate angiosperm lignin subunit composition in a predictable manner. PMID:9401123

  4. Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

    PubMed Central

    Pepin, M C; Barden, N

    1991-01-01

    Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids. Images PMID:1996114

  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. Transcriptome analysis of antigenic variation in Plasmodium falciparum - var silencing is not dependent on antisense RNA

    PubMed Central

    Ralph, Stuart A; Bischoff, Emmanuel; Mattei, Denise; Sismeiro, Odile; Dillies, Marie-Agnès; Guigon, Ghislaine; Coppee, Jean-Yves; David, Peter H; Scherf, Artur

    2005-01-01

    Background Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36. Results In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript. Conclusion Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci. PMID:16277748

  7. 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

  8. 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.

  9. 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

  10. Increased amplification success from forensic samples with locked nucleic acids.

    PubMed

    Ballantyne, Kaye N; van Oorschot, Roland A H; Mitchell, R John

    2011-08-01

    Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples.

  11. Rapid genotyping using pyrene−perylene locked nucleic acid complexes

    PubMed Central

    Kumar, T. Santhosh; Myznikova, Anna; Samokhina, Evgeniya; Astakhova, Irina Kira

    2013-01-01

    We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene−perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2′-amino group of 2′-amino-LNA in position 4 allows for the first time to efficiently utilize dipole−dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene−perylene FRET pairs, e.g., in imaging and clinical diagnostics. PMID:24044052

  12. 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

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

    PubMed

    Kaur, Parvinder; 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. 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

  15. Cis-encoded non-coding antisense RNAs in streptococci and other low GC Gram (+) bacterial pathogens

    PubMed Central

    Cho, Kyu Hong; Kim, Jeong-Ho

    2015-01-01

    Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies. PMID:25859258

  16. 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

  17. Biochemical responses to dietary α-linolenic acid restriction proceed differently among brain regions in mice.

    PubMed

    Miyazawa, Daisuke; Yasui, Yuko; Yamada, Kazuyo; Ohara, Naoki; Okuyama, Harumi

    2011-08-01

    Previously, we noted that the dietary restriction of α-linolenic acid (ALA, n-3) for 4 weeks after weaning brought about significant decreases in the BDNF content and p38 MAPK activity in the striatum of mice, but not in the other regions of the brain, compared with an ALA- and linoleic acid (LNA, n-6)-adequate diet. In this study, we examined whether a prolonged dietary manipulation induces biochemical changes in other regions of the brain as well. Mice were fed a safflower oil (SAF) diet (ALA-restricted, LNA-adequate) or a perilla oil (PER) diet (containing adequate amounts of ALA and LNA) for 8 weeks from weaning. The docosahexaenoic acid (DHA, 22:6n-3) contents and p38 MAPK activities in the cerebral cortex, striatum and hippocampus were significantly lower in the SAF group. The BDNF contents and protein kinase C (PKC) activities in the cerebral cortex as well as in the striatum, but not in the hippocampus, were significantly lower in the SAF group. These data indicate that the biochemical changes induced by the dietary restriction of ALA have a time lag in the striatum and cortex, suggesting that the signal is transmitted through decreased p38 MAPK activity and BDNF content and ultimately decreased PKC activity.

  18. Variability of HHV8 LNA-1 Immunohistochemical Staining Across the 3 Histologic Stages of HIV-Associated Mucocutaneous Kaposi Sarcoma: Is There a Relationship to Patients' CD4 Counts?

    PubMed

    Mohanlal, Reena D; Pather, Sugeshnee

    2015-07-01

    The histologic diagnosis of Kaposi sarcoma (KS) can be confirmed with human herpes virus 8 (HHV8) latency-associated nuclear antigen (LNA)-1 immunohistochemistry, which may show variability in distribution and intensity. This retrospective study was aimed at addressing the factors that may contribute to this variability. All cases of mucocutaneous KS diagnosed in a 5-year period at the histopathology department at a tertiary hospital in South Africa with available patients' CD4 counts and HHV8 LNA-1 immunohistochemically stained slides were reviewed, and the biopsy stages of KS (patch/plaque/nodular), CD4 counts, immunohistochemistry staining method (manual vs. automated), and distribution (diffuse/focal) and intensity (strong/weak) of HHV8 LNA-1 staining were recorded. A total of 127 cases were reviewed. No relationship was demonstrated between the median CD4 count and the histologic stages of KS (P = 0.701) or the intensity and distribution of HHV8 immunohistochemical staining using either staining method. Multivariate analysis showed that method of immunohistochemical staining was a significant predictor of distribution (P = 0.006) and intensity (P = 0.044) of staining, and that stage was a significant predictor of distribution of staining (P = 0.033).

  19. Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites.

    PubMed

    Guo, Junli; Zheng, Wenyu; Wang, Yuehua; Li, Yao; Lu, Siqi; Feng, Xianmin

    2014-02-14

    A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches.

  20. 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

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

    PubMed

    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).

  2. 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.

  3. The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia.

    PubMed

    Dürig, J; Dührsen, U; Klein-Hitpass, L; Worm, J; Hansen, J B Rode; Ørum, H; Wissenbach, M

    2011-04-01

    SPC2996 is a novel locked nucleic acid phosphorothioate antisense molecule targeting the mRNA of the Bcl-2 oncoprotein. We investigated the mechanism of action of SPC2996 and the basis for its clinically observed immunostimulatory effects in chronic lymphocytic leukemia (CLL). Patients with relapsed CLL were treated with a maximum of six doses of SPC2996 (0.2-6 mg/kg) in a multicenter phase I trial. Microarray-based transcriptional profiling of circulating CLL cells was carried out before and after the first infusion of SPC2996 in 18 patients. Statistically significant transcriptomic changes were observed at doses 4 mg/kg and occurred as early as 24 h after the first infusion of the oligonucleotide. SPC2996 induced the upregulation of 466 genes including a large number of immune response and apoptotic regulator molecules, which were enriched for Toll-like receptor response genes. Serum measurements confirmed the release of pro-inflammatory cytokines including chemokine (C-C motif) ligand 3 (macrophage inflammatory protein 1α) and tumor necrosis factor-α, thereby validating the in vivo transcriptomic data at the protein level. SPC2996 caused a 50% reduction of circulating lymphocytes in five of 18 (28%) patients, which was found to be independent of its immunostimulatory and anti-Bcl-2 effects.

  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. A potential suppressive effect of natural antisense IL-1β RNA on lipopolysaccharide-induced IL-1β expression

    PubMed Central

    Lu, Jiawei; Wu, Xiurong; Hong, Mao; Tobias, Peter; Han, Jiahuai

    2013-01-01

    Although more than half of genomic loci are believed to have antisense transcription, whether antisense transcription is involved in cytokine expression has not been studied. Here we show that some loci of innate immunity related genes do have antisense transcripts. We investigated the effect of several antisense RNAs, including anti-4-1BBL, anti-p100 and anti-IL-1β, on their cognate sense gene’s expression in macrophages. We found that overexpression of antisense IL-1β transcript suppressed IL-1β expression. Anti-IL-1β is complementary to the sequence in the 5′ upstream region of the IL-1β promoter. Its mediated inhibition of IL-1β production occurred at the transcriptional level. Anti-IL-1β did not alter the methylation status of the IL-1β promoter. However, chromatin immunoprecipitation (ChIP) assays revealed that the anti-IL-1β transcript can change the chromatin structure of the IL-1β promoter by decreasing H3K4 trimethylation on the promoter, which is at least part of the mechanism underlying the reduced binding of RNA polymerase II to the IL-1β promoter upon anti-IL-1β expression. Our data suggest that some antisense-transcripts of innate immunity related genes play a role by regulating cytokine expression. PMID:23677478

  6. Downregulation of brain mineralocorticoid and glucocorticoid receptor by antisense oligodeoxynucleotide treatment fails to alter spatial navigation in rats.

    PubMed

    Engelmann, M; Landgraf, R; Lörscher, P; Conzelmann, C; Probst, J C; Holsboer, F; Reul, J M

    1998-11-13

    Adult male Brown Norway rats were long-term intracerebroventricularly (i.c.v.) infused with antisense oligodeoxynucleotides (18-mer, double endcapped phosphorothioate protected) targeting either mineralocorticoid or glucocorticoid receptor mRNA, or received the respective mixed bases sequence or vehicle. Mineralocorticoid receptor-mixed bases and glucocorticoid receptor-mixed bases oligodeoxynucleotide infusion (1 microg/0.5 microl/h) over a time period of seven days did not alter hippocampal mineralocorticoid receptor and glucocorticoid receptor binding when compared to vehicle treatment. In contrast, i.c.v. administration of mineralocorticoid receptor, as well as glucocorticoid receptor-antisense over the same time period resulted in a significantly reduced binding of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus [mineralocorticoid receptor-antisense group approx. 72% of mineralocorticoid receptor-mixed bases and vehicle groups (100%); glucocorticoid receptor antisense group approx. 77% of glucocorticoid receptor-mixed bases and vehicle]. The specificity of these antisense effects is indicated by the finding that rats treated with mineralocorticoid receptor-antisense did not show any changes in glucocorticoid receptor and vice versa. Animals treated according to this infusion protocol and tested in the Morris water maze for their spatial navigation abilities failed to show significant differences among the groups. These data indicate that a reduction of hippocampal mineralocorticoid receptor or glucocorticoid receptor binding capacity by 20-30% does not interfere with spatial navigation.

  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. Mining SAGE data allows large-scale, sensitive screening of antisense transcript expression.

    PubMed

    Quéré, Ronan; Manchon, Laurent; Lejeune, Mireille; Clément, Oliver; Pierrat, Fabien; Bonafoux, Béatrice; Commes, Thérèse; Piquemal, David; Marti, Jacques

    2004-11-23

    As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.

  9. Antisense-RNA mediated control of plasmid replication - pIP501 revisited.

    PubMed

    Brantl, Sabine

    2015-03-01

    Over the past decade, a wealth of small noncoding RNAs (sRNAs) have been discovered in the genomes of almost all bacterial species, where they constitute the most abundant class of posttranscriptional regulators. These sRNAs are key-players in prokaryotic metabolism, stress response and virulence. However, the first bona-fide antisense RNAs had been found already in 1981 in plasmids, where they regulate replication or maintenance. Antisense RNAs involved in plasmid replication control - meanwhile investigated in depth for almost 35 years - employ a variety of mechanisms of action: They regulate primer maturation, inhibit translation of essential replication initiator proteins (Rep proteins) as well as leader peptides or the formation of activator pseudoknots required for efficient rep translation. Alternatively they attenuate transcription or translation of rep mRNAs. Some antisense RNAs collaborate with transcriptional repressors to ensure proper copy-number control. Here, I summarize our knowledge on replication control of the broad-host range plasmid pIP501 that was originally isolated from Streptococcus agalactiae. Plasmid pIP501 uses two copy number-control elements, RNAIII, a cis-encoded antisense RNA, and transcriptional repressor CopR. RNA III mediates transcription attenuation, a rather widespread concept that found its culmination in the recent discovery of riboswitches. A peculiarity of pIP501 is the unusual stability of RNA III, which requires a second function of CopR: CopR does not only repress transcription from the essential repR promoter, but also prevents convergent transcription between rep mRNA and RNAIII, thereby indirectly increasing the amount of RNAIII. The concerted action of these two control elements is necessary to prevent plasmid loss at dangerously low copy numbers.

  10. 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

  11. Naturally occurring antisense RNA of histone H2a in mouse cultured cell lines

    PubMed Central

    Nishida, Hiromi; Tomaru, Yasuhiro; Oho, Yuko; Hayashizaki, Yoshihide

    2005-01-01

    Background An antisense transcript of histone H2a that has no significant protein-coding region has been cloned from a mouse full-length cDNA library. In the present study, we evaluated this transcript by using RT-PCR and compared the expression patterns of the sense and antisense transcripts by using quantitative RT-PCR (qRT-PCR). Results This antisense RNA was expressed in three mouse cell lines. We call it ASH2a. ASH2a includes not only the complementary sequence of the transcript of Hist2h2aa2 (a replication-dependent histone H2a gene), but also that of the promoter of Hist2h2aa2. The upstream genomic sequence of the transcription start site of the ASH2a-coding gene (ASH2a) lacks both CCAAT and TATA boxes. This absence suggests that the regulation of ASH2a is different from that of the replication-dependent histone H2a genes. Findings from qRT-PCR indicated that the expression pattern of ASH2a was different from that of Hist2h2aa2. Expression of Hist2h2aa2 peaked at 2 to 4 h during S-phase, but that of ASH2a peaked at 1 h. Conclusion We showed the existence of ASH2a, a histone H2a antisense RNA, in mouse cultured cells. The expression pattern of ASH2a is different from that of the sense RNA. PMID:15892893

  12. Bulged-out nucleotides protect an antisense RNA from RNase III cleavage.

    PubMed Central

    Hjalt, T A; Wagner, E G

    1995-01-01

    Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo. Images PMID:7534906

  13. Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin–related liver disease in mice

    PubMed Central

    Guo, Shuling; Booten, Sheri L.; Aghajan, Mariam; Hung, Gene; Zhao, Chenguang; Blomenkamp, Keith; Gattis, Danielle; Watt, Andrew; Freier, Susan M.; Teckman, Jeffery H.; McCaleb, Michael L.; Monia, Brett P.

    2013-01-01

    Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease. PMID:24355919

  14. Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression.

    PubMed

    Mourtada-Maarabouni, Mirna; Kirkham, Lucy; Farzaneh, Farzin; Williams, Gwyn T

    2004-12-16

    Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways independently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone' by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis.

  15. Antisense-mediated depletion of tomato GDP-L-galactose phosphorylase increases susceptibility to chilling stress.

    PubMed

    Wang, Li-Yan; Li, Dong; Deng, Yong-Sheng; Lv, Wei; Meng, Qing-Wei

    2013-02-15

    The GDP-L-galactose phosphorylase (GGP), which converts GDP-l-galactose to l-Gal-1-phosphate, is generally considered to be a key enzyme of the major ascorbate biosynthesis pathways in higher plants, but experimental evidence for its role in tomato is lacking. In the present study, the GGP gene was isolated from tomato (Solanum lycopersicum) and transient expression of SlGGP-GFP (green fluorescent protein) fusion protein in onion cells revealed the cytoplasmic and nucleus localization of the protein. Antisense transgenic tomato lines with only 50-75% ascorbate level of the wild type (WT) were obtained. Chilling treatment induced lower increase in AsA levels and redox ratio of ascorbate in antisense transgenic plants compared with WT plants. Under chilling stress, transgenic plants accumulated more malendialdehyde (MDA) and more O(2)(·-), leaked more electrolytes and showed lower maximal photochemical efficiency of PSII (Fv/Fm), net photosynthetic rate (Pn), and oxidizable P700 compared with WT plants. Furthermore, the antisense transgenic plants exhibited significantly higher H(2)O(2) level and lower ascorbate peroxidase (APX) activity. Our results suggested that GGP plays an important role in protecting plants against chilling stress by maintaining ascorbate pool and ascorbate redox state.

  16. A noncoding RNA antisense to moesin at 5p14.1 in autism.

    PubMed

    Kerin, Tara; Ramanathan, Anita; Rivas, Kasey; Grepo, Nicole; Coetzee, Gerhard A; Campbell, Daniel B

    2012-04-04

    People with autism spectrum disorder (ASD) are characterized by deficits in social interaction, language, and behavioral flexibility. Rare mutations and copy number variations have been identified in individuals with ASD, but in most patients, the causal variants remain unknown. A genome-wide association study (GWAS), designed to identify genes and pathways that contribute to ASD, indicated a genome-wide significant association of ASD with the single-nucleotide polymorphism (SNP) rs4307059 (P = 10⁻¹⁰), which is located in a gene-poor region of chromosome 5p14.1. We describe here a 3.9-kb noncoding RNA that is transcribed from the region of the chromosome 5p14.1 ASD GWAS association SNP. The noncoding RNA was encoded by the opposite (antisense) strand of moesin pseudogene 1 (MSNP1), and we therefore designated it as MSNP1AS (moesin pseudogene 1, antisense). Chromosome 5p14.1 MSNP1AS was 94% identical and antisense to the X chromosome transcript of MSN, which encodes a protein (moesin) that regulates neuronal architecture. Individuals who carry the ASD-associated rs4307059 T allele showed increased expression of MSNP1AS. The MSNP1AS noncoding RNA bound to MSN, was highly overexpressed (12.7-fold) in postmortem cerebral cortex of individuals with ASD, and could regulate levels of moesin protein in human cell lines. These data reveal a biologically functional element that may contribute to ASD risk.

  17. 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.

  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. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    PubMed

    Riboldi, Giulietta; Zanetta, Chiara; Ranieri, Michela; Nizzardo, Monica; Simone, Chiara; Magri, Francesca; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-12-01

    Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of "toxic" RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon.

  20. ASBEL, an ANA/BTG3 antisense transcript required for tumorigenicity of ovarian carcinoma.

    PubMed

    Yanagida, Satoshi; Taniue, Kenzui; Sugimasa, Hironobu; Nasu, Emiko; Takeda, Yasuko; Kobayashi, Mana; Yamamoto, Tadashi; Okamoto, Aikou; Akiyama, Tetsu

    2013-01-01

    Mammalian genomes encode numerous antisense non-coding RNAs, which are assumed to be involved in the regulation of the sense gene expression. However, the mechanisms of their action and involvement in the development of diseases have not been well elucidated. The ANA/BTG3 protein is an antiproliferative protein whose expression is downregulated in prostate and lung cancers. Here we show that an antisense transcript of the ANA/BTG3 gene, termed ASBEL, negatively regulates the levels of ANA/BTG3 protein, but not of ANA/BTG3 mRNA and is required for proliferation and tumorigenicity of ovarian clear cell carcinoma. We further show that knockdown of ANA/BTG3 rescues growth inhibition caused by ASBEL knockdown. Moreover, we demonstrate that ASBEL forms duplexes with ANA/BTG3 mRNA in the nucleus and suppresses its cytoplasmic transportation. Our findings illustrate a novel function for an antisense transcript that critically promotes tumorigenesis by suppressing translation of the sense gene by inhibiting its cytoplasmic transportation.

  1. Antisense-mediated FLC transcriptional repression requires the P-TEFb transcription elongation factor

    PubMed Central

    Wang, Zhi-Wei; Wu, Zhe; Raitskin, Oleg; Sun, Qianwen; Dean, Caroline

    2014-01-01

    The functional significance of noncoding transcripts is currently a major question in biology. We have been studying the function of a set of antisense transcripts called COOLAIR that encompass the whole transcription unit of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Alternative polyadenylation of COOLAIR transcripts correlates with different FLC sense expression states. Suppressor mutagenesis aimed at understanding the importance of this sense–antisense transcriptional circuitry has identified a role for Arabidopsis cyclin-dependent kinase C (CDKC;2) in FLC repression. CDKC;2 functions in an Arabidopsis positive transcription elongation factor b (P-TEFb) complex and influences global RNA polymerase II (Pol II) Ser2 phosphorylation levels. CDKC;2 activity directly promotes COOLAIR transcription but does not affect an FLC transgene missing the COOLAIR promoter. In the endogenous gene context, however, the reduction of COOLAIR transcription by cdkc;2 disrupts a COOLAIR-mediated repression mechanism that increases FLC expression. This disruption then feeds back to indirectly increase COOLAIR expression. This tight interconnection between sense and antisense transcription, together with differential promoter sensitivity to P-TEFb, is central to quantitative regulation of this important floral repressor gene. PMID:24799695

  2. 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

    2012-09-07

    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.

  3. Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice.

    PubMed

    Guo, Shuling; Booten, Sheri L; Aghajan, Mariam; Hung, Gene; Zhao, Chenguang; Blomenkamp, Keith; Gattis, Danielle; Watt, Andrew; Freier, Susan M; Teckman, Jeffery H; McCaleb, Michael L; Monia, Brett P

    2014-01-01

    Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.

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

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-02

    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.

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

    PubMed Central

    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

  6. Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development.

    PubMed

    Wu, Bo; Benrashid, Ehsan; Lu, Peijuan; Cloer, Caryn; Zillmer, Allen; Shaban, Mona; Lu, Qi Long

    2011-01-01

    Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

  7. Apolipoprotein E-deficient mice created by systemic administration of antisense oligodeoxynucleotides: a new model for lipoprotein metabolism studies.

    PubMed

    Morishita, R; Gibbons, G H; Kaneda, Y; Zhang, L; Ogihara, T; Dzau, V J

    2002-11-01

    Atherosclerotic cardiovascular disease results from complex interactions among multiple genetic and environmental factors. Thus, it is important to elucidate the influence of each factor on cholesterol metabolism. For this purpose, transgenic/gene-targeting technology is a powerful tool for studying gene functions. However, this technology has several disadvantages such as being time consuming and expensive. Accordingly, we established new animal models using in vivo gene transfer technology. In this study, we examined the feasibility of the creation of a new animal model for the study of atherosclerosis. We hypothesized that apolipoprotein (apo) E-deficient mice can be created by systemic administration of antisense apo E oligodeoxynucleotides (ODN) coupled to the HVJ-liposome complex. Initially, we examined the localization and cellular fate of FITC-labeled antisense ODN administered intravenously. FITC-labeled ODN transfection by the HVJ-liposome method resulted in fluorescence in the liver, spleen and kidney, but not in other organs such as brain. Moreover, fluorescence with the HVJ-liposome method was sustained for up to 2 weeks after transfection, which resulted in a striking difference from transfection of ODN alone or ODN in liposomes without HVJ, which showed rapid disappearance of fluorescence (within 1 day). Given these unique characteristics of the HVJ-liposome method, we next examined transfection of antisense apo E ODN by intravenous administration. Transfection of antisense apo E ODN resulted in a marked reduction of apo E mRNA levels in the liver, but no change in apo B and beta-actin mRNA levels. In mice fed a normal diet, a transient increase in cholesterol and triglyceride levels was observed in the antisense apo E-treated group, but they returned to normal levels by 6 days after transfection. Similar findings were also found in mice fed a high cholesterol diet. Neither scrambled nor mismatched ODN resulted in any increase in cholesterol. To make

  8. The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid.

    PubMed

    Xu, You; Villa, Alessandra; Nilsson, Lennart

    2017-01-19

    Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  9. Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription

    PubMed Central

    Poole, Rebecca L; Barker, Gary LA; Werner, Kay; Biggi, Gaia F; Coghill, Jane; Gibbings, J George; Berry, Simon; Dunwell, Jim M; Edwards, Keith J

    2008-01-01

    Background Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat. Results Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of

  10. 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

  11. Genetic modification of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in "hairy root" cultures.

    PubMed

    Carron, T R; Robbins, M P; Morris, P

    1994-03-01

    An antisense dihydroflavonol reductase (DFR) gene-construct made using the cDNA for DFR from Antirrhinum majus was introduced into the genome of a series of clonal genotypes of Lotus corniculatus via Agrobacterium rhizogenes. After initial screening, 17 antisense and 11 control transformation events were analysed and tannin levels found to be reduced in antisense root cultures. The effect of this antisense construct, (pMAJ2), which consisted of the 5' half of the DFR cDNA sequence, was compared in three different recipient Lotus genotypes. This construct effectively down-regulated tannin biosynthesis in two of the recepient genotypes (s33 and s50); however, this construct was relatively ineffective in a third genotype (s41) which accumulated high levels of condensed tannins in derived transgenic root cultures. Four pMAJ2 antisense and three control lines derived from clonal genotypes s33 and s50 were selected and studied in greater detail. The antisense DFR construct was found to be integrated into the genome of the antisense "hairy root" cultures, and the antisense RNA was shown to be expressed. Tannin levels were much lower in antisense roots compared to the controls and this reduction in tannin levels was accompanied by a change in condensed tannin subunit composition.

  12. Water-absorbent polymer as a carrier for a discrete deposit of antisense oligodeoxynucleotides in the central nervous system.

    PubMed

    Bannai, M; Ichikawa, M; Nishimura, F; Nishihara, M; Takahashi, M

    1998-09-01

    One of the problems of introducing antisense oligodeoxynucleotides (ODN) into the central nervous system (CNS) is their rapid disappearance from the target site due to their dispersion and diffusion, which results in poor uptake and/or retention in cells (M. Morris, A.B. Lucion, Antisense oligonucleotides in the study of neuroendocrine systems, J. Neuroendocrinol. 7 (1995) 493-500; S. Ogawa, H.E. Brown, H.J. Okano, D.W. Pfaff, Cellular uptake of intracerebrally administrated oligodeoxynucleotides in mouse brain, Regul. Pept. 59 (1995) 143-149) [2,5]. Recently, we adapted a new method using water-absorbent polymer (WAP; internally cross-linked starch-grafted-polyacrylates) as a carrier for antisense ODN. The polymer forms a hydro-gel after absorbing water which is chemically and biologically inert. In these studies, the polymer (powder-form) is fully swollen by physiological saline containing antisense ODN (0.2 micromol/ml) to make 80-fold volume gel. Hydro-gel (1 microliter) is injected into the target site, and water solutes are assumed to be diffused stoichiometrically into CNS from the surface of the gel. Histological studies indicate that 24 h after the injection, antisense ODN (5'biotinylated-S-oligos of 15 mer) are distributed to within 800 micrometer from the edge of the area where the gel is located and then gradually disappear from this area within days, but still remain within 300-micrometer distance 7 days later. Antisense ODN are effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias, and suppress the synthesis of the target protein. This method can be adapted to slow delivery of antisense ODN and other water soluble substances into the CNS.

  13. 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

  14. Utilization of milk fatty acids by the suckling Iberian piglets.

    PubMed

    Aguinaga, M A; Haro, A; Lara, L; Gómez-Carballar, F; Nieto, R; Aguilera, J F

    2016-11-01

    A total of 16 pure-bred Iberian (IB) sows, all of them suckling six piglets, were used, eight of them in each of the two consecutive trials (1 and 2). Daily milk yield and composition were determined weekly over a 34-day lactation period. Within each litter, one piglet at birth and four piglets on day 35 of life were slaughtered. Milk intake per piglet tended to be greater in trial 2 (832 v. 893 g/day; P=0.066), but piglets grew at 168±3.3 g/day, irrespective of the trial. In the IB sow milk, the linoleic (LA) : linolenic (LNA) acid ratio averaged 14.6 and 15.2 in trial 1 and trial 2, respectively. A fivefold increase in piglet body fat content was observed over lactation (P<0.001). Most of this fat (81.4%) was present in the carcass. After 34 days of lactation, whole-body relative content of palmitic, palmitoleic, stearic and oleic acids were very close to those in the milk consumed, suggesting direct deposition. Daily deposition of LA derivatives and of LNA and its derivatives was found to be extremely low (<0.02 g, on average). Moreover, some of the arachidonic acid (ARA) in tissues of the IB piglet at birth disappeared throughout the lactating period. An overall fractional deposition for total fatty acids (FA) was 0.409. Fractional oxidation (disappearance) rates were 0.939 and 0.926 for n-6 and n-3 polyunsaturated FA. The overall rate of disappearance for the major non-essential FA (myristic, palmitic, palmitoleic, stearic and oleic acids), estimated as 1-the overall fractional deposition rate, was 0.546. It is concluded that the high degree of FA unsaturation, high oxidation rate of LA and LNA, and poor synthesis of ARA from LA and of docosahexaenoic acid from LNA found in the suckling piglet might increase the energy cost of whole-body fat accretion, a contributor to the observed low efficiency of use of milk energy for growth.

  15. A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide.

    PubMed

    Denys, Barbara; El Housni, Hakim; Nollet, Friedel; Verhasselt, Bruno; Philippé, Jan

    2010-07-01

    The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

  16. Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers

    PubMed Central

    Puckett, Susan E.; Reese, Kaleb A.; Mitev, Georgi M.; Mullen, Valerie; Johnson, Rudd C.; Pomraning, Kyle R.; Mellbye, Brett L.; Tilley, Lucas D.; Iversen, Patrick L.; Freitag, Michael

    2012-01-01

    Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)3RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)3RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)3RXB-AcpP was 4 × 10−7 mutations/cell division. A spontaneous (RFF)3RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)3RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)3RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)3RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)3RXB-AcpP. Deletion of sbmA caused resistance to (RFF)3RXB-AcpP. We conclude that resistance to (RFF)3RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)3R-XB PPMOs may be transported across the plasma membrane by SbmA. PMID:22985881

  17. Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers.

    PubMed

    Puckett, Susan E; Reese, Kaleb A; Mitev, Georgi M; Mullen, Valerie; Johnson, Rudd C; Pomraning, Kyle R; Mellbye, Brett L; Tilley, Lucas D; Iversen, Patrick L; Freitag, Michael; Geller, Bruce L

    2012-12-01

    Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)(3)RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)(3)RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)(3)RXB-AcpP was 4 × 10(-7) mutations/cell division. A spontaneous (RFF)(3)RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)(3)RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)(3)RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)(3)RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)(3)RXB-AcpP. Deletion of sbmA caused resistance to (RFF)(3)RXB-AcpP. We conclude that resistance to (RFF)(3)RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)(3)R-XB PPMOs may be transported across the plasma membrane by SbmA.

  18. 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.

  19. Long antisense non-coding RNAs function to direct epigenetic complexes that regulate transcription in human cells

    PubMed Central

    Morris, Kevin V.

    2009-01-01

    Epigenetic silencing of tumor suppressor gene promoters is one of the most common observations found in cancer. Despite the plethora of observed epigenetically silenced cancer related genes little is known about what is guiding the silencing to these particular loci. Two recent articles suggest that long antisense non-coding RNAs function as epigenetic regulators of transcription in human cells. These reports, along with previous observations that small antisense non-coding RNAs can epigenetically regulate transcription, imply that long antisense non-coding RNAs function as endogenous transcriptional regulatory RNAs in humans. Mechanistically, these long antisense non-coding RNAs may be involved in maintaining balanced transcription at bidirectionally transcribed loci as a method to modulate gene expression according to the selective pressures placed on the cell. The loss of this intricate bidirectional RNA based regulatory network can result in overt epigenetic silencing of gene expression. In the case of tumor suppressor genes, this silencing can lead to the loss of cellular regulation and be a contributing factor in cancer. This perspective will highlight the endogenous effector RNAs and mechanism of action whereby long antisense non-coding RNAs transcriptionally regulate gene expression in human cells. PMID:19633414

  20. The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery.

    PubMed

    Eckenfelder, Agathe; Tordo, Julie; Babbs, Arran; Davies, Kay E; Goyenvalle, Aurélie; Danos, Olivier

    2012-06-26

    Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD). Yet, clinical translation of these approaches is limited by the amounts of vector to be administered. In this respect, maximizing the amount of snRNA antisense shuttle delivered by the vector is essential. Here, we have used a muscle- and heart-specific enhancer (MHCK) to drive the expression of U7 snRNA shuttles carrying antisense sequences against the human or murine DMD pre-mRNAs. Although antisense delivery and subsequent exon skipping were improved both in tissue culture and in vivo, we observed the formation of additional U7 snRNA by-products following gene transfer. These included aberrantly 3' processed as well as unprocessed species that may arise because of the saturation of the cellular processing capacity. Future efforts to increase the amounts of functional U7 shuttles delivered into a cell will have to take this limitation into account.

  1. The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery

    PubMed Central

    Eckenfelder, Agathe; Tordo, Julie; Babbs, Arran; Davies, Kay E; Goyenvalle, Aurélie; Danos, Olivier

    2012-01-01

    Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD). Yet, clinical translation of these approaches is limited by the amounts of vector to be administered. In this respect, maximizing the amount of snRNA antisense shuttle delivered by the vector is essential. Here, we have used a muscle- and heart-specific enhancer (MHCK) to drive the expression of U7 snRNA shuttles carrying antisense sequences against the human or murine DMD pre-mRNAs. Although antisense delivery and subsequent exon skipping were improved both in tissue culture and in vivo, we observed the formation of additional U7 snRNA by-products following gene transfer. These included aberrantly 3′ processed as well as unprocessed species that may arise because of the saturation of the cellular processing capacity. Future efforts to increase the amounts of functional U7 shuttles delivered into a cell will have to take this limitation into account. PMID:23344083

  2. 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.

  3. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    PubMed Central

    2011-01-01

    Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP. PMID:21929758

  4. 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.

  5. Antisense-Mediated RNA Targeting: Versatile and Expedient Genetic Manipulation in the Brain

    PubMed Central

    Zalachoras, Ioannis; Evers, Melvin M.; van Roon-Mom, Willeke M. C.; Aartsma-Rus, Annemieke M.; Meijer, Onno C.

    2011-01-01

    A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized. PMID:21811437

  6. 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

  7. Targeting CAG repeat RNAs reduces Huntington's disease phenotype independently of huntingtin levels.

    PubMed

    Rué, Laura; Bañez-Coronel, Mónica; Creus-Muncunill, Jordi; Giralt, Albert; Alcalá-Vida, Rafael; Mentxaka, Gartze; Kagerbauer, Birgit; Zomeño-Abellán, M Teresa; Aranda, Zeus; Venturi, Veronica; Pérez-Navarro, Esther; Estivill, Xavier; Martí, Eulàlia

    2016-11-01

    Huntington's disease (HD) is a polyglutamine disorder caused by a CAG expansion in the Huntingtin (HTT) gene exon 1. This expansion encodes a mutant protein whose abnormal function is traditionally associated with HD pathogenesis; however, recent evidence has also linked HD pathogenesis to RNA stable hairpins formed by the mutant HTT expansion. Here, we have shown that a locked nucleic acid-modified antisense oligonucleotide complementary to the CAG repeat (LNA-CTG) preferentially binds to mutant HTT without affecting HTT mRNA or protein levels. LNA-CTGs produced rapid and sustained improvement of motor deficits in an R6/2 mouse HD model that was paralleled by persistent binding of LNA-CTG to the expanded HTT exon 1 transgene. Motor improvement was accompanied by a pronounced recovery in the levels of several striatal neuronal markers severely impaired in R6/2 mice. Furthermore, in R6/2 mice, LNA-CTG blocked several pathogenic mechanisms caused by expanded CAG RNA, including small RNA toxicity and decreased Rn45s expression levels. These results suggest that LNA-CTGs promote neuroprotection by blocking the detrimental activity of CAG repeats within HTT mRNA. The present data emphasize the relevance of expanded CAG RNA to HD pathogenesis, indicate that inhibition of HTT expression is not required to reverse motor deficits, and further suggest a therapeutic potential for LNA-CTG in polyglutamine disorders.

  8. Measurement of phosphorothioate oligodeoxynucleotide antisense transport across the blood-brain barrier.

    PubMed

    Banks, William A

    2011-01-01

    Phosphorothioate oligodeoxynucleotides (PODNs) can act as antisense molecules, knocking down proteins. Many PODNs have the unusual characteristic of being transported across the blood-brain barrier by a saturable system. This means that PODNs injected intravenously can accumulate in the central nervous system in quantities sufficient to knock down proteins in brain and the blood-brain barrier. A critical step in the development of PODNs that can be administered peripherally and knockdown proteins in the central nervous system is to determine the relation to the blood-brain barrier, specifically, does the PODN cross the blood-brain barrier and, if so, how fast and to what degree.

  9. Welcome to the splice age: antisense oligonucleotide–mediated exon skipping gains wider applicability

    PubMed Central

    McNally, Elizabeth M.; Wyatt, Eugene J.

    2016-01-01

    Exon skipping uses antisense oligonucleotides (ASOs) to alter transcript splicing for the purpose of rescuing or modulating protein expression. In this issue of the JCI, Lee and colleagues developed and evaluated an ASO-dependent method for treating certain molecularly defined diseases associated with alterations in lamin A/C (LMNA) splicing. Exon skipping by ASOs is gaining traction as a therapeutic strategy, and the use of ASOs is now being applied to bypass mutations and generate modified but functional proteins for an array of genetic disorders. PMID:26999602

  10. 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.

  11. Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties.

    PubMed

    Ogata, Aya; Maeda, Yusuke; Ueno, Yoshihito

    2017-04-01

    The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d-ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3'-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

  12. Administration of antisense DNA for GPR39-1b causes anxiolytic-like responses and appetite loss in rats.

    PubMed

    Ishitobi, Yoshinobu; Akiyoshi, Jotaro; Honda, Shuhei; Ninomiya, Taiga; Kanehisa, Masayuki; Tanaka, Yoshihiro; Tsuru, Jusen; Isogawa, Koichi; Kitamura, Hirokazu; Fujikura, Yoshihisa

    2012-03-01

    The G protein-coupled receptor 39-b (GPR39-1b) is a splice variant of which is expressed in the central nervous and gastrointestinal systems. Previously, GPR39-1b was proposed to be the receptor for obestatin, but current evidence does not support this hypothesis. The purpose of the present work was to identify the role of GPR39-1b in anxiety and eating behaviors. Antisense oligonucleotides were infused at a constant rate into the cerebral lateral ventricles of rats and their effect on anxiety-like behavior and food intake was monitored. GPR39-1b antisense oligonucleotides produced anxiolytic-like effects in the elevated-plus maze test and in the black and white box test. Antisense oligonucleotides also decreased food intake. These results indicate that inhibition of GPR39-1b induces a decrease in anxiety-related behaviors and disturbs appetite.

  13. Upregulation of long noncoding RNA zinc finger antisense 1 enhances epithelial-mesenchymal transition in vitro and predicts poor prognosis in glioma.

    PubMed

    Lv, Qiao-Li; Chen, Shu-Hui; Zhang, Xue; Sun, Bao; Hu, Lei; Qu, Qiang; Huang, Yuan-Tao; Wang, Gui-Hua; Liu, Yan-Ling; Zhang, Ying-Ying; Zhou, Hong-Hao

    2017-03-01

    Increasing evidence indicates that long noncoding RNAs play important roles in development and progression of various cancers. Zinc finger antisense 1 is a novel long noncoding RNA whose clinical significance, biological function, and underlying mechanism are still undetermined in glioma. In this study, we reported that zinc finger antisense 1 expression was markedly upregulated in glioma and tightly correlated with clinical stage. Moreover, patients with high zinc finger antisense 1 expression had shorter survival. Multivariate Cox regression analysis provided a clue that, probably, zinc finger antisense 1 level could serve as an independent prognostic factor for glioma. Functionally, zinc finger antisense 1 acted as an oncogene in glioma because its knockdown could promote apoptosis and significantly inhibit cell proliferation, migration, and invasion. Furthermore, zinc finger antisense 1 silencing could result in cell cycle arrest at the G0/G1 phase and correspondingly decrease the percentage of S phase cells in both U87 and U251 cell lines. Moreover, it was found that silenced zinc finger antisense 1 could impair migration and invasion by inhibiting the epithelial-mesenchymal transition through reducing the expression of MMP2, MMP9, N-cadherin, Integrin β1, ZEB1, Twist, and Snail as well as increasing E-cadherin level in glioma. Taken together, our data identified that zinc finger antisense 1 might act as a valuable prognostic biomarker and potential therapeutic target for glioma.

  14. Class switch recombination signals induce lymphocyte-derived Spo11 expression and Spo11 antisense oligonucleotide inhibits class switching.

    PubMed

    Tokuyama, H; Tokuyama, Y

    2001-08-01

    Recently, we showed that mouse Spo11 is induced in normal mu(+) B cells by class switch recombination (CSR) stimuli, by RT-PCR using primers based on the reported cDNA sequence of testis-derived Spo11 (test-Spo11) cDNA. In the present study, we first determined the cDNA sequence of lymphocyte-derived Spo11 (lym-Spo11). The 5' upstream portion had an as yet unreported sequence but the remaining part from exons 2 to 12 and the subsequent 3'UTR was completely identical to that of test-Spo11. RT-PCR analysis indicated that lymphocytes express lym-Spo11 but not test-Spo11. Second, we showed that lym-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS-stimulated mu(+)B cells in the presence of all-trans retinoic acid and IL-4. Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) can inhibit CSR reactions in three in vitro CSR systems, IgA,IgG1, and IgE. Lym-Spo11 AS or the sense oligonucleotide was added to the cultures at the start, and total RNA was extracted after 4 days. IgA, IgG1, and IgE mRNAs (J(H)C(H)) and mature germline C(H) transcripts (I(H)C(H)) were quantitatively assayed by RT-PCR. AS inhibited J(H)C(H) expression dose-dependently. In all three systems, the maximum inhibition by 20 microM AS was in the range of 60 to 90%. Interestingly, I(H)C(H) was also inhibited by AS to a similar extent as J(H)C(H). These results suggested that lym-Spo11 plays an important role in the initiation step of CSR.

  15. Cocaine and amphetamine elicit differential effects in rats with a unilateral injection of dopamine transporter antisense oligodeoxynucleotides.

    PubMed

    Silvia, C P; Jaber, M; King, G R; Ellinwood, E H; Caron, M G

    1997-02-01

    We have developed an antisense oligodeoxynucleotide to the dopamine transporter and used it to discriminate the behavioral properties of amphetamine and cocaine. In SK-N-MC cells permanently transfected with the dopamine transporter complementary DNA, treatment with 5 mM antisense oligodeoxynucleotide reduced dopamine uptake by 25% when compared to sense control. Unilateral intranigral administration of dopamine transporter antisense (50 microM) twice daily in freely moving rats for 2.5 days was sufficient to reduce dopamine transporter messenger RNA by 70% as measured by in situ hybridization, but not protein levels as measured by [3H]mazindol binding. However, intranigral treatment via implanted osmotic minipump over a period of seven days produced reductions in both dopamine transporter messenger RNA and protein levels (32%) at a dose of 500 pmol/day. These results indicate a longer half-life for the dopamine transporter than expected. Potassium chloride depolarization of ipsilateral striatal slices showed a greater than 200% increase in dopamine overflow on the antisense-treated side compared to the control side. Since imbalance of dopamine tone is known to induce rotational activity, we tested this behavioral paradigm in rats treated with various oligodeoxynucleotides at different doses and time-points. We have found that antisense-treated animals did not rotate spontaneously under any experimental conditions. Using various psychostimulants that target the dopamine transporter and increase dopamine levels, we found that the antisense-treated animals consistently rotated contralaterally in response to amphetamine (2 mg/kg), but not to cocaine (10 mg/kg) or nomifensine (10 mg/kg). These results bring in vivo evidence for a different mode of action of amphetamine and cocaine on the dopamine transporter and lend direct support to the view that amphetamine acts as a dopamine releaser, whereas cocaine acts by blocking dopamine transport.

  16. Engineering of gibberellin levels in citrus by sense and antisense overexpression of a GA 20-oxidase gene modifies plant architecture.

    PubMed

    Fagoaga, Carmen; Tadeo, Francisco R; Iglesias, Domingo J; Huerta, Laura; Lliso, Ignacio; Vidal, Ana M; Talon, Manuel; Navarro, Luís; García-Martínez, José L; Peña, Leandro

    2007-01-01

    Carrizo citrange (Citrus sinensisxPoncirus trifoliata) is a citrus hybrid widely used as a rootstock, whose genetic manipulation to improve different growth characteristics is of high agronomic interest. In this work, transgenic Carrizo citrange plants have been produced overexpressing sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) under control of the 35S promoter to modify plant architecture. As expected, taller (sense) and shorter (antisense) phenotypes correlated with higher and lower levels, respectively, of active GA1 in growing shoots. In contrast, other phenotypic characteristics seemed to be specific to citrus, or different from those described for similar transgenics in other species. For instance, thorns, typical organs of citrus at juvenile stages, were much longer in sense and shorter in antisense plants, and xylem tissue was reduced in leaf and internode of sense plants. Antisense plants presented a bushy phenotype, suggesting a possible effect of GAs on auxin biosynthesis and/or transport. The main foliole of sense plants was longer, although total leaf area was reduced. Leaf thickness was smaller in sense and larger in antisense plants due to changes in the spongy parenchyma. Internode cell length was not altered in transgenic plants, indicating that, in citrus, GAs regulate cell division rather than cell elongation. Interestingly, the phenotypes described were not apparent when transgenic plants were grafted on non-transgenic rootstock. This suggests that roots contribute to the GA economy of aerial parts in citrus and opens the possibility of using the antisense plants as dwarfing rootstocks.

  17. 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

  18. Interleukin-6 antisense oligonucleotides inhibit the growth of human myeloma cell lines.

    PubMed Central

    Levy, Y; Tsapis, A; Brouet, J C

    1991-01-01

    IL-6 has been shown to be a plasmacytoma growth factor in mice and is believed to play a key role in the development of human multiple myeloma. We investigated the IL-6 requirements for the growth of two human myeloma cell lines, U 266 and RPMI 8226. These cell lines secreted minute amounts of IL-6 (20 U/ml) and featured IL-6 mRNA. IL-6 receptors were detectable at the surface of malignant cells by immunofluorescence. Antibodies to IL-6 did not alter the proliferation of these myeloma cells. There was a dose-dependent decrease, however, in [3H]-thymidine uptake in the presence of IL-6 antisense (and not sense) oligodeoxynucleotides; in the presence of 20 microM IL-6 antisense, an 80 and 95% inhibition of the proliferation of U 266 and RPMI 8226 cells was observed, respectively. These results provide strong evidence for an IL-6 autocrine proliferation of myeloma cells which may occur via internal interaction between IL-6 and the IL-6 receptor. Images PMID:1864979

  19. Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

    PubMed Central

    Georg, Jens; Voß, Björn; Scholz, Ingeborg; Mitschke, Jan; Wilde, Annegret; Hess, Wolfgang R

    2009-01-01

    Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks. PMID:19756044

  20. In vitro characterization of two novel biodegradable vectors for the delivery of radiolabeled antisense oligonucleotides.

    PubMed

    von Guggenberg, Elisabeth; Shahhosseini, Soraya; Koslowsky, Ingrid; Lavasanifar, Afsaneh; Murray, David; Mercer, John

    2010-12-01

    The development of antisense oligonucleotides suitable for tumor targeting applications is hindered by low stability and bioavailability of oligonucleotides in vivo and by the absence of efficient and safe vectors for oligonucleotide delivery. Stabilization in vivo has been achieved through chemical modification of oligonucleotides by various means, but effective approaches to enhance their intracellular delivery are lacking. This study reports on the characterization in vitro of a fully phosphorothioated 20-mer oligonucleotide, complementary to p21 mRNA, radiolabeled with fluorine-18 using a thiol reactive prosthetic group. The potential of two novel synthetic block copolymers containing grafted polyamines on their hydrophobic blocks for vector-assisted cell delivery was studied in vitro. Extensive cellular uptake studies were performed in human colon carcinoma cell lines with enhanced or deficient p21 expression to evaluate and compare the uptake mechanism of naked and vectorized radiolabeled formulations. Uptake studies with the two novel biodegradable vectors showed a moderate increase in cell uptake of the radiofluorinated antisense oligonucleotide. The two vectors show, however, promising advantages over conventional lipidic vectors regarding their biocompatibility and subcellular distribution.

  1. 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.

  2. Antisense suppression of donor splice site mutations in the dystrophin gene transcript

    PubMed Central

    Fletcher, Sue; Meloni, Penny L; Johnsen, Russell D; Wong, Brenda L; Muntoni, Francesco; Wilton, Stephen D

    2013-01-01

    We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis. PMID:24498612

  3. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic.

    PubMed

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-10-11

    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.

  4. 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

  5. Chicken GHR natural antisense transcript regulates GHR mRNA in LMH cells

    PubMed Central

    An, Lilong; Ma, Jinge; Qiu, Fengfang; Jia, Rumin; Nie, Qinghua; Zhang, Dexiang; Luo, Qingbin; Li, Ting; Wang, Zhang; Zhang, Xiquan

    2016-01-01

    Growth hormone receptor (GHR) played key roles in human and animal growth. Both human laron type dwarfism and sex linked dwarf chicken were caused by the mutation of GHR gene. In this study, we identified an endogenously expressed long non-coding natural antisense transcript, GHR-AS, which overlapped with the GHR mRNA (GHR-S) in a tail to tail manner. Spatial and temporal expression analyses indicated that GHR-AS were highly expressed in chicken liver and displayed ascending with the development of chicken from E10 to 3 w of age. Interfering GHR-AS caused GHR-S decreasing, accompanied with increasing of the inactive gene indicator, H3K9me2, in the GHR-S promoter regions in LMH cells. RNase A experiment exhibited that GHR-AS and GHR-S can form double strand RNAs at the last exon of GHR gene in vivo and in vitro, which hinted they could act on each other via the region. In addition, the levels of GHR-S and GHR-AS can be affected by DNA methylation. Compared the normal chicken with the dwarfs, the negative correlation trends were showed between the GHR-S promoter methylation status and the GHR-AS levels. This is the first report of that GHR gene possessed natural antisense transcript and the results presented here further highlight the fine and complicated regulating mechanism of GHR gene in chicken development. PMID:27713155

  6. Treatment of experimental autoimmune encephalomyelitis with antisense oligonucleotides against the low affinity neurotrophin receptor.

    PubMed

    Soilu-Hänninen, M; Epa, R; Shipham, K; Butzkueven, H; Bucci, T; Barrett, G; Bartlett, P F; Kilpatrick, T J

    2000-03-15

    Upregulated expression of the low-affinity neurotrophin receptor (p75) in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) has recently been demonstrated. To investigate whether p75 plays a role in disease pathogenesis, we adopted a gene therapy approach, utilizing antisense oligonucleotides to downregulate p75 expression during EAE. Phosphorothioate antisense oligonucleotides (AS), nonsense oligonucleotides (NS) or phosphate buffered saline (PBS) were injected daily for 18 days after immunization of SJL/J (H-2s)-mice with myelin proteolipid protein (PLP) peptide 139-151. In the AS group, there was a statistically significant reduction in both the mean maximal disease score (1.85 in the AS, 2.94 in the NS and 2.75 in the PBS-groups, respectively, P < 0.025) and in the cumulative disease incidence ( approximately 60% in the AS group and approximately 90% in the control groups). Histological and immunohistochemical analysis showed reduced inflammation and demyelination, as well as reduced p75 expression at the blood-brain barrier (BBB) in the AS-treated mice in comparison with both control groups. There was no difference, however, in p75 expression on neural cells within the CNS between the three groups of mice. We conclude that p75 could play a proactive role in the pathogenesis of EAE and may exert its effect at the level of the BBB.

  7. 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.

  8. In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation.

    PubMed Central

    Noonberg, S B; Scott, G K; Garovoy, M R; Benz, C C; Hunt, C A

    1994-01-01

    Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences. Images PMID:8052538

  9. Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing.

    PubMed

    Koh, Chee Guan; Zhang, Xulang; Liu, Shujun; Golan, Sharon; Yu, Bo; Yang, Xiaojuan; Guan, Jingjiao; Jin, Yan; Talmon, Yeshayahu; Muthusamy, Natarajan; Chan, Kenneth K; Byrd, John C; Lee, Robert J; Marcucci, Guido; Lee, L James

    2010-01-04

    A multi-inlet microfluidic hydrodynamic focusing (MF) system to prepare lipopolyplex (LP) containing Bcl-2 antisense deoxyoligonucleotide (ODN) was developed and evaluated. The lipopolyplex nanoparticles consist of ODN:protamine:lipids (1:0.3:12.5wt/wt ratio) and the lipids included DC-Chol:egg PC:PEG-DSPE (40:58:2mol/mol%). Using K562 human erythroleukemia cells, which contain an abundance of Bcl-2 and overexpression of transferrin receptors (TfR), and G3139 (oblimerson sodium or Genasense(TM)) as a model cell line and drug, respectively, the Bcl-2 down-regulation at the mRNA and protein levels as well as cellular uptake and apoptosis was compared between the conventional bulk mixing (BM) method and the MF method. The lipopolyplex size and surface charge were characterized by dynamic light scattering (DLS) and zeta potential (zeta) measurement, respectively, while the ODN encapsulation efficiency was determined by gel electrophoresis. Cryogenic transmission electron microscopy (Cryo-TEM) was used to determine the morphology of LPs. Our results demonstrated that MF produced LP nanoparticles had similar structures but smaller size and size distribution compared to BM LP nanoparticles. MF LP nanoparticles had higher level of Bcl-2 antisense uptake and showed more efficient down-regulation of Bcl-2 protein level than BM LP nanoparticles.

  10. Natural antisense RNAs are involved in the regulation of CD45 expression in autoimmune diseases.

    PubMed

    Rong, J; Yin, J; Su, Z

    2015-03-01

    CD45 is a transmembrane protein tyrosine phosphatase that is specifically expressed in hematopoietic cells and can initiate signal transduction via the dephosphorylation of tyrosine. Alternatively spliced transcript variants of this gene encode distinct isoforms, which indicate different functional states of CD45. Among these variants, CD45RO, which contains neither exon 4, 5, or 6, is over-expressed in lymphocytes in autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type I diabetes. The CD45 RO serves as a marker of the immune response activity and lymphocyte development. Previous studies have indicated that exon splicing is generally correlated with local hypermethylated DNA and acetylated histone modification, while autoimmune diseases are commonly associated with global hypomethylation and histone deacetylation in lymphocytes. Thus, the question arises of how exons 4, 5, and 6 of CD45RO are excluded under the status of global DNA hypomethylation and histone deacetylation in these autoimmune diseases. On the basis of the analyses of the context sequence of CD45 and its natural antisense RNA in GenBank, we proposed that the long noncoding RNA encoded by the natural antisense gene of CD45 contributes to the expressional regulation of the CD45RO splicing variant via recruitment of DNA methyltransferase and histone modification modulators specific to the sense gene CD45; thus, it is associated with the over-expression of CD45RO and the functional regulation of lymphocytes in the pathogenic development of autoimmune diseases.

  11. 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.

  12. Selective suppression of antisense transcription by Set2-mediated H3K36 methylation

    PubMed Central

    Venkatesh, Swaminathan; Li, Hua; Gogol, Madelaine M.; Workman, Jerry L.

    2016-01-01

    Maintenance of a regular chromatin structure over the coding regions of genes occurs co-transcriptionally via the ‘chromatin resetting' pathway. One of the central players in this pathway is the histone methyltransferase Set2. Here we show that the loss of Set2 in yeast, Saccharomyces cerevisiae, results in transcription initiation of antisense RNAs embedded within body of protein-coding genes. These RNAs are distinct from the previously identified non-coding RNAs and cover 11% of the yeast genome. These RNA species have been named Set2-repressed antisense transcripts (SRATs) since the co-transcriptional addition of the H3K36 methyl mark by Set2 over their start sites results in their suppression. Interestingly, loss of chromatin resetting factor Set2 or the subsequent production of SRATs does not affect the abundance of the sense transcripts. This difference in transcriptional outcomes of overlapping transcripts due to a strand-independent addition of H3K36 methylation is a key regulatory feature of interleaved transcriptomes. PMID:27892455

  13. 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

  14. 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

  15. Retroposition as a source of antisense long non-coding RNAs with possible regulatory functions.

    PubMed

    Bryzghalov, Oleksii; Szcześniak, Michał Wojciech; Makałowska, Izabela

    2016-01-01

    Long non-coding RNAs (lncRNAs) are a class of intensely studied, yet enigmatic molecules that make up a substantial portion of the human transcriptome. In this work, we link the origins and functions of some lncRNAs to retroposition, a process resulting in the creation of intronless copies (retrocopies) of the so-called parental genes. We found 35 human retrocopies transcribed in antisense and giving rise to 58 lncRNA transcripts. These lncRNAs share sequence similarity with the corresponding parental genes but in the sense/antisense orientation, meaning they have the potential to interact with each other and to form RNA:RNA duplexes. We took a closer look at these duplexes and found that 10 of the lncRNAs might regulate parental gene expression and processing at the pre-mRNA and mRNA levels. Further analysis of the co-expression and expression correlation provided support for the existence of functional coupling between lncRNAs and their mate parental gene transcripts.

  16. 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

  17. PAAn-1b and PAAn-E: two phosphorothioate antisense oligodeoxynucleotides inhibit human aromatase gene expression.

    PubMed

    Auvray, P; Sourdaine, P; Séralini, G E

    1998-12-09

    Estrogen-dependent diseases, especially breast cancers, are frequently treated with aromatase inhibitors. Another more recent strategy is the antisense technology. In this study, after predicting aromatase mRNA secondary structure, we describe the design, the efficiency, and the toxicity of two antisense phosphorothioate oligodeoxynucleotides (PAAn-1b and PAAn-E) directed toward aromatase mRNA. Indeed, 2 microM PAAn-1b and PAAn-E encapsulated with 54 microM polyethylenimine inhibit aromatase activity by 71 and 79%, respectively, in transfected 293 cells, with IC50 values of 0.2 and 0.6 microM. The mechanism of inhibition appears to be specific after using sense and scramble oligodeoxynucleotides as controls and largely decreases aromatase mRNA and protein amounts. Moreover, PAAn-1b and PAAn-E are not cytotoxic for 293 cells. This study finally provides a new strategy for aromatase inhibition. It offers new tools for studying aromatase gene expression and its role in cancer for instance, and this could be of help for the therapy of estrogen-dependent diseases.

  18. Selective suppression of antisense transcription by Set2-mediated H3K36 methylation.

    PubMed

    Venkatesh, Swaminathan; Li, Hua; Gogol, Madelaine M; Workman, Jerry L

    2016-11-28

    Maintenance of a regular chromatin structure over the coding regions of genes occurs co-transcriptionally via the 'chromatin resetting' pathway. One of the central players in this pathway is the histone methyltransferase Set2. Here we show that the loss of Set2 in yeast, Saccharomyces cerevisiae, results in transcription initiation of antisense RNAs embedded within body of protein-coding genes. These RNAs are distinct from the previously identified non-coding RNAs and cover 11% of the yeast genome. These RNA species have been named Set2-repressed antisense transcripts (SRATs) since the co-transcriptional addition of the H3K36 methyl mark by Set2 over their start sites results in their suppression. Interestingly, loss of chromatin resetting factor Set2 or the subsequent production of SRATs does not affect the abundance of the sense transcripts. This difference in transcriptional outcomes of overlapping transcripts due to a strand-independent addition of H3K36 methylation is a key regulatory feature of interleaved transcriptomes.

  19. 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.

  20. Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

    PubMed

    Masters, Thao L; Wachter, Jenny; Hill, Stuart A

    2016-11-01

    Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

  1. Genome-wide analysis of expression modes and DNA methylation status at sense-antisense transcript loci in mouse.

    PubMed

    Watanabe, Yutaka; Numata, Koji; Murata, Shinya; Osada, Yuko; Saito, Rintaro; Nakaoka, Hajime; Yamamoto, Naoyuki; Watanabe, Kazufumi; Kato, Hidemasa; Abe, Kuniya; Kiyosawa, Hidenori

    2010-12-01

    The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.

  2. [Connection of magnetic antisense probe with SK-Br-3 oncocyte mRNA nucleotide detected by high resolution atomic force microscope].

    PubMed

    Tan, Shude; Ouyang, Yu; Li, Xinyou; Wen, Ming; Li, Shaolin

    2011-06-01

    The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.

  3. Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene-Silencing Strategy to Study Gene Functions in Rice

    PubMed Central

    Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-01-01

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice. PMID:21408609

  4. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    PubMed

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  5. Targeted Knockdown of Hepatic SOAT2 with Antisense Oligonucleotides Stabilizes Atherosclerotic Plaque in ApoB100-only LDLr−/− Mice

    PubMed Central

    Melchior, John T.; Olson, John D.; Kelley, Kathryn L.; Wilson, Martha D.; Sawyer, Janet K.; Link, Kerry M.; Rudel, Lawrence L.

    2015-01-01

    Objective To test the hypothesis that the attenuation of CO packaging into apoB-containing lipoproteins will arrest progression of pre-existing atherosclerotic lesions. Approach and Results Atherosclerosis was induced in apoB-100 only, LDLr−/− mice by feeding a diet enriched in cis-monounsaturated fatty acids (cis-MUFAs) for 24 weeks. A subset of mice was then sacrificed to quantify the extent of atherosclerosis. The remaining mice were continued on the same diet (controls) or assigned to the following treatments for 16 weeks: (1) a diet enriched in n-3 polyunsaturated fatty acids, (2) the cis-MUFA diet plus bi-weekly injections of an antisense oligonucleotide (ASO) specific to hepatic SOAT2; or (3) the cis-MUFA diet and bi-weekly injections of a non-targeting hepatic ASO. Extent of atherosclerotic lesions in the aorta was monitored morphometrically in vivo with magnetic resonance imaging (MRI) and ex vivo histologically and immunochemically. Hepatic knockdown of SOAT2 via ASO treatment arrested lesion growth and stabilized lesions. Conclusions Hepatic knockdown of SOAT2 in apoB100-only, LDLr−/− mice resulted in remodeling of aortic atherosclerotic lesions into a stable phenotype, suggesting SOAT2 is a viable target for treatment of atherosclerosis. PMID:26229140

  6. C-type starch from high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme.

    PubMed

    Wei, Cunxu; Xu, Bin; Qin, Fengling; Yu, Huaguang; Chen, Chong; Meng, Xianglen; Zhu, Lijia; Wang, Youping; Gu, Minghong; Liu, Qiaoquan

    2010-06-23

    High-amylose starch is a source of resistant starch (RS) which has a great benefit on human health. A transgenic rice line (TRS) enriched amylose and RS had been developed by antisense RNA inhibition of starch branching enzymes. In this study, the native starch granules were isolated from TRS grains as well as the wild type, and their crystalline type was carefully investigated before and after acid hydrolysis. In high-amylose TRS rice, the C-type starch, which might result from the combination of both A-type and B-type starch, was observed and subsequently confirmed by multiple physical techniques, including X-ray powder diffraction, solid-state nuclear magnetic resonance, and Fourier transform infrared. Moreover, the change of starch crystalline structure from C- to B-type during acid hydrolysis was also observed in this RS-rich rice. These data could add to our understanding of not only the polymorph structure of cereal starch but also why high-amylose starch is more resistant to digestion.

  7. Antisense RNA modulation of alkyl hydroperoxide reductase levels in Helicobacter pylori correlates with organic peroxide toxicity but not infectivity.

    PubMed

    Croxen, Matthew A; Ernst, Peter B; Hoffman, Paul S

    2007-05-01

    Much of the gene content of the human gastric pathogen Helicobacter pylori ( approximately 1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an approximately 72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

  8. BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity.

    PubMed

    Zheng, Lu; Liang, Ping; Zhou, JianBo; Huang, XiaoBing; Wen, Yu; Wang, Zheng; Li, Jing

    2012-02-01

    The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG(2) cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG(2) cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG(2) cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G(1) phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

  9. Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication.

    PubMed

    Lavigne, C; Yelle, J; Sauvé, G; Thierry, A G

    2001-01-01

    We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the "cocktail" might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.

  10. Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increases turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

    PubMed Central

    Igarashi, Miki; Kim, Hyung-Wook; Gao, Fei; Chang, Lisa; Ma, Kaizong; Rapoport, Stanley I.

    2012-01-01

    Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15 weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-14C]DPAn-6 was infused intravenously for 5 min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2–7.7 fold, while turnover rates were increased 2.1–4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. PMID:22142872

  11. 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.

  12. Antisense Oligonucleotides: Rising Stars in Eliminating RNA Toxicity in Myotonic Dystrophy

    PubMed Central

    Gao, Zhihua

    2013-01-01

    Abstract Myotonic dystrophy (DM) is a dominantly inherited, multisystemic disease caused by expanded CTG (type 1, DM1) or CCTG (type 2, DM2) repeats in untranslated regions of the mutated genes. Pathogenesis results from expression of RNAs from the mutated alleles that are toxic because of the expanded CUG or CCUG repeats. Increased understanding of the repeat-containing RNA (C/CUGexp RNA)-induced toxicity has led to the development of multiple strategies targeting the toxic RNA. Among these approaches, antisense oligonucleotides (ASOs) have demonstrated high potency in reversing the RNA toxicity in both cultured DM1 cells and DM1 animal models, thus offering great promise for the potential treatment of DM1. ASO targeting approaches will also provide avenues for the treatment of other repeat RNA-mediated diseases. PMID:23252746

  13. Assessment of biofilm formation in Pseudomonas aeruginosa by antisense mazE-PNA.

    PubMed

    Valadbeigi, Hassan; Sadeghifard, Nourkhoda; Salehi, Majid Baseri

    2017-03-01

    The hallmark patogenicity in Pseudomonas aeruginosa (P. aeruginosa) is biofilm formation that is not easy to eradicate, because it has variety mechanisms for antibiotic resistance. In addition, toxin-antitoxin (TA) system may play role in biofilm formation. The current study aimed to evaluate the role of TA loci in biofilm formation. Therefore, 18 P. aeruginosa clinical isolates were collected and evaluated for specific biofilm and TA genes. The analysis by RT-qPCR demonstrated that expression of mazE antitoxin in biofilm formation was increase. On the other hand, mazE antitoxin TA system was used as target for antisense PNA. mazE-PNA was able to influence in biofilm formation and was inhibit at 5,10 and 15 μM concentrations biofilm formation in P. aeruginosa. Therefore, it could be highlighted target for anti-biofilm target to eradicate P. aeruginosa biofilm producer.

  14. Engineering Resistance Against Mungbean yellow mosaic India virus Using Antisense RNA.

    PubMed

    Haq, Q M I; Ali, Arif; Malathi, V G

    2010-06-01

    Yellow mosaic disease of cultivated legumes in South-East Asia, is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Efforts to engineer resistance against the genus Begomovirus are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a complementary-sense gene (ACI) encoding Replication initiation Protein (Rep) to develop resistance against soybean isolate of Mungbean yellow mosaic India virus-[India:New Delhi:Soybean 2:1999], a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that the legume host plants co-agroinoculated with infectious constructs of soybean isolate of Mungbean yellow mosaic India virus [India:New Delhi:Soybean 2:1999] along with this antisense Rep gene construct show resistance to the virus.

  15. Discovery and antibacterial activity of glabramycin A-C from Neosartorya glabra by an antisense strategy.

    PubMed

    Jayasuriya, Hiranthi; Zink, Deborah; Basilio, Angela; Vicente, Francisca; Collado, Javier; Bills, Gerald; Goldman, Mary Lee; Motyl, Mary; Huber, Joann; Dezeny, Gabe; Byrne, Kevin; Singh, Sheo B

    2009-05-01

    Treatment of drug-resistant bacteria is a significant unmet medical need. This challenge can be met only by the discovery and development of new antibiotics. Antisense technology is one of the newest discovery tools that provides enhanced sensitivity for detection of antibacterials, and has led to the discovery of a number of interesting new antibacterial natural products. Continued utilization of this technology led to the discovery of three new bicyclic lactones, glabramycins A-C, from a Neosartorya glabra strain. Glabramycin C showed strong antibiotic activity against Streptococcus pneumoniae (MIC 2 microg ml(-1)) and modest antibiotic activity against Staphylococcus aureus (MIC 16 microg ml(-1)). The isolation, structure, relative configuration and antibacterial activity, and plausible biogenesis of these compounds have been discussed.

  16. 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.

  17. Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis.

    PubMed

    Okada, Kazunori; Kawaide, Hiroshi; Kuzuyama, Tomohisa; Seto, Haruo; Curtis, Ian S; Kamiya, Yuji

    2002-06-01

    Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.

  18. Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture[W][OA

    PubMed Central

    Araújo, Wagner L.; Nunes-Nesi, Adriano; Osorio, Sonia; Usadel, Björn; Fuentes, Daniela; Nagy, Réka; Balbo, Ilse; Lehmann, Martin; Studart-Witkowski, Claudia; Tohge, Takayuki; Martinoia, Enrico; Jordana, Xavier; DaMatta, Fábio M.; Fernie, Alisdair R.

    2011-01-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell–specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture. PMID:21307286

  19. Murine transcription factor alpha A-crystallin binding protein I. Complete sequence, gene structure, expression, and functional inhibition via antisense RNA.

    PubMed

    Brady, J P; Kantorow, M; Sax, C M; Donovan, D M; Piatigorsky, J

    1995-01-20

    alpha A-crystallin binding protein I (alpha A-CRYBP1) is a ubiquitously expressed DNA binding protein that was previously identified by its ability to interact with a functionally important sequence in the mouse alpha A-crystallin gene promoter. Here, we have cloned a single copy gene with 10 exons spanning greater than 70 kb of genomic DNA that encodes alpha A-CRYBP1. The mouse alpha A-CRYBP1 gene specifies a 2,688-amino acid protein with 72% amino acid identity to its human homologue, PRDII-BF1. Both the human and the mouse proteins contain two sets of consensus C2H2 zinc fingers at each end as well a central nonconsensus zinc finger. The alpha A-CRYBP1 gene produces a 9.5-kb transcript in 11 different tissues as well as a testis-specific, 7.7-kb transcript. alpha A-CRYBP1 cDNA clones were isolated from adult mouse brain and testis as well as from cell lines derived from mouse lens (alpha TN4-1) and muscle (C2C12). A single clone isolated from the muscle C2C12 library contains an additional exon near the 5'-end that would prevent production of a functional protein if the normal translation start site were utilized; however, there is another potential initiation codon located downstream that is in frame with the rest of the coding region. In addition, we identified multiple cDNAs from the testis in which the final intron is still present. Finally, we used an antisense expression construct derived from an alpha A-CRYBP1 cDNA clone to provide the first functional evidence that alpha A-CRYBP1 regulates gene expression. When introduced into the alpha TN4-1 mouse lens cell line, the antisense construct significantly inhibited expression from a heterologous promoter that utilized the alpha A-CRYBP1 binding site as an enhancer.

  20. 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.

  1. 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.

  2. 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

  3. Inhibition of keratin 17 expression with antisense and RNAi strategies: exploring novel therapy for psoriasis.

    PubMed

    Chang, Ting; Sun, Linchao; Wang, Yan; Wang, Datai; Li, Wei; Li, Chunying; Gao, Tianwen; Liu, Yufeng; Wang, Gang

    2011-07-01

    Psoriasis is now considered to be a chronic, immune-mediated and inflammatory skin disease. As the precise cause of psoriasis remains unknown, its treatment is challenging for dermatologists. Keratin 17 (K17), an intermediate filament protein, is highly expressed in psoriatic lesions, while not normally expressed in healthy epidermis. Studies have suggested that K17 plays a role in the pathogenesis of psoriasis. However, no study has been performed to determine the potential application of K17 down-regulation as a treatment option for psoriatic lesions. We hypothesized that anti-K17 interference may suppress the development and progression of psoriasis and potentially serve as a novel strategy for the treatment of psoriasis. Therefore, we down-regulated and silenced K17 gene expression in keratinocytes (KCs) using antisense and RNA interference (RNAi) techniques. We found that K17-specific antisense oligonucleotides (ASODN) or siRNAs inhibited proliferation and induced apoptosis in KCs as well as down-regulated K17 expression at both mRNA and protein levels. For our in vivo study, we constructed the SCID-hu xenogeneic transplantation psoriasis mouse model by grafting psoriatic lesions onto SCID mice and topically applied K17-specific ASODN and liposome-encapsulated siRNA to the grafts. We observed morphological and histological improvement in the treated psoriatic grafts. As a result, K17 mRNA and protein expression was significantly decreased in the grafts of the mouse model. Taken together, we conclude that anti-K17 therapy is an effective treatment option for psoriasis, and the K17 molecule, as a new target, may hold tremendous potential for the treatment of psoriasis in the future.

  4. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.

  5. Therapeutic potentialities of EWS-Fli-1 mRNA-targeted vectorized antisense oligonucleotides.

    PubMed

    Maksimenko, A; Lambert, G; Bertrand, J R; Fattal, E; Couvreur, P; Malvy, C

    2003-12-01

    We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli-1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS-Fli-1 cDNA under the control of the LTR retroviral promoter--which do not undergo apoptosis and which became tumoral--were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS-Fli-1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS-Fli-1. Both types of AON-loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS-Fli-1 was specifically down-regulated, confirming the antisense activity of the targeted AON.

  6. Circadian Rhythms of Sense and Antisense Transcription in Sugarcane, a Highly Polyploid Crop

    PubMed Central

    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. PMID:23936527

  7. Granule structure and distribution of allomorphs in C-type high-amylose rice starch granule modified by antisense RNA inhibition of starch branching enzyme.

    PubMed

    Wei, Cunxu; Qin, Fengling; Zhou, Weidong; Yu, Huaguang; Xu, Bin; Chen, Chong; Zhu, Lijia; Wang, Youping; Gu, Minghong; Liu, Qiaoquan

    2010-11-24

    C-type starch, which is a combination of both A-type and B-type crystal starch, is usually found in legumes and rhizomes. We have developed a high-amylose transgenic line of rice (TRS) by antisense RNA inhibition of starch branching enzymes. The starch in the endosperm of this TRS was identified as typical C-type crystalline starch, but its fine granular structure and allomorph distribution remained unclear. In this study, we conducted morphological and spectroscopic studies on this TRS starch during acid hydrolysis to determine the distribution of A- and B-type allomorphs. The morphology of starch granules after various durations of acid hydrolysis was compared by optical microscopy, scanning electron microscopy, and transmission electron microscopy. The results showed that amorphous regions were located at the center part of TRS starch subgranules. During acid hydrolysis, starch was degraded from the interior of the subgranule to the outer surface, while the peripheral part of the subgranules and the surrounding band of the starch granule were highly resistant to acid hydrolysis. The spectroscopic changes detected by X-ray powder diffraction, 13C cross-polarization magic-angle spinning NMR, and attenuated total reflectance Fourier transform infrared showed that the A-type allomorph was hydrolyzed more rapidly than the B-type, and that the X-ray diffraction profile gradually changed from a native C-type to a CB-type with increasing hydrolysis time. Our results showed that, in TRS starch, the A-type allomorph was located around the amorphous region, and was surrounded by the B-type allomorph located in the peripheral region of the subgranules and the surrounding band of the starch granule. Thus, the positions of A- and B-type allomorphs in the TRS C-type starch granule differ markedly from those in C-type legume and rhizome starch.

  8. Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer ▿

    PubMed Central

    Mitev, Georgi M.; Mellbye, Brett L.; Iversen, Patrick L.; Geller, Bruce L.

    2009-01-01

    Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3′ end of each is attached the membrane-penetrating peptide (RXR)4XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and β-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3+Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3+Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 μM (7 μg/ml), 0.156 μM (0.94 μg/ml), and >160 μM (>900 μg/ml), respectively. 3+Pip-AcpP PPMO at 1.25 or 2.5 μM significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3+Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 μM 3+Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3+Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3+Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3+Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages. PMID:19581453

  9. 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

  10. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

    PubMed Central

    Landschütze, V; Willmitzer, L; Müller-Röber, B

    1995-01-01

    The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistinguishable from wild-type plants in the greenhouse during vegetative growth. A major change, however, was seen upon initiation of the generative phase (flower formation). In the case of transgenic plants with a strong reduction in citrate synthase activity (< 30% of wild-type levels), flower buds formed > 2 weeks later as compared with wild-type plants. Furthermore, flower buds from these plants did not develop into mature flowers but rather were aborted at an early stage of development. Microscopic analysis showed that in these cases ovaries disintegrated during flower development. We conclude that the TCA cycle is of major importance during the transition from the vegetative to the generative phase. Images PMID:7882969

  11. 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.

  12. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    PubMed

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy.

  13. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  14. [The creation of transgenic tobacco plants expressing fragments of the ARGOS and NtEXPA4 genes in antisense orientation].

    PubMed

    Kuluev, B R; Kniazev, A V; Postrigan', B N; Chemeris, A V

    2014-01-01

    Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stalk sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.

  15. Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration.

    PubMed Central

    Biro, S; Fu, Y M; Yu, Z X; Epstein, S E

    1993-01-01

    Smooth muscle cell (SMC) proliferation and migration play pivotal roles in restenosis following angioplasty. c-myc is an immediate early response gene induced by various mitogens, and several lines of evidence derived from experiments using transformed or hematopoietic cell lines, or transgenic mice, suggest its protein product plays a role in numerous signaling transduction pathways, including those modulating cell division. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODNs) complementary to c-myc mRNA (antisense ODNs) might be potent inhibitors of SMC proliferation and, perhaps, of SMC migration. To evaluate this concept, we tested several antisense ODNs targeted to c-myc mRNA (15- or 18-mer ODNs complementary to different c-myc mRNA sequences) by introducing them individually into the medium of cultured rat aortic SMCs. Phosphoroamidate-modified ODNs were employed to retard degradation. Antisense ODNs inhibited, in a concentration-dependent manner, SMC proliferation and SMC migration. Maximal inhibitory effect was 50% for proliferation and > 90% for migration. These effects were associated with decreased SMC expression of c-myc-encoded protein by Western immunoblotting and immunocytochemical staining. ODNs with the same nucleotides but a scrambled sequence caused no effect. These results indicate that the c-myc gene product is involved in the signal transduction pathways mediating SMC proliferation and migration in the in vitro model we employed. The results also suggest a potential role of antisense strategies designed to inhibit c-myc expression for the prevention of coronary restenosis. Images PMID:8421701

  16. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

    PubMed

    Koty, P P; Zhang, H; Levitt, M L

    1999-02-01

    Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC.

  17. Effects of salt, polyethylene glycol, and locked nucleic acids on the thermodynamic stabilities of consecutive terminal adenosine mismatches in RNA duplexes.

    PubMed

    Gu, Xiaobo; Nguyen, Mai-Thao; Overacre, Abigail; Seaton, Samantha; Schroeder, Susan J

    2013-04-04

    Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

  18. 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

  19. 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

  20. Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene

    PubMed Central

    Kralovicova, Jana; Moreno, Pedro M.D.; Cross, Nicholas C.P.; Pêgo, Ana Paula

    2016-01-01

    ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs. PMID:27658045

  1. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

    PubMed

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O; Burzio, Verónica A

    2016-09-06

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

  2. Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

    PubMed

    Bajpai, Arun K; Park, Jeong-Hoh; Moon, Ik-Jae; Kang, Hyungu; Lee, Yun-Han; Doh, Kyung-Oh; Suh, Seong-Il; Chang, Byeong-Churl; Park, Jong-Gu

    2005-09-29

    Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

  3. Novel antisense therapeutics delivery systems: In vitro and in vivo studies of liposomes targeted with anti-CD20 antibody.

    PubMed

    Meissner, Justyna M; Toporkiewicz, Monika; Czogalla, Aleksander; Matusewicz, Lucyna; Kuliczkowski, Kazimierz; Sikorski, Aleksander F

    2015-12-28

    Antisense gene therapy using molecules such as antisense oligodeoxynucleotides, siRNA or miRNA is a very promising strategy for the treatment of neoplastic diseases. It can be combined with other treatment strategies to enhance therapeutic effect. In acute leukemias, overexpression of the antiapoptotic gene BCL2 is observed in more than 70% of cases. Therefore, reduction of the Bcl-2 protein level could, in itself, prevent the development of cancer or could possibly help sensitize cancer cells to apoptosis inducers. The main objective of our work is to develop therapeutic liposome formulations characterized by high transfection efficiency, stability in the presence of serum, as well as specificity and toxicity for target (leukemic) cells. Each of our liposomal formulations consists of a core composed of antisense oligonucleotides complexed by either cationic lipid, DOTAP, or a synthetic polycation, polyethyleneimine, encapsulated within liposomes modified with polyethylenoglycol. In addition, the liposomal shells are enriched with covalently-bound antibodies recognizing a well characterized bio-marker, CD20, exposed on the surface of leukemia cells. The resulting immunoliposomes selectively and effectively reduced the expression of BCL2 in target cells. Model animal experiments carried out on mice-engrafted tumors expressing the specific marker showed high efficiency of the liposome formulations against specific tumor development. In conclusion, we show that lipid formulations based on a polyplex or lipoplex backbone additionally equipped with antibodies are promising non-viral vectors for specific oligonucleotide transfer into human tumor cells.

  4. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors

    PubMed Central

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O.; Burzio, Verónica A.

    2016-01-01

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy. PMID:27507060

  5. 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.

  6. Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides.

    PubMed

    Gong, Qiuming; Stump, Matthew R; Zhou, Zhengfeng

    2014-11-01

    The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study, we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of Kv11.1a mRNA, protein and channel current. More importantly, blocking these elements by antisense morpholino oligonucleotides shifted the alternative processing of KCNH2 intron 9 from the polyadenylation to the splicing pathway, leading to the predominant production of Kv11.1a and a significant increase in Kv11.1 current. Our findings indicate that the expression of the Kv11.1a isoform can be upregulated by an antisense approach. Antisense inhibition of KCNH2 intronic polyadenylation represents a novel approach to increase Kv11.1 channel function.

  7. Antisense Morpholino-Oligomers Directed against the 5′ End of the Genome Inhibit Coronavirus Proliferation and Growth†

    PubMed Central

    Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Paulino, Amy D.; Moulton, Hong M.; Iversen, Patrick L.; Buchmeier, Michael J.

    2004-01-01

    Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection. PMID:15140987

  8. Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

    PubMed Central

    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. PMID:21829656

  9. Conjugated linoleic acid (CLA) and fatty acid composition of milk, curd and Grana Padano cheese in conventional and organic farming systems.

    PubMed

    Prandini, Aldo; Sigolo, Samantha; Piva, Gianfranco

    2009-08-01

    CLA levels and fatty acid composition were measured to compare the fat composition in organic bulk milk, destined to the production of Grana Padano cheese, with those produced by conventional system. The curds and Grana Padano cheeses were also analysed to evaluate the effects of the production technology on the CLA content. All analysed organic samples were characterized by higher annual means of CLA, vaccenic acid (TVA) and linolenic acid (LNA) in comparison with conventional samples (with P<0.05). Nevertheless, no particular effect of the production technology was seen on the CLA content. The animal diet appears to be the factor which has the highest effect on the CLA concentration in milk and milk products and an organic diet based on fresh or dried forage, that is rich in CLA precursory fatty acids, may improve the yield of fatty acids with beneficial effects on health.

  10. Spray-dried milk supplemented with alpha-linolenic acid or eicosapentaenoic acid and docosahexaenoic acid decreases HMG Co A reductase activity and increases biliary secretion of lipids in rats.

    PubMed

    Ramaprasad, Talahalli R; Srinivasan, Krishnapura; Baskaran, Vallikannan; Sambaiah, Kari; Lokesh, Belur R

    2006-05-01

    In our earlier study, we have shown that rats fed spray-dried milk containing alpha-linolenic acid (LNA 18:3 n-3) or eicosapentaenoic acid (EPA 20:5 n-3) and docosahexaenoic acid (DHA 22:6 n-3) had significantly lower amounts of serum and liver cholesterol. To evaluate the mechanism for hypocholesterolemic effect of n-3 fatty acids containing milk formulation, we fed male Wistar rats with spray-dried milk containing linseed oil (LSO) (source of LNA) or fish oil (FO) (source of EPA+DHA) for 8 weeks. Feeding n-3 fatty acid containing milk formulation lowered the hepatic 3-hydroxy-methylglutaryl coenzyme A (HMG Co A) activity by 17-22% compared to rats given control diet devoid of n-3 fatty acids. The cholesterol level in liver microsomes was found to be decreased by 16% and 20%, respectively, in LSO and FO containing formulation fed rats. The bile flow was enhanced to an extent of 19-23% in experimental groups compared to control animals. The biliary cholesterol and phospholipid secretion was increased to an extent of 49-55% and 140-146%, respectively, in rats fed n-3 fatty acid containing formulation. The increase in the total bile acids secretion in bile was mainly reflected on an increase in the levels of taurine conjugated bile acids. These results indicated that n-3 fatty acid containing spray-dried milk formulation would bring about the hypocholesterolemic effect by lowering HMG Co A reductase activity in liver and by increasing the secretion of bile constituents.

  11. Defining Synphenotype Groups in Xenopus tropicalis by Use of Antisense Morpholino Oligonucleotides

    PubMed Central

    Rana, Amer Ahmed; Collart, Clara; Gilchrist, Michael J; Smith, J. C

    2006-01-01

    To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis. PMID:17112317

  12. siRNA-mediated heterochromatin establishment requires HP1 and is associated with antisense transcription

    PubMed Central

    Iida, Tetsushi; Nakayama, Jun-ichi; Moazed, Danesh

    2008-01-01

    Summary Heterochromatic gene silencing at the pericentromeric DNA repeats in fission yeast requires the RNA interference (RNAi) machinery. The RNA-Induced Transcriptional Silencing (RITS) complex mediates histone H3 lysine 9 (H3K9) methylation and recruits the RNA-Dependent RNA Polymerase Complex (RDRC) to promote double-strand RNA (dsRNA) synthesis and siRNA generation. Here we show that ectopic expression of a long hairpin RNA bypasses the requirement for chromatin-dependent steps in siRNA generation. The ability of hairpin-produced siRNAs to silence homologous sequences in trans is subject to local chromatin structure, requires HP1, and correlates with antisense transcription at the target locus. Furthermore, although hairpin siRNAs can be produced in the absence of RDRC, trans-silencing of reporter genes by hairpin-produced siRNAs is completely dependent on the dsRNA synthesis activity of RDRC. These results provide new insights into the regulation of siRNA action and reveal roles for cis-dsRNA synthesis and HP1 in siRNA-mediated heterochromatin assembly. PMID:18657501

  13. Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

    PubMed Central

    Bonifert, Tobias; Gonzalez Menendez, Irene; Battke, Florian; Theurer, Yvonne; Synofzik, Matthis; Schöls, Ludger; Wissinger, Bernd

    2016-01-01

    Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2′O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (~55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (~16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ~3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs. PMID:27874857

  14. 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

  15. Identification of three novel antisense RNAs in the fur locus from unicellular cyanobacteria.

    PubMed

    Sevilla, Emma; Martín-Luna, Beatriz; González, Andrés; Gonzalo-Asensio, Jesús A; Peleato, María Luisa; Fillat, María F

    2011-12-01

    The interplay between Fur (ferric uptake regulator) proteins and small, non-coding RNAs has been described as a key regulatory loop in several bacteria. In the filamentous cyanobacterium Anabaena sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and an α-furA RNA is involved in the modulation of the global regulator FurA. In this work we report the existence of three novel antisense RNAs in cyanobacteria and show that a cis α-furA RNA is conserved in very different genomic contexts, namely in the unicellular cyanobacteria Microcystis aeruginosa PCC 7806 and Synechocystis sp. PCC 6803. Syα-fur RNA covers only part of the coding sequence of the fur orthologue sll0567, whose flanking genes encode two hypothetical proteins. Transcriptional analysis of fur and its adjacent genes in Microcystis unravels a highly compact organization of this locus involving overlapping transcripts. Maα-fur RNA spans the whole Mafur CDS and part of the flanking dnaJ and sufE sequences. In addition, Mafur seems to be part of a dicistronic operon encoding this regulator and an α-sufE RNA. These results allow new insights into the transcriptomes of two unicellular cyanobacteria and suggest that in M. aeruginosa PCC 7806, the α-fur and α-sufE RNAs might participate in a regulatory connection between the genes of the dnaJ-fur-sufE locus.

  16. 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.

  17. Trans-splicing improvement by the combined application of antisense strategies.

    PubMed

    Koller, Ulrich; Hainzl, Stefan; Kocher, Thomas; Hüttner, Clemens; Klausegger, Alfred; Gruber, Christina; Mayr, Elisabeth; Wally, Verena; Bauer, Johann W; Murauer, Eva M

    2015-01-06

    Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  18. 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

  19. 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.

  20. 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.

  1. Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.

    PubMed

    Lentz, Jennifer J; Jodelka, Francine M; Hinrich, Anthony J; McCaffrey, Kate E; Farris, Hamilton E; Spalitta, Matthew J; Bazan, Nicolas G; Duelli, Dominik M; Rigo, Frank; Hastings, Michelle L

    2013-03-01

    Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in approximately 1 in 1,000 newborns. Hereditary deafness is often mediated by the improper development or degeneration of cochlear hair cells. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre-mRNA splicing of transcripts from the USH1C gene with the c.216G>A mutation, which causes human Usher syndrome, the leading genetic cause of combined deafness and blindness. Treatment of neonatal mice with a single systemic dose of ASO partially corrects Ush1c c.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and low-frequency hearing in mice. These effects were sustained for several months, providing evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression and demonstrating the therapeutic potential of ASOs in the treatment of deafness.

  2. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models.

    PubMed

    Osman, Erkan Y; Miller, Madeline R; Robbins, Kate L; Lombardi, Abby M; Atkinson, Arleigh K; Brehm, Amanda J; Lorson, Christian L

    2014-09-15

    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 (E1(MO)-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, E1(MO)-ASO treatment in an intermediate SMA mouse (SMN(RT) 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.

  3. PlantNATsDB: a comprehensive database of plant natural antisense transcripts

    PubMed Central

    Chen, Dijun; Yuan, Chunhui; Zhang, Jian; Zhang, Zhao; Bai, Lin; Meng, Yijun; Chen, Ling-Ling; Chen, Ming

    2012-01-01

    Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a ‘Gene Set Analysis’ module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/. PMID:22058132

  4. Antisense oligonucleotides delivered to the amniotic cavity in utero modulate gene expression in the postnatal mouse

    PubMed Central

    Depreux, Frederic F.; Wang, Lingyan; Jiang, Han; Jodelka, Francine M.; Rosencrans, Robert F.; Rigo, Frank; Lentz, Jennifer J.; Brigande, John V.; Hastings, Michelle L.

    2016-01-01

    Congenital diseases account for a large portion of pediatric illness. Prenatal screening and diagnosis permit early detection of many genetic diseases. Fetal therapeutic strategies to manage disease processes in utero represent a powerful new approach for clinical care. A safe and effective fetal pharmacotherapy designed to modulate gene expression ideally would avoid direct mechanical engagement of the fetus and present an external reservoir of drug. The amniotic cavity surrounding the fetus could serve as an ideal drug reservoir. Antisense oligonucleotides (ASOs) are an established tool for the therapeutic modulation of gene expression. We hypothesize that ASOs administered to the amniotic cavity will gain entry to the fetus and modulate gene expression. Here, we show that an ASO targeting MALAT1 RNA, delivered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13-13.5, reduces target RNA expression for up to 4 weeks after birth. A similarly delivered ASO targeting a causal splice site mutation for Usher syndrome corrects gene expression in the inner ear, a therapeutically relevant target tissue. We conclude that intra-amniotic delivery of ASOs is well tolerated and produces a sustained effect on postnatal gene expression. Transuterine delivery of ASOs is an innovative platform for developing fetal therapeutics to efficaciously treat congenital disease. PMID:27683224

  5. Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs

    PubMed Central

    Behrens, Sebastian; Widder, Stefanie; Mannala, Gopala Krishna; Qing, Xiaoxing; Madhugiri, Ramakanth; Kefer, Nathalie; Mraheil, Mobarak Abu; Rattei, Thomas; Hain, Torsten

    2014-01-01

    Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40–150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes. PMID:24498259

  6. 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.

  7. Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization

    PubMed Central

    Csorba, Tibor; Questa, Julia I.; Sun, Qianwen; Dean, Caroline

    2014-01-01

    Long noncoding RNAs (lncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for lncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation. PMID:25349421

  8. In vivo correction of a Menkes disease model using antisense oligonucleotides.

    PubMed

    Madsen, Erik C; Morcos, Paul A; Mendelsohn, Bryce A; Gitlin, Jonathan D

    2008-03-11

    Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish orthologue of the ATP7A gene. Embryonic-recessive lethal mutants have impaired copper homeostasis that results in absent melanin pigmentation, impaired notochord formation, and hindbrain neurodegeneration. In this current study, we have attempted to rescue these striking phenotypic alterations by using a series of antisense morpholino oligonucleotides directed against the splice-site junctions of two mutant calamity alleles. Our findings reveal a robust and complete correction of the copper-deficient defects of calamity in association with the generation of the WT Menkes protein in all rescued mutants. Interestingly, a quantitative analysis of atp7a-specific transcripts suggests that competitive translational regulation may account for the synthesis of WT protein in these embryos. This in vivo correction of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the potential for zebrafish models of human genetic disease in the development of treatments based on the principles of interactions of synthetic oligonucleotide analogues with mRNA.

  9. The Effects of Aerosolized STAT1 Antisense Oligodeoxynucleotides on Rat Pulmonary Fibrosis

    PubMed Central

    Wang, Wenjun; Liao, Bin; Zeng, Ming; Zhu, Chen; Fan, Xianming

    2009-01-01

    Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages (AMs) and decreased the concentrations of TGF-β, PDGF and TNF-α in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are needed to determine whether there is an effect from administration of STAT1 ASON on fibrosis. This study investigated the effect of aerosolized STAT1 ASON on the expressions of inflammatory mediators, hydroxyproline and type I and type III collagen mRNA in BLM-induced rat pulmonary fibrosis. The results showed that STAT1 ASON applied by aerosolization could ameliorate alveolitis and fibrosis, inhibit the expressions of inflammatory mediators, decrease the content of hydroxyproline, and suppress the expressions of type I and type III collagen mRNA in lung tissue in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis. PMID:19254480

  10. 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.

  11. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    NASA Astrophysics Data System (ADS)

    Zhao, Ying-Zheng; Lu, Cui-Tao

    2008-03-01

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C3F8, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C3F8 microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C3F8-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  12. Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.

    PubMed

    van Deutekom, J C; Bremmer-Bout, M; Janson, A A; Ginjaar, I B; Baas, F; den Dunnen, J T; van Ommen, G J

    2001-07-15

    Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

  13. 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

  14. 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.

  15. Transcription termination within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA.

    PubMed

    Stork, Michiel; Di Lorenzo, Manuela; Welch, Timothy J; Crosa, Jorge H

    2007-05-01

    The iron transport-biosynthesis (ITB) operon in Vibrio anguillarum includes four genes for ferric siderophore transport, fatD, -C, -B, and -A, and two genes for siderophore biosynthesis, angR and angT. This cluster plays an important role in the virulence mechanisms of this bacterium. Despite being part of the same polycistronic mRNA, the relative levels of transcription for the fat portion and for the whole ITB message differ profoundly, the levels of the fat transcript being about 17-fold higher. Using S1 nuclease mapping, lacZ transcriptional fusions, and in vitro studies, we were able to show that the differential gene expression within the ITB operon is due to termination of transcription between the fatA and angR genes, although a few transcripts proceeded beyond the termination site to the end of this operon. This termination process requires a 427-nucleotide antisense RNA that spans the intergenic region and acts as a novel transcriptional terminator.

  16. Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves.

    PubMed

    Cheng, Lailiang; Zhou, Rui; Reidel, Edwin J; Sharkey, Thomas D; Dandekar, Abhaya M

    2005-03-01

    Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, 'Greensleeves' apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15-30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. (14)CO(2) pulse-chase labeling at ambient CO(2) demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO(2) assimilation in response to photon flux density or intercellular CO(2) concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis

  17. Functional tissue engineering for ligament healing: potential of antisense gene therapy.

    PubMed

    Woo, Savio L Y; Jia, Fengyan; Zou, Li; Gabriel, Mary T

    2004-03-01

    histomorphological properties of ligaments have been investigated in recent years. These therapeutic strategies include growth factor stimulation (Conti, N. A., and L. E. Dahners. Presented at Orthopaedic Research Society, San Francisco, CA; Deie, M., T. Marui, C. R. Allen, K. A. Hildebrand, H. I. Georgescu, et al. Mech. Ageing Dev. 97:121-130, 1997), cell therapy (Menetrey, J., C. Kasemkijwattana, C. S. Day, P. Bosch, F. H. Fu, et al. Tissue Eng. 5:435-442, 1999; Watanabe, N., S. L-Y. Woo, C. Papageorgiou, C. Celechovsky, and S. Takai. Microsc. Res. Tech. 58:39-44, 2002), as well as gene stherapy (Nakamura N., D. A. Hart, R. S. Boorman, Y. Kaneda, N. G. Shrive, et al. J. Orthop. Res. 18:517-523, 2000; Shimomura, T., F. Jia, C. Niyibizi, and S. L-Y. Woo. Connect. Tissue Res.:2003). The knowledge gained by studying these therapeutic strategies could potentially be applied to other ligaments and tendons. In this article, antisense gene therapy to alter gene expression by using antisense oligonucleotides will be examined as a possible solution.

  18. Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

    PubMed Central

    Laitala, T; Väänänen, H K

    1994-01-01

    The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

  19. Medial prefrontal cortical injections of c-fos antisense oligonucleotides transiently lower c-Fos protein and mimic amphetamine withdrawal behaviours.

    PubMed

    Persico, A M; Schindler, C W; Davis, S C; Ambrosio, E; Uhl, G R

    1998-02-01

    Prefrontal cerebral cortical areas display decreased expression of several transcription factor/immediate-early genes, including c-fos, during amphetamine withdrawal. Antisense strategies can help to test possible roles for this prefrontal c-fos down-regulation in the behavioural correlates of amphetamine withdrawal. Medial prefrontal cortical injections delivering 1.7 nmoles of anti c-fos oligonucleotides revealed an approximately 3 h half-life for phosphothioate and a 15 min half-life for phosphodiester oligonucleotides. Antisense phosphothioates complementary to the c-fos translational start site reduced levels of c-Fos protein, while exerting modest and variable effects on c-fos messenger RNA levels. Neither missense phosphorothioate nor antisense phosphodiester oligonucleotides significantly reduced levels of either c-fos messenger RNA or protein. Animals injected with anti c-fos phosphothioate oligonucleotides into the medial prefrontal cortex displayed marked reductions in linear locomotor activity and repetitive movements measured in a novel environment, effects not seen when missense oligonucleotides were used or when animals were accustomed to the activity monitor prior to antisense oligonucleotide injection. Behavioural changes produced by prefrontal cortical injections of c-fos antisense oligonucleotides closely mimic alterations recorded during amphetamine withdrawal. Prefrontal c-fos could thus conceivably play roles in the neurobiological underpinnings of psychostimulant withdrawal and of responses to stressors such as exposure to novel environments.

  20. 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

  1. 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.

  2. Sense-antisense gene pairs: sequence, transcription, and structure are not conserved between human and mouse

    PubMed Central

    Wood, Emily J.; Chin-Inmanu, Kwanrutai; Jia, Hui; Lipovich, Leonard

    2013-01-01

    Previous efforts to characterize conservation between the human and mouse genomes focused largely on sequence comparisons. These studies are inherently limited because they don't account for gene structure differences, which may exist despite genomic sequence conservation. Recent high-throughput transcriptome studies have revealed widespread and extensive overlaps between genes, and transcripts, encoded on both strands of the genomic sequence. This overlapping gene organization, which produces sense-antisense (SAS) gene pairs, is capable of effecting regulatory cascades through established mechanisms. We present an evolutionary conservation assessment of SAS pairs, on three levels: genomic, transcriptomic, and structural. From a genome-wide dataset of human SAS pairs, we first identified orthologous loci in the mouse genome, then assessed their transcription in the mouse, and finally compared the genomic structures of SAS pairs expressed in both species. We found that approximately half of human SAS loci have single orthologous locations in the mouse genome; however, only half of those orthologous locations have SAS transcriptional activity in the mouse. This suggests that high human-mouse gene conservation overlooks widespread distinctions in SAS pair incidence and expression. We compared gene structures at orthologous SAS loci, finding frequent differences in gene structure between human and orthologous mouse SAS pair members. Our categorization of human SAS pairs with respect to mouse conservation of expression as well as structure points to limitations of mouse models. Gene structure differences, including at SAS loci, may account for some of the phenotypic distinctions between primates and rodents. Genes in non-conserved SAS pairs may contribute to evolutionary lineage-specific regulatory outcomes. PMID:24133500

  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. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides.

    PubMed

    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.

  5. 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

  6. Long antisense non-coding RNAs and the epigenetic regulation of gene expression.

    PubMed

    Vadaie, Nadia; Morris, Kevin V

    2013-08-01

    Shortly after the completion of the human genome project in 2003, the Encode project was launched. The project was set out to identify the functional elements in the human genome, and unexpectedly it was found that >80% of the genome is transcribed. The Encode project identified those transcribed regions of the genome to be encoded by non-coding RNAs (ncRNAs). With only 2% of the genome carrying gene-encoding proteins, the conundrum was then, what is the function, if any, of these non-coding regions of the genome? These ncRNAs included both short and long RNAs. The focus of this review will be on antisense long non-coding RNAs (lncRNAs), as these transcripts have been observed to play a role in gene expression of protein-coding genes. Some lncRNAs have been found to regulate protein-coding gene transcription at the epigenetic level, whereby they suppress transcription through the recruitment of protein complexes to target loci in the genome. Conversely, there are lncRNAs that have a positive role in gene expression with less known about mechanism, and some lncRNAs have been shown to be involved in post-transcriptional processes. Additionally, lncRNAs have been observed to regulate their own expression in a positive feedback loop by functioning as a decoy. The biological significance of lncRNAs is only just now becoming evident, with many lncRNAs found to play a significant role in several human diseases.

  7. Distinct Expression Patterns of Natural Antisense Transcripts in Arabidopsis1[C][W

    PubMed Central

    Henz, Stefan R.; Cumbie, Jason S.; Kasschau, Kristin D.; Lohmann, Jan U.; Carrington, James C.; Weigel, Detlef; Schmid, Markus

    2007-01-01

    It has been shown that overlapping cis-natural antisense transcripts (cis-NATs) can form a regulatory circuit in which small RNAs derived from one transcript regulate stability of the other transcript, which manifests itself as anticorrelated expression. However, little is known about how widespread antagonistic expression of cis-NATs is. We have determined how frequently cis-NAT pairs, which make up 7.4% of annotated transcription units in the Arabidopsis (Arabidopsis thaliana) genome, show anticorrelated expression patterns. Indeed, global expression profiles of pairs of cis-NATs on average have significantly lower pairwise Pearson correlation coefficients than other pairs of neighboring genes whose transcripts do not overlap. However, anticorrelated expression that is greater than expected by chance is found in only a small number of cis-NAT pairs. The degree of anticorrelation does not depend on the length of the overlap or on the distance of the 5′ ends of the transcripts. Consistent with earlier findings, cis-NATs do not exhibit an increased likelihood to give rise to small RNAs, as determined from available small RNA sequences and massively parallel signature sequencing tags. However, the overlapping regions of cis-NATs appeared to be enriched for small RNA loci compared to nonoverlapping regions. Furthermore, expression of cis-NATs was not disproportionately affected in various RNA-silencing mutants. Our results demonstrate that there is a trend toward anticorrelated expression of cis-NAT pairs in Arabidopsis, but currently available data do not produce a strong signature of small RNA-mediated silencing for this process. PMID:17496106

  8. Antisense suppression of potassium channel expression demonstrates its role in maturation of the action potential.

    PubMed

    Vincent, A; Lautermilch, N J; Spitzer, N C

    2000-08-15

    A developmental increase in delayed rectifier potassium current (I(Kv)) in embryonic Xenopus spinal neurons is critical for the maturation of excitability and action potential waveform. Identifying potassium channel genes that generate I(Kv) is essential to understanding the mechanisms by which they are controlled. Several Kv genes are upregulated during embryogenesis in parallel with increases in I(Kv) and produce delayed rectifier current when heterologously expressed, indicating that they could encode channels underlying this current. We used antisense (AS) cRNA to test the contribution of xKv3.1 to the maturation of I(Kv), because xKv3.1 AS appears to suppress specifically heterologous expression of potassium current by xKv3.1 mRNA. The injection of xKv3.1 AS into embryos reduces endogenous levels of xKv3.1 mRNA in the developing spinal cord and reduces the amplitude and rate of activation of I(Kv) in 40% of cultured neurons, similar to the percentage of neurons in which endogenous xKv3.1 transcripts are detected. The current in these mature neurons resembles that at an earlier stage of differentiation before the appearance of xKv3.1 mRNA. Furthermore, AS expression increases the duration of the action potential in 40% of the neurons. No change in voltage-dependent calcium current is observed, suggesting that the decrease in I(Kv) is sufficient to account for lengthening of the action potential. Computer-simulated action potentials incorporating observed reductions in amplitude and rate of activation of I(Kv) exhibit an increase in duration similar to that observed experimentally. Thus xKv3.1 contributes to the maturation of I(Kv) in a substantial percentage of these developing spinal neurons.

  9. Antisense inhibition of apolipoprotein (a) to lower plasma lipoprotein (a) levels in humans

    PubMed Central

    Graham, Mark J.; Viney, Nick; Crooke, Rosanne M.; Tsimikas, Sotirios

    2016-01-01

    Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis. PMID:26538546

  10. 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

  11. Antisense Repression of the Medicago truncatula Nodule-Enhanced Sucrose Synthase Leads to a Handicapped Nitrogen Fixation Mirrored by Specific Alterations in the Symbiotic Transcriptome and Metabolome1[W

    PubMed Central

    Baier, Markus C.; Barsch, Aiko; Küster, Helge; Hohnjec, Natalija

    2007-01-01

    We analyzed the role of the sucrose (Suc) synthase MtSucS1 during nodulation of the model legume Medicago truncatula, integrating data for the developmental, transcriptional, and metabolic processes affected downstream of an impaired Suc cleavage in root nodules. To reduce carbohydrate supply to nodule tissues, transgenic plants expressing a p35S-driven MtSucS1-antisense fusion were constructed. These plants displayed an up to 90% reduction of MtSucS1 proteins in roots and nodules. Phenotypic studies of two independent MtSucS1-reduced lines demonstrated that only under conditions depending on nodulation, these plants appeared to be impaired in above-ground growth. Specifically plant height, shoot weight, leaf development, flowering, as well as seed maturation were reduced, and the efficiency of photosynthesis was affected. Concomitantly, a significantly enhanced root to shoot ratio with a marked increase in root tip numbers was observed. Root nodule formation was found retarded and the impaired nodulation was accompanied by a less efficient nitrogen (N) acquisition. The decreased total N content of MtSucS1-antisense lines and an enhanced carbon to N ratio in roots, nodules, and shoots correlated with the extent of MtSucS1 knockdown. On the level of transcription, effects of an MtSucS1 reduction were evident for genes representing important nodes of the nodule carbon and N metabolism, while metabolite profiling revealed significantly lower levels of amino acids and their derivatives particularly in strongly MtSucS1-reduced nodules. Our results support the model that nodule-enhanced Suc synthase 1 of the model legume M. truncatula is required for the establishment and maintenance of an efficient N-fixing symbiosis. PMID:17951459

  12. Modulation of γ2-MSH hepatoprotection by antisense peptides and melanocortin subtype 3 and 4 receptor antagonists.

    PubMed

    Turcic, Petra; Stambuk, Nikola; Konjevoda, Pasko; Kelava, Tomislav; Gabricevic, Mario; Stojkovic, Ranko; Aralica, Gorana

    2015-01-01

    Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

  13. Putative mitochondrial polypeptides coded by expanded quadruplet codons, decoded by antisense tRNAs with unusual anticodons.

    PubMed

    Seligmann, Hervé

    2012-11-01

    Weak triplet codon-anticodon interactions render ribosome-free translation unlikely. Some modern tRNAs read quadruplet codons (tetracodons), suggesting vestigial ribosome-free translation. Here, mitochondrial genomes are explored for tetracoded overlapping protein coding (tetra)genes. Occasional single tetracodons within regular mitochondrial genes coevolve positively/negatively with antisense tRNAs with predicted reduced/expanded anticodons (depending on taxon), suggesting complex tetra-decoding mechanisms. Transcripts of antisense tRNAs with unusual anticodons are more abundant than of homologues with regular anticodons. Assuming overlapping tetracoding with silent 4th tetracodon position, BLAST aligns 10 putative tetragenes spanning 17% of regular human mitochondrial protein coding tricodons with 14 GenBank proteins. Various tests including predicted peptide secondary structures, 3rd codon position (of the regular main frame of the protein coding gene) conservation against replicational deamination mutation gradients, and circular code usage (overlapping genes avoid using circular code codons) confirm tetracoding in these overlapping tetragenes with silent 4th position, but not for BLAST-predicted tetragenes assuming silent 2nd or 3rd positions. This converges with tetradecoding mechanisms that are more compatible with silent 4th, than at other, tetracodon positions. Tetracoding increases with (a) GC-contents, perhaps conserved or switched on in high temperature conditions; (b) usage of theoretically predicted 'tessera' tetracodons; (c) 12s rRNA stability; and d) antisense tRNA numbers with predicted expanded anticodons. Most detected tetragenes are not evolutionarily conserved, apparently reflect specific, transient adaptations. Tetracoding increases with mammal longevity.

  14. Down-regulation of specific members of the glutamine synthetase gene family in alfalfa by antisense RNA technology.

    PubMed

    Temple, S J; Bagga, S; Sengupta-Gopalan, C

    1998-06-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. In plants GS is an octameric enzyme and is located either in the cytoplasm (GS1) or in the chloroplast (GS2). Two distinct classes of GS1 genes with unique 3'-untranslated region (3'UTR) have been identified in alfalfa. We have demonstrated that the two classes exhibit differential expression pattern in the different plant organs suggesting different functional roles for the different isozymes. To determine the functional significance of the two classes of GS1 genes in alfalfa, we have utilized antisense gene constructs aimed specifically at the 3'UTR of the two GS1 genes and introduced them individually into alfalfa. Our data show that the gene constructs are effective in lowering the corresponding transcript level very effectively though there were organ-specific differences in the level of reduction. No transcript corresponding to the antisense gene construct was detected in any of the alfalfa transformants though they accumulated to significant levels in transgenic tobacco containing the same construct. This suggests that the antisense transcript was not stable in the presence of the homologous target sequence. Transgenic alfalfa with up to 80% reduction in the transcript level corresponding to each gene class, however, showed no reduction in GS activity or GS1 polypeptide level. The results suggest that GS1 mRNA levels are not rate-limiting for GS1 polypeptide synthesis and that GS levels are controlled both at the transcriptional and translational/post-translational level.

  15. 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.

  16. Magnetic force microscopy analysis of apoptosis of HL-60 cells induced by complex of antisense oligonucleotides and magnetic nanoparticles.

    PubMed

    Shen, He-bai; Long, De-hong; Zhu, Long-zhang; Li, Xing-Yu; Dong, Ya-ming; Jia, Neng-qin; Zhou, Hai-qing; Xin, Xi; Sun, Yang

    2006-06-20

    Magnetic force microscopy (MFM) has been employed to observe antisense oligonucleotides (ASOs)-coupled silica-coated magnetic iron oxide nanoparticles (SMNPs) internalized into human leukemia (HL-60) cells. The experiment demonstrated that the ASOs-coupled SMNPs delivery into the cells really occurred. The nanoparticles were internalized into the cells and the apoptotic topography can be directly visualized simultaneously with MFM technology. These present observations offer direct morphology evidence on studying the apoptosis of tumor cells and provide useful information for better design of new diagnostic and therapeutic tools in tumor treatment.

  17. Antisense long non-coding RNA PCNA-AS1 promotes tumor growth by regulating proliferating cell nuclear antigen in hepatocellular carcinoma.

    PubMed

    Yuan, Sheng-Xian; Tao, Qi-Fei; Wang, Jie; Yang, Fu; Liu, Lei; Wang, Li-Li; Zhang, Jin; Yang, Yuan; Liu, Hui; Wang, Fang; Sun, Shu-Han; Zhou, Wei-Ping

    2014-07-10

    About 61-72% of transcribed regions possess long noncoding RNAs in antisense orientation (Antisense long noncoding RNAs, aslncRNAs). However, the function of aslncRNAs in HCC remains unclear. We found numerous aslncRNAs were deregulated and might be involved in regulatory gene-net of HCC. The PCNA-AS1, antisense to PCNA, is significantly up-regulated in HCC and could promote tumor growth in vitro and in vivo. The effects of PCNA-AS1 rely on regulation of PCNA via forming RNA hybridization to increase PCNA mRNA stability. We concluded that aslncRNAs might act as upstream regulators in HCC and PCNA-AS1 could serve as a novel therapeutic target.

  18. 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.

  19. Exploring Differentially Expressed Genes and Natural Antisense Transcripts in Sheep (Ovis aries) Skin with Different Wool Fiber Diameters by Digital Gene Expression Profiling.

    PubMed

    Yue, Yaojing; Guo, Tingting; Liu, Jianbin; Guo, Jian; Yuan, Chao; Feng, Ruilin; Niu, Chune; Sun, Xiaoping; Yang, Bohui

    2015-01-01

    Wool fiber diameter (WFD) is the most important economic trait of wool. However, the genes specifically controlling WFD remain elusive. In this study, the expression profiles of skin from two groups of Gansu Alpine merino sheep with different WFD (a super-fine wool group [FD = 18.0 ± 0.5 μm, n=3] and a fine wool group [FD=23.0 ± 0.5 μm, n=3]) were analyzed using next-generation sequencing-based digital gene expression profiling. A total of 40 significant differentially expressed genes (DEGs) were detected, including 9 up-regulated genes and 31 down-regulated genes. Further expression profile analysis of natural antisense transcripts (NATs) showed that more than 30% of the genes presented in sheep skin expression profiles had NATs. A total of 7 NATs with significant differential expression were detected, and all were down-regulated. Among of 40 DEGs, 3 DEGs (AQP8, Bos d2, and SPRR) had significant NATs which were all significantly down-regulated in the super-fine wool group. In total of DEGs and NATs were summarized as 3 main GO categories and 38 subcategories. Among the molecular functions, cellular components and biological processes categories, binding, cell part and metabolic process were the most dominant subcategories, respectively. However, no significant enrichment of GO terms was found (corrected P-value >0.05). The pathways that were significantly enriched with significant DEGs and NATs were mainly the lipoic acid metabolism, bile secretion, salivary secretion and ribosome and phenylalanine metabolism pathways (P < 0.05). The results indicated that expression of NATs and gene transcripts were correlated, suggesting a role in gene regulation. The discovery of these DEGs and NATs could facilitate enhanced selection for super-fine wool sheep through gene-assisted selection or targeted gene manipulation in the future.

  20. 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

  1. Characterization of a Streptococcus mutans intergenic region containing a small toxic peptide and its cis-encoded antisense small RNA antitoxin.

    PubMed

    Koyanagi, Stephanie; Lévesque, Céline M

    2013-01-01

    Toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a protein toxin and an antitoxin, which may be in the form of either a labile protein or an antisense small RNA. Here we describe, to the best of our knowledge, the first functional chromosomal type I TA system in streptococci. Our model organism is the oral pathogen Streptococcus mutans. Our results showed that the genome of S. mutans UA159 reference strain harbors a previously unannotated Fst-like toxin (Fst-Sm) and its cis-encoded small RNA antitoxin (srSm) converging towards the end of the toxin gene in IGR176, a small intergenic region of 318 nt. Fst-Sm is a small hydrophobic peptide of 32 amino acid residues with homology to the Fst toxin family. Transcripts of ∼200 nt and ∼70 nt specific to fst-Sm mRNA and srSm RNA, respectively, were detected by Northern blot analysis throughout S. mutans growth. The toxin mRNA was considerably more stable than its cognate antitoxin. The half-life of srSm RNA was determined to be ∼30 min, while fst-Sm mRNA had a half-life of ∼90 min. Both fst-Sm and srSm RNAs were transcribed across direct tandem repeats providing a region of complementarity for inhibition of toxin translation. Overproduction of Fst-Sm had a toxic effect on E. coli and S. mutans cells which can be neutralized by coexpression of srSm RNA. Deletion of fst-Sm/srSm locus or overexpression of Fst-Sm/srSm had no effect on S. mutans cell growth in liquid medium and no differences in the total biofilm biomass were noted. In contrast, mild-overproduction of Fst-Sm/srSm type I TA system decreases the levels of persister cells tolerant to bacterial cell wall synthesis inhibitors.

  2. 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.

  3. A c-myc antisense oligonucleotide inhibits human retinal pigment epithelial cell proliferation.

    PubMed

    Capeáns, C; Piñeiro, A; Domínguez, F; Loidi, L; Buceta, M; Carneiro, C; Garcia-Caballero, T; Sanchez-Salorio, M

    1998-05-01

    The purpose of this work was to investigate if MYC-dependent intracellular mitogenic pathway is active in cultures of human retinal pigment epithelial (hRPE) cells and whether myc antisense phosphorotioate oligonucleotides (c-myc-AS-ODN) are useful tools for inhibiting the proliferation of hRPE cells. Cultures of hRPE cells were established from adult human corneal donors. These cells were positively stained for cytokeratins and vimentin. Myc mRNA expression was determined by Northern blot analysis and it was determined by means of immunofluorescence if MYC was expressed. C-myc-AS-ODN effect on cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine into cellular DNA. Cell number was estimated by using a tetrazolium bromide based colorimetric method. Human RPE cells in culture expressed MYC and myc mRNA as well as prothymosin alpha mRNA--a gene whose transcription is under MYC control--indicating that MYC-dependent intracellular mitogenic pathway is active in these cells. In accordance with this, we found that blocking the expression of myc by the addition of c-myc-AS-ODN to the culture medium inhibited hRPE cell proliferation. The effect of the c-myc-AS-ODN was found to be sequence specific (the use of a control oligonucleotide with the same sequence but in an opposite direction had no effect) and dose-dependent (4 microM was the lowest effective dose tested). By using RT-PCR we found that the c-myc-AS-ODN inhibition of cell proliferation was related to a diminution in c-myc mRNA expression, and by immunofluorescence we detected a diminution in c-MYC protein staining in RPE cells after 48 hr of treatment with c-myc-AS-ODN. Furthermore, growth inhibition remained for at least 5 days after addition of a single dose of the c-myc-AS-ODN to the culture. We conclude that hRPE cell proliferation is under MYC control. Blocking the expression of myc by c-myc-AS-ODN inhibited hRPE cell proliferation. These findings establish a rationale

  4. [Anionic long circulation liposomes mediated antisense scintigraphy in tumor-bearing rats].

    PubMed

    Ma, Chao; Kuang, Anren; Huang, Rui; Tang, Gongshun

    2011-04-01

    This paper was aimed to investigate the biodistribution and ability of free 131-bcl-2/bcl-xl ASON (FA) and anionic long circulation liposomes encapsulated with 131I-bcl-2/bcl-xlASON (NA), in tumor-bearing rats, to image breast cancer. We investigated the tissue distribution of NA in virgin female Sprague-Dawley (SD) rats with n-methyl nitrosourea (MNU)-induced breast cancers in situ. The percentage of the injected dose per gram (%ID/g) was calculated, with the maximum ratios of tumor to blood and tumor to muscle, after injections of NA and FA for 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h and 24 h, respectively. The ability of NA to image breast cancer in tumor-bearing rats was determined using emission computed tomography (ECT). Seventy percent (90/130) SD rats in the study developed mammary tumors after MNU injection with the average latency (NA) (96 +/- 1.2)days. The %ID/g of NA in breast cancer tissue, tumor bearing rats in liver and spleen tumor tissues after 10 hours were (6.23 +/- 0.23) %ID/g, (12.00 +/- 0.26) %ID/g and (18.25 +/- 1.33)% ID/g, respectively. The ratios of tumor to blood 6.29 +/- 0.76 and tumor to muscle 10.55 +/- 0.68 in tumor bearing rats slowly maximized at 10 h post injection of NA, most probably due to the enhanced permeability and retention effect. Hence in radionuclide antisense scintigraphy, the breast cancer in rat was clearly displayed at 10h after iv administration of NA-D. However, tumors were not visualized in rats with the iv injection of NS and NN even at the delayed time. Due to the inhibition of rapid uptake of NA by the reticulo-endothelial system, NA displays valuable pharmacologic properties characterized by the enhanced accumulation in tumor.

  5. Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

    PubMed Central

    Agrawal, S; Ikeuchi, T; Sun, D; Sarin, P S; Konopka, A; Maizel, J; Zamecnik, P C

    1989-01-01

    Antisense oligodeoxynucleotides, both the phosphorothioate analogues and unmodified oligomers of the same sequence, inhibit replication and expression of human immunodeficiency virus already growing in tissue cultures of MOLT-3 cells with much greater efficacy than do mismatched ("random") oligomers and homooligomers of the same length and with the same internucleotide modification. This preferential inhibitory effect is elicited in as short a time as 4-24 hr postinfection. Likewise, antisense oligomers exhibit greater inhibitory effects on human immunodeficiency virus in chronically infected cells than do mismatched oligomers and homooligomers. Phosphorothioate antisene oligomers are up to 100 times more potent than unmodified oligomers of the same sequence in these inhibitory assays. These results, in major respects, confirm and extend those recently published by Matsukura et al. [Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitsuza, H., Wong-Staal, F., Cohen, J. S. & Broder, S. (1989) Proc. Natl. Acad. Sci. USA 86, 4244-4248]. They also point out the importance of computer analysis of sequences though to be random but that in reality contain significant areas of likely hybridization, either to the viral genome or to the complementary DNA strand synthesized from it. They thus reinforce the concept that specific base pairing is a crucial feature of oligonucleotide inhibition of human immunodeficiency virus. PMID:2682627

  6. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

    PubMed

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E; Habib, Abdella M; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L; Woods, C Geoffrey; Wood, John N; Cox, James J

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

  7. Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches.

    PubMed

    Guzowski, John F

    2002-01-01

    In the 14 years since it was discovered that specific genes could be dynamically regulated in the brain by neural activity, there has been a substantial research focus attempting to understand the role immediate-early genes (IEGs) play in various brain functions. This article examines the involvement of IEGs in hippocampal synaptic plasticity and in memory consolidation processes performed by the hippocampus. Studies employing conventional IEG detection methodologies and a novel gene-imaging approach that provides temporal and cellular resolution (cellular compartment analysis of emporal activity by fluorescence in situ hybridization or catFISH) provide evidence supporting the assertion that IEG expression reflects the integration of information processed by hippocampal neurons. However, IEG expression is not merely correlated with neural activity, but also plays a pivotal role in stabilizing recent changes in synaptic efficacy. As such, localized disruption of IEGs Arc or c-fos by intrahippocampal administration of antisense oligonucleotides or germline disruption of the IEGs c-fos, tissue plasminogen activator, or zif268 impairs consolidation of long-term memory formation, without affecting learning or short-term memory. Further investigation into the expression and function of IEGs using catFISH and antisense approaches will likely increase understanding of the molecular and cellular bases of information processing involving the hippocampus.

  8. 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.

  9. 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.

  10. Enhancement of infectivity and persistence in vivo by HBZ, a natural antisense coded protein of HTLV-1.

    PubMed

    Arnold, Joshua; Yamamoto, Brenda; Li, Min; Phipps, Andrew J; Younis, Ihab; Lairmore, Michael D; Green, Patrick L

    2006-05-15

    Natural antisense viral transcripts have been recognized in retroviruses, including human T-cell leukemia virus type 1 (HTLV-1), HIV-1, and feline immunodeficiency virus (FIV), and have been postulated to encode proteins important for the infection cycle and/or pathogenesis of the virus. The antisense strand of the HTLV-1 genome encodes HBZ, a novel nuclear basic region leucine zipper (b-ZIP) protein that in overexpression assays down-regulates Tax oncoprotein-induced viral transcription. Herein, we investigated the contribution of HBZ to HTLV-1-mediated immortalization of primary T lymphocytes in vitro and HTLV-1 infection in a rabbit animal model. HTLV-1 HBZ mutant viruses were generated and evaluated for viral gene expression, protein production, and immortalization capacity. Biologic properties of HBZ mutant viruses in vitro were indistinguishable from wild-type HTLV-1, providing the first direct evidence that HBZ is dispensable for viral replication and cellular immortalization. Rabbits inoculated with irradiated cells expressing HTLV-1 HBZ mutant viruses became persistently infected. However, these rabbits displayed a decreased antibody response to viral gene products and reduced proviral copies in peripheral blood mononuclear cells (PBMCs) as compared with wild-type HTLV-1-infected animals. Our findings indicated that HBZ was not required for in vitro cellular immortalization, but enhanced infectivity and persistence in inoculated rabbits. This study demonstrates that retroviruses use negative-strand-encoded proteins in the establishment of chronic viral infections.

  11. SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation

    PubMed Central

    Zucchelli, S; Cotella, D; Takahashi, H; Carrieri, C; Cimatti, L; Fasolo, F; Jones, MH; Sblattero, D; Sanges, R; Santoro, C; Persichetti, F; Carninci, P; Gustincich, S

    2015-01-01

    Over the past 10 years, it has emerged that pervasive transcription in mammalian genomes has a tremendous impact on several biological functions. Most of transcribed RNAs are lncRNAs and repetitive elements. In this review, we will detail the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies. PMID:26259533

  12. Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides.

    PubMed

    Epa, W R; Rong, P; Bartlett, P F; Coulson, E J; Barrett, G L

    1998-12-01

    The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo. However, only a minor degree of downregulation of p75 protein was obtained with 5' cholesteryl oligos. Three different linkers was used to attach the 5' cholesteryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregulation (31%) and p75 protein downregulation (28%) than occurred with the 5' cholesteryl oligos. The biggest improvement in antisense efficacy, both at the mRNA and protein levels, was obtained from the conjugation of cholesterol to both ends of the oligo. One of the bischolesteryl oligos was nearly as effective as cycloheximide at decreasing synthesis of p75. The bis-cholesteryl oligos also displayed significant efficacy at 1 microM, whereas the other oligos required 5 microM to be effective. The enhanced efficacy of bis-cholesteryl oligos is likely to be due to a combination of enhanced cellular uptake and resistance to both 5' and 3' exonucleases.

  13. 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 cou