Science.gov

Sample records for macromolecules dna rna

  1. Nonlinear focusing of DNA macromolecules

    NASA Astrophysics Data System (ADS)

    Frumin, Leonid L.; Peltek, Sergey E.; Zilberstein, Gleb V.

    2001-08-01

    The present paper reports the nonlinear electrophoretic focusing techniques developed after an original idea by Chacron and Slater [Phys. Rev. E 56, 3436 (1997)]. Focusing of DNA molecules is achieved in an alternating nonuniform electric field, created in a wedge gel with hyperbolic boundaries. The fractions separated on such a wedge retained their rectilinear shape during the electrophoresis. Experiments with gel electrophoresis confirm the possibility of a noticeable nonlinear focusing of DNA molecules.

  2. Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules.

    PubMed

    Sparks, Sarah M; Waite, Carolyn L; Harmon, Alexander M; Nusblat, Leora M; Roth, Charles M; Uhrich, Kathryn E

    2011-09-01

    New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N

  3. Diffraction in resonant electron scattering from helical macromolecules: A- and B-type DNA

    SciTech Connect

    Caron, Laurent; Sanche, Leon

    2004-09-01

    We elaborate our theoretical framework to treat low-energy electron scattering from helical macromolecules. A model of simple scatterers organized into a helical structure is proposed with application to base-pair arrangements in DNA. We extend our analysis to A-type DNA as well as nonperiodic arrangements in B-type DNA. Diffraction patterns due to base-pair spacing are observed under all conditions. We discuss the role of electron diffraction in electron attachment to the bases leading to the formation of shape and core-excited resonances. The decay of such transient anions into dissociative-state channels producing DNA strand breaks is also described. We find A-type DNA to be much more sensitive to low-energy electrons than B type.

  4. Target Biological Structures: The Cell, Organelles, DNA and RNA

    NASA Astrophysics Data System (ADS)

    van Holst, Marcelis; Grant, Maxine P.; Aldrich-Wright, Janice

    Living organisms are self replicating molecular factories of staggering complexity [1]. As a result, we are often overwhelmed when trying to identify potential targets for therapeutics. Water, inorganic ions and a large array of relatively small organic molecules (e.g., sugars, vitamins and fatty acids) account for approximately 80% of living matter, with water being the most abundant. Macromolecules such as proteins, polysaccharides, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) constitute the rest. The majority of potential therapeutic targets are found within the cell. Small molecules which are vital for cellular function are imported into the cell by a variety of mechanisms but unlike smaller molecules, macromolecules are assembled within the cell itself. Drugs are usually designed to target cellular macromolecules, as they perform very specific roles in the metabolic processes.

  5. Diffraction in resonant electron scattering from helical macromolecules: Effects of the DNA backbone

    SciTech Connect

    Caron, Laurent; Sanche, Leon

    2005-09-15

    We recently developed a theoretical framework to treat low-energy electron scattering from helical macromolecules. In this article, we use this framework to extend our previous model of simple base-pair scatterers, organized into the DNA structure, to include the backbone. The internal diffraction pattern due to base pairs is still present, but addition of the backbone screens the base pairs by a factor of 2. More interestingly, the effect of constructive interference on the phosphate groups within the backbone itself is seen to be strong at lower energies. We perform a calculation for electrons incident perpendicular and parallel to the axis of a fragment and find comparable electron patterns on the phosphate groups at the surface of films consisting of vertically or horizontally arranged segments relative to the substrate.

  6. Hairpins under tension: RNA versus DNA

    PubMed Central

    Bercy, Mathilde; Bockelmann, Ulrich

    2015-01-01

    We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity. PMID:26323319

  7. RNA Study Using DNA Nanotechnology.

    PubMed

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. PMID:26970193

  8. Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

    PubMed Central

    Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

    2007-01-01

    Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription. PMID:17356699

  9. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  10. Initiator RNA in Discontinuous Polyoma DNA Synthesis*

    PubMed Central

    Reichard, Peter; Eliasson, Rolf; Söderman, Gunilla

    1974-01-01

    During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5′ ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5′ end with 32P from β-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released 32P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name “initiator RNA” for it. The covalent linkage of initiator RNA to 5′ ends of growing DNA chains was substantiated by the finding that 32P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [α-32P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5′ end with 32P released a variety of different [32P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence. PMID:4373733

  11. The RNA Response to DNA Damage.

    PubMed

    Giono, Luciana E; Nieto Moreno, Nicolás; Cambindo Botto, Adrián E; Dujardin, Gwendal; Muñoz, Manuel J; Kornblihtt, Alberto R

    2016-06-19

    Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields. PMID:26979557

  12. A DNA enzyme that cleaves RNA

    NASA Technical Reports Server (NTRS)

    Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

    1994-01-01

    BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

  13. DNA and RNA technology in soil biodiversity

    NASA Astrophysics Data System (ADS)

    Pereg, Lily

    2016-04-01

    DNA technology has come a long way and state of the art techniques are currently used in the analysis of soil biodiversity. Current methods will be presented and their strengths and limitations discussed. RNA technology, for the study of gene expression and potential activity of functional groups in the soil, is lagging behind, mostly due to the difficulties of extracting stable RNA from the soil. The potentials and challenges of adopting RNA technology for soil analysis will be discussed.

  14. Interaction of Sulforaphane with DNA and RNA

    PubMed Central

    Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

    2015-01-01

    Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

  15. MicroRNA response to DNA damage

    PubMed Central

    Wan, Guohui; Mathur, Rohit; Hu, Xiaoxiao; Zhang, Xinna; Lu, Xiongbin

    2011-01-01

    Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution, but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways, composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that microRNAs (miRNAs) play a critical role in regulation of DNA damage response. Here, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage. PMID:21741842

  16. Free-energy calculations for semi-flexible macromolecules: applications to DNA knotting and looping.

    PubMed

    Giovan, Stefan M; Scharein, Robert G; Hanke, Andreas; Levene, Stephen D

    2014-11-01

    We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases. PMID:25381542

  17. Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas; Levene, Stephen D.

    2014-11-01

    We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

  18. Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

    SciTech Connect

    Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas; Levene, Stephen D.

    2014-11-07

    We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

  19. RNA-directed DNA methylation in plants.

    PubMed

    Movahedi, Ali; Sun, Weibu; Zhang, Jiaxin; Wu, Xiaolong; Mousavi, Mohaddesseh; Mohammadi, Kourosh; Yin, Tongming; Zhuge, Qiang

    2015-11-01

    In plants, many small interfering RNAs (siRNAs) direct de novo methylation by DNA methyltransferase. DNA methylation typically occurs by RNA-directed DNA methylation (RdDM), which directs transcriptional gene silencing of transposons and endogenous transgenes. RdDM is driven by non-coding RNAs (ncRNAs) produced by DNA-dependent RNA polymerases IV and V (PolIV and PolV). The production of siRNAs is initiated by PolIV and ncRNAs produced by PolIV are precursors of 24-nucleotide siRNAs. In contrast, ncRNAs produced by PolV are involved in scaffolding RNAs. In this review, we summarize recent studies of RdDM. In particular, we focus on the mechanisms involved in chromatin remodeling by PolIV and PolV. PMID:26183954

  20. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  1. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  2. Isothermal amplified detection of DNA and RNA.

    PubMed

    Yan, Lei; Zhou, Jie; Zheng, Yue; Gamson, Adam S; Roembke, Benjamin T; Nakayama, Shizuka; Sintim, Herman O

    2014-05-01

    This review highlights various methods that can be used for a sensitive detection of nucleic acids without using thermal cycling procedures, as is done in PCR or LCR. Topics included are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated amplification (LAMP), Invader assay, rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking endonuclease signal amplification (NESA) and nicking endonuclease assisted nanoparticle activation (NENNA), exonuclease-aided target recycling, Junction or Y-probes, split DNAZyme and deoxyribozyme amplification strategies, template-directed chemical reactions that lead to amplified signals, non-covalent DNA catalytic reactions, hybridization chain reactions (HCR) and detection via the self-assembly of DNA probes to give supramolecular structures. The majority of these isothermal amplification methods can detect DNA or RNA in complex biological matrices and have great potential for use at point-of-care. PMID:24643211

  3. Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

    PubMed Central

    2015-01-01

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. PMID:25521794

  4. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  5. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD) simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15, and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS) P-test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein. PMID:26594643

  6. Synergistic self-assembly of RNA and DNA molecules

    NASA Astrophysics Data System (ADS)

    Ko, Seung Hyeon; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

    2010-12-01

    DNA has recently been used as a programmable 'smart' building block for the assembly of a wide range of nanostructures. It remains difficult, however, to construct DNA assemblies that are also functional. Incorporating RNA is a promising strategy to circumvent this issue as RNA is structurally related to DNA but exhibits rich chemical, structural and functional diversities. However, only a few examples of rationally designed RNA structures have been reported. Herein, we describe a simple, general strategy for the de novo design of nanostructures in which the self-assembly of RNA strands is programmed by DNA strands. To demonstrate the versatility of this approach, we have designed and constructed three different RNA-DNA hybrid branched nanomotifs (tiles), which readily assemble into one-dimensional nanofibres, extended two-dimensional arrays and a discrete three-dimensional object. The current strategy could enable the integration of the precise programmability of DNA with the rich functionality of RNA.

  7. DNA and RNA Quadruplex-Binding Proteins

    PubMed Central

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C. C.; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  8. DNA and RNA quadruplex-binding proteins.

    PubMed

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C C; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  9. Polynucleotide 3'-terminal phosphate modifications by RNA and DNA ligases.

    PubMed

    Zhelkovsky, Alexander M; McReynolds, Larry A

    2014-11-28

    RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3'-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3'p substrate to generate an RNA 2',3'-cyclic phosphate or convert DNA3'p to ssDNA(3')pp(5')A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3'p. These modifications of RNA and DNA 3'-phosphates are similar to the activities of RtcA, an RNA 3'-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3'p or DNA 3'p to generate the adenylated intermediate. For RNA (3')pp(5')A, the third step involves attack of the adjacent 2' hydroxyl to generate the RNA 2',3'-cyclic phosphate. These steps are analogous to those in classical 5' phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3'p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3'-phosphorylated nicks in double-stranded DNA to produce a 3'-adenylated product. These 3'-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5'Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3'-terminal phosphates. PMID:25324547

  10. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  11. DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

    PubMed Central

    Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

    2016-01-01

    Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

  12. Evolution of DNA and RNA as catalysts for chemical reactions.

    PubMed

    Jäschke, A; Seelig, B

    2000-06-01

    In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA molecules that have catalytic properties. Catalyzed reactions now go far beyond self-modifying reactions of nucleic acid molecules. The future application of in vitro selected RNA and DNA catalysts in bioorganic synthesis appears promising. PMID:10826969

  13. Unusual Thermal Stability of RNA/[RP-PS]-DNA/RNA Triplexes Containing a Homopurine DNA Strand

    PubMed Central

    Guga, Piotr; Boczkowska, Małgorzata; Janicka, Magdalena; Maciaszek, Anna; Kuberski, Sławomir; Stec, Wojciech J.

    2007-01-01

    Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2′-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4–6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process. PMID:17218459

  14. Synthesis of Amplified DNA That Codes for Ribosomal RNA

    PubMed Central

    Crippa, Marco; Tocchini-Valentini, Glauco P.

    1971-01-01

    During the amplification stage in ovaries, the complete repetitive unit of the DNA that codes for ribosomal RNA in Xenopus appears to be transcribed. This large RNA transcript is found in a complex with DNA. Substitution experiments with 5-bromodeoxyuridine do not show any evidence that a complete amplified cistron is used as a template for further amplification. A derivative of rifampicin, 2′,5′-dimethyl-N(4′)benzyl-N(4′)[desmethyl] rifampicin, preferentially inhibits the DNA synthesis responsible for ribosomal gene amplification. These results are consistent with the hypothesis that RNA-dependent DNA synthesis is involved in gene amplification. PMID:5288254

  15. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    PubMed Central

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual’s DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  16. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    PubMed

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  17. Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World

    PubMed Central

    Siegel, Robert W.; Bellon, Laurent; Beigelman, Leonid; Kao, C. Cheng

    1999-01-01

    All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes. PMID:10400735

  18. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus. PMID:24571086

  19. RNA-directed DNA methylation and demethylation in plants

    PubMed Central

    Viswanathan, CHINNUSAMY; Jian-Kang, ZHU

    2011-01-01

    RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit complementary siRNAs and the associated RdDM effector complex to the target loci in a transcription-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct target-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling enzymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Locus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses. PMID:19381459

  20. Biological Macromolecule Crystallization Database

    National Institute of Standards and Technology Data Gateway

    SRD 21 Biological Macromolecule Crystallization Database (Web, free access)   The Biological Macromolecule Crystallization Database and NASA Archive for Protein Crystal Growth Data (BMCD) contains the conditions reported for the crystallization of proteins and nucleic acids used in X-ray structure determinations and archives the results of microgravity macromolecule crystallization studies.

  1. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  2. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  3. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  4. RNA intrusions change DNA elastic properties and structure

    NASA Astrophysics Data System (ADS)

    Chiu, Hsiang-Chih; Koh, Kyung Duk; Evich, Marina; Lesiak, Annie L.; Germann, Markus W.; Bongiorno, Angelo; Riedo, Elisa; Storici, Francesca

    2014-08-01

    The units of RNA, termed ribonucleoside monophosphates (rNMPs), have been recently found as the most abundant defects present in DNA. Despite the relevance, it is largely unknown if and how rNMPs embedded in DNA can change the DNA structure and mechanical properties. Here, we report that rNMPs incorporated in DNA can change the elastic properties of DNA. Atomic force microscopy (AFM)-based single molecule elasticity measurements show that rNMP intrusions in short DNA duplexes can decrease - by 32% - or slightly increase the stretch modulus of DNA molecules for two sequences reported in this study. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy identify a series of significant local structural alterations of DNA containing embedded rNMPs, especially at the rNMPs and nucleotide 3' to the rNMP sites. The demonstrated ability of rNMPs to locally alter DNA mechanical properties and structure may help in understanding how such intrusions impact DNA biological functions and find applications in structural DNA and RNA nanotechnology.The units of RNA, termed ribonucleoside monophosphates (rNMPs), have been recently found as the most abundant defects present in DNA. Despite the relevance, it is largely unknown if and how rNMPs embedded in DNA can change the DNA structure and mechanical properties. Here, we report that rNMPs incorporated in DNA can change the elastic properties of DNA. Atomic force microscopy (AFM)-based single molecule elasticity measurements show that rNMP intrusions in short DNA duplexes can decrease - by 32% - or slightly increase the stretch modulus of DNA molecules for two sequences reported in this study. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy identify a series of significant local structural alterations of DNA containing embedded rNMPs, especially at the rNMPs and nucleotide 3' to the rNMP sites. The demonstrated ability of rNMPs to locally alter DNA mechanical properties and structure

  5. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  6. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  7. Folic acid binds DNA and RNA at different locations.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2015-03-01

    We located multiple binding sites for folic acid on DNA and tRNA at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural analysis revealed that folic acid binds DNA and tRNA at multiple sites via hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of Kfolic acid-DNA=1.1 (±0.3)×10(4) M(-1) and Kfolic acid-tRNA=6.4 (±0.5)×10(3) M(-1). Molecular modeling showed the participation of several nucleobases in folic acid complexes with DNA and tRNA, stabilized by H-bonding network. Two types of complexes were located for folic acid-tRNA adducts, one at the major groove and the other with TΨC loop, while acid binding occurs at major and minor grooves of DNA duplex. Folic acid complexation induced more alterations of DNA structure than tRNA. PMID:25555838

  8. Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes.

    PubMed

    Heyduk, T; Niedziela-Majka, A

    Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated. PMID:11987181

  9. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small angle X-ray scattering

    PubMed Central

    Rambo, Robert P.; Tainer, John A.

    2010-01-01

    Summary Small-Angle X-ray Scattering (SAXS) is changing how we perceive biological structures, because it reveals dynamic macromolecular conformations and assemblies in solution. SAXS information captures thermodynamic ensembles, enhances static structures detailed by high-resolution methods, uncovers commonalities among diverse macromolecules, and helps define biological mechanisms. SAXS-based experiments on RNA riboswitches and ribozymes and on DNA-protein complexes including DNA-PK and p53 discover flexibilities that better define structure-function relationships. Furthermore, SAXS results suggest conformational variation is a general functional feature of macromolecules. Thus, accurate structural analyses will require a comprehensive approach that assesses both flexibility, as seen by SAXS, and detail, as determined by X-ray crystallography and NMR. Here, we review recent SAXS computational tools, technologies, and applications to nucleic acids and related structures. PMID:20097063

  10. RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

    PubMed Central

    Altmann, C R; Solow-Cordero, D E; Chamberlin, M J

    1994-01-01

    In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein. Images PMID:7513426

  11. Quantum-mechanical predictions of DNA and RNA ionization by energetic proton beams

    NASA Astrophysics Data System (ADS)

    Galassi, M. E.; Champion, C.; Weck, P. F.; Rivarola, R. D.; Fojón, O.; Hanssen, J.

    2012-04-01

    Among the numerous constituents of eukaryotic cells, the DNA macromolecule is considered as the most important critical target for radiation-induced damages. However, up to now ion-induced collisions on DNA components remain scarcely approached and theoretical support is still lacking for describing the main ionizing processes. In this context, we here report a theoretical description of the proton-induced ionization of the DNA and RNA bases as well as the sugar-phosphate backbone. Two different quantum-mechanical models are proposed: the first one based on a continuum distorted wave-eikonal initial state treatment and the second perturbative one developed within the first Born approximation with correct boundary conditions (CB1). Besides, the molecular structure information of the biological targets studied here was determined by ab initio calculations with the Gaussian 09 software at the restricted Hartree-Fock level of theory with geometry optimization. Doubly, singly differential and total ionization cross sections also provided by the two models were compared for a large range of incident and ejection energies and a very good agreement was observed for all the configurations investigated. Finally, in comparison with the rare experiment, we have noted a large underestimation of the total ionization cross sections of uracil impacted by 80 keV protons, whereas a very good agreement was shown with the recently reported ionization cross sections for protons on adenine, at both the differential and the total scale.

  12. Quantum-mechanical predictions of DNA and RNA ionization by energetic proton beams.

    PubMed

    Galassi, M E; Champion, C; Weck, P F; Rivarola, R D; Fojón, O; Hanssen, J

    2012-04-01

    Among the numerous constituents of eukaryotic cells, the DNA macromolecule is considered as the most important critical target for radiation-induced damages. However, up to now ion-induced collisions on DNA components remain scarcely approached and theoretical support is still lacking for describing the main ionizing processes. In this context, we here report a theoretical description of the proton-induced ionization of the DNA and RNA bases as well as the sugar-phosphate backbone. Two different quantum-mechanical models are proposed: the first one based on a continuum distorted wave-eikonal initial state treatment and the second perturbative one developed within the first Born approximation with correct boundary conditions (CB1). Besides, the molecular structure information of the biological targets studied here was determined by ab initio calculations with the Gaussian 09 software at the restricted Hartree-Fock level of theory with geometry optimization. Doubly, singly differential and total ionization cross sections also provided by the two models were compared for a large range of incident and ejection energies and a very good agreement was observed for all the configurations investigated. Finally, in comparison with the rare experiment, we have noted a large underestimation of the total ionization cross sections of uracil impacted by 80 keV protons,whereas a very good agreement was shown with the recently reported ionization cross sections for protons on adenine, at both the differential and the total scale. PMID:22433314

  13. Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

    PubMed Central

    Goguel, V; Delahodde, A; Jacq, C

    1992-01-01

    The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase

  14. Telomerase RNA is more than a DNA template.

    PubMed

    Webb, Christopher J; Zakian, Virginia A

    2016-08-01

    The addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE). (1) The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target. PMID:27245259

  15. Comparing Charge Transport in Oligonucleotides: RNA:DNA Hybrids and DNA Duplexes.

    PubMed

    Li, Yuanhui; Artés, Juan M; Qi, Jianqing; Morelan, Ian A; Feldstein, Paul; Anantram, M P; Hihath, Joshua

    2016-05-19

    Understanding the electronic properties of oligonucleotide systems is important for applications in nanotechnology, biology, and sensing systems. Here the charge-transport properties of guanine-rich RNA:DNA hybrids are compared to double-stranded DNA (dsDNA) duplexes with identical sequences. The conductance of the RNA:DNA hybrids is ∼10 times higher than the equivalent dsDNA, and conformational differences are determined to be the primary reason for this difference. The conductance of the RNA:DNA hybrids is also found to decrease more rapidly than dsDNA when the length is increased. Ab initio electronic structure and Green's function-based density of states calculations demonstrate that these differences arise because the energy levels are more spatially distributed in the RNA:DNA hybrid but that the number of accessible hopping sites is smaller. These combination results indicate that a simple hopping model that treats each individual guanine as a hopping site is insufficient to explain both a higher conductance and β value for RNA:DNA hybrids, and larger delocalization lengths must be considered. PMID:27145167

  16. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.

    PubMed

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-02-13

    The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape. PMID:14963331

  17. Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.

    PubMed

    Avrameas, A; Ternynck, T; Nato, F; Buttin, G; Avrameas, S

    1998-05-12

    Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes. PMID:9576929

  18. Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

    PubMed

    Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. PMID:23968861

  19. DNA homologies of ribosomal RNA genes of Neurospora species

    SciTech Connect

    Mukhopadhyay, D.K.; Mimiko, R.; Dutta, S.K.

    1980-01-01

    Ribosomal RNA genes (rDNAs) of Neurospora crassa contain DNA sequences which code for 17S, 5.8S, and 26S rRNAs, in addition to internal and external spacers. As has been reported for many eukaryotes, the DNA sequences which code for 17S, 5.8S, and 26S rRNAs in Neurospora species are probably conserved while the internal and external spacer regions are probably variable sequences. Extensive electron microscopic studies of 45S precursor rRNA of several cold and warm blooded animals confirm that spacer regions vary extensively from species to species. It was desirable to know whether such differences in rDNA sequences exist between Neurospora species. Any such difference should be detectable using standard procedures for DNA homology studies rDNA sequences were isolated from N. crassa mycelial cells using the procedure described previously. The purified rDNA was /sup 3/H-labeled (by nick translation) and reassociated with total DNA isolated from the heterothallic species N. crassa and from three homothalliospecies: N. dodgei, N. lineolata, and N. africana. In addition, /sup 32/P-labeled total DNA of N. crassa was reannealed with unlabeled bulk DNA from N. crassa, N. dodgei, and N. lineolata.

  20. Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA.

    PubMed

    Samanta, Biswajit; Seikowski, Jan; Höbartner, Claudia

    2016-01-26

    5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA. PMID:26679556

  1. Effects of DNA replication on mRNA noise.

    PubMed

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  2. Effects of DNA replication on mRNA noise

    PubMed Central

    Peterson, Joseph R.; Cole, John A.; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A.

    2015-01-01

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript’s half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories. PMID:26669443

  3. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  4. ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

    NASA Astrophysics Data System (ADS)

    Birrento, Monica L.; Bryan, Tracy M.; Samosorn, Siritron; Beck, Jennifer L.

    2015-07-01

    Electrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine ( 1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of ( 1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand ( 1) for binding to the bimolecular qDNA over the DNA/RNA hybrid positions it as a useful compound for probing the role(s), if any, of antiparallel qDNA in the telomerase mechanism.

  5. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

    PubMed Central

    Thompson, David B.; Cronican, James J.; Liu, David R.

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

  6. Molecular docking of the anticancer bioactive compound proceraside with macromolecules involved in the cell cycle and DNA replication.

    PubMed

    Gurung, A B; Ali, M A; Bhattacharjee, A; AbulFarah, M; Al-Hemaid, F; Abou-Tarboush, F M; Al-Anazi, K M; Al-Anazi, F S M; Lee, J

    2016-01-01

    The bioactive compounds proceraside A, frugoside and calotropin, which were extracted from the root bark of Calotropis procera (Aiton) W.T. Aiton (family Asclepiadaceae), were recently reported to inhibit the growth of inhibition against various human cancer cell lines in vitro. However, their modes of action have not been clearly defined. Therefore, we attempted an in silico approach to gain insights into their binding modes against the following selected molecular targets: CDK-2, CDK-6, topoisomerase I, BCL-2, VEGFR-2, telomere: G-quadruplex, and topoisomerase II. These targets were selected based on their key roles in cancer progression via the regulation of the cell cycle and DNA replication. Molecular-docking analyses revealed that proceraside A was the best docked ligand against all the targets, with the exception of telomere-G: quadruplex. Furthermore, it displayed the lowest binding energies and inhibition constants, and critical hydrogen bonds and hydrophobic interactions with the targets were also revealed. The present study may aid in the identification of possible targets for proceraside A, and might provide a plausible explanation for its proven anti-tumor activities. Moreover, the result of this study may further guide structure-activity relationship studies used to generate more potent target-specific inhibitors. PMID:27173346

  7. siRNA-directed DNA Methylation in Plants

    PubMed Central

    Xie, Meng; Yu, Bin

    2015-01-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  8. A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

    PubMed

    Finerty, P J; Bass, B L

    1997-08-15

    Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix. PMID:9268652

  9. DNA?RNA: What Do Students Think the Arrow Means?

    ERIC Educational Resources Information Center

    Wright, L. Kate; Fisk, J. Nick; Newman, Dina L.

    2014-01-01

    The central dogma of molecular biology, a model that has remained intact for decades, describes the transfer of genetic information from DNA to protein though an RNA intermediate. While recent work has illustrated many exceptions to the central dogma, it is still a common model used to describe and study the relationship between genes and protein…

  10. Protein localization with flexible DNA or RNA.

    PubMed

    Bernhardsson, Sebastian; Mitarai, Namiko; Sneppen, Kim

    2012-01-01

    Localization of activity is ubiquitous in life, and also within sub-cellular compartments. Localization provides potential advantages as different proteins involved in the same cellular process may supplement each other on a fast timescale. It might also prevent proteins from being active in other regions of the cell. However localization is at odds with the spreading of unbound molecules by diffusion. We model the cost and gain for specific enzyme activity using localization strategies based on binding to sites of intermediate specificity. While such bindings in themselves decrease the activity of the protein on its target site, they may increase protein activity if stochastic motion allows the acting protein to touch both the intermediate binding site and the specific site simultaneously. We discuss this strategy in view of recent suggestions on long non-coding RNA as a facilitator of localized activity of chromatin modifiers. PMID:22347995

  11. DNA/RNA Detection Using DNA-Templated Few-Atom Silver Nanoclusters.

    PubMed

    Obliosca, Judy M; Liu, Cong; Batson, Robert Austin; Babin, Mark C; Werner, James H; Yeh, Hsin-Chih

    2013-01-01

    DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors. PMID:25586126

  12. DNA/RNA Detection Using DNA-Templated Few-Atom Silver Nanoclusters

    PubMed Central

    Obliosca, Judy M.; Liu, Cong; Batson, Robert Austin; Babin, Mark C.; Werner, James H.; Yeh, Hsin-Chih

    2013-01-01

    DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors. PMID:25586126

  13. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.

    PubMed

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P; Low, Audrey; Yoshida, Masayuki; Bennett, C Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  14. DNA Targeting by a Minimal CRISPR RNA-Guided Cascade.

    PubMed

    Hochstrasser, Megan L; Taylor, David W; Kornfeld, Jack E; Nogales, Eva; Doudna, Jennifer A

    2016-09-01

    Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms. PMID:27588603

  15. Roles of RNA-Binding Proteins in DNA Damage Response

    PubMed Central

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  16. Roles of RNA-Binding Proteins in DNA Damage Response.

    PubMed

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  17. Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

    PubMed Central

    Schneck, P K; Staudenbauer, W L; Hofschneider, P H

    1976-01-01

    Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

  18. Single-Molecule Electrical Random Resequencing of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2012-07-01

    Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

  19. Single-Molecule Electrical Random Resequencing of DNA and RNA

    PubMed Central

    Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2012-01-01

    Two paradigm shifts in DNA sequencing technologies—from bulk to single molecules and from optical to electrical detection—are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5′-UGAGGUA-3′ from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes. PMID:22787559

  20. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi.

    1989-12-26

    Hydrophilic polyacids are described, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests. 2 figs.

  1. Polyacid macromolecule primers

    DOEpatents

    Sugama, Toshifumi

    1989-01-01

    Hydrophylic polyacids, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests.

  2. An Oligonucleotide Affinity Column for RNA-Dependent DNA Polymerase from RNA Tumor Viruses

    PubMed Central

    Gerwin, Brenda I.; Milstien, Julie B.

    1972-01-01

    Columns of (dT)12-18-cellulose provide a one-step enrichment procedure for RNA-dependent DNA polymerase. The enzyme of the virus from RD-114 cells, as well as that from Rauscher murine leukemia virus, have been purified in this way. The preference of viral as compared to cellular DNA polymerases for (dT)12-18 as a primer is reflected in the fact that the DNA polymerases of uninfected cells do not bind to this column. Viral enzymes have been purified and identified from crude cellular extracts. PMID:4506781

  3. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  4. Detection of RNA-DNA association by a proximity ligation-based method.

    PubMed

    Petruk, Svetlana; Fenstermaker, Tyler K; Black, Kathryn L; Brock, Hugh W; Mazo, Alexander

    2016-01-01

    We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30-45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis. PMID:27256324

  5. Detection of RNA-DNA association by a proximity ligation-based method

    PubMed Central

    Petruk, Svetlana; Fenstermaker, Tyler K.; Black, Kathryn L.; Brock, Hugh W.; Mazo, Alexander

    2016-01-01

    We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30–45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis. PMID:27256324

  6. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    PubMed Central

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R.; Pradhan, Sriharsa

    2015-01-01

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

  7. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation.

    PubMed

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R; Pradhan, Sriharsa

    2015-07-13

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

  8. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

    PubMed Central

    Götte, M; Fackler, S; Hermann, T; Perola, E; Cellai, L; Gross, H J; Le Grice, S F; Heumann, H

    1995-01-01

    Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA. Images PMID:7533725

  9. Redefining regulation of DNA methylation by RNA interference

    PubMed Central

    Muthusamy, Viswanathan; Bosenberg, Marcus; Wajapeyee, Narendra

    2013-01-01

    Epigenetic changes refer to heritable changes that may modulate gene expression without affecting DNA sequence. DNA methylation is one such heritable epigenetic change, which is causally associated with the transcription regulation of many genes in the mammalian genome. Altered DNA methylation has been implicated in a wide variety of human diseases including cancer. Understanding the regulation of DNA methylation is likely to improve the ability to diagnose and treat these diseases. With the advent of high-throughput RNA interference (RNAi) screens, answering epigenetic questions on a genomic scale is now possible. Two recent genome-wide RNAi screens have addressed the regulation of DNA methylation in cancer, leading to the identification of the regulators of epigenetic silencing by oncogenic RAS and how epigenetic silencing of the tumor suppressor RASSF1A is maintained. These RNAi screens have much wider applications, since similar screens can now be adapted to identify the mechanism of silencing of any human disease-associated gene that is epigenetically regulated. In this review, we discuss two recent genome-wide RNAi screens for epigenetic regulators and explore potential applications in understanding DNA methylation and gene expression regulation in mammalian cells. We also discuss some of the key unanswered questions in the field of DNA methylation and suggest genome-wide RNAi screens designed to answer them. PMID:20620207

  10. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

    PubMed

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences. PMID:26500066

  11. RNA FISH, DNA FISH and Chromosome Painting of Chicken Oocytes.

    PubMed

    Guioli, Silvana; Lovell-Badge, Robin

    2016-01-01

    Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique. It identifies the location of DNA loci and RNAs, including nascent RNAs in the process of being transcribed, within individual cells. Great advances in fluorescent dye technology and technique sensitivity, combined with developments in light microscopy and imaging software have made it widely accessible and have expanded the range of applications in basic research as well as in diagnostics. Being able to perform RNA hybridization, DNA hybridization, and protein immunofluorescence consecutively on the same sample is an invaluable tool to study RNA expression in relation to their gene loci and to map RNA and DNA in relation to nuclear or cellular structures. This has contributed to enormous progress in understanding basal mechanisms of male and female meiosis in different animal model systems. In this chapter we describe in detail the protocols for FISH based techniques applied to study gene expression dynamics and nuclear architecture of chicken oocytes during meiotic prophase I. These techniques can be easily performed in any molecular and cell biology laboratory and be adapted to different systems and to different phases of gametogenesis. PMID:27557582

  12. A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.

    PubMed

    Pélissier, T; Wassenegger, M

    2000-01-01

    In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed. PMID:10668798

  13. Did the Pre-RNA World Rest Upon DNA Molecules?

    NASA Technical Reports Server (NTRS)

    Lazcano, Antonio; Dworkin, Jason P.; Miller, Stanley L.

    2004-01-01

    The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the

  14. Spectrofluorometric determination of DNA and RNA with berberine

    NASA Astrophysics Data System (ADS)

    Gong, Guo-Quan; Zong, Zhi-Xin; Song, Yu-Min

    1999-08-01

    On binding to nucleic acids, the dye berberine increases its fluorescence quantum efficiency by a factor of 25-30. Based on this, an easy, rapid and accurate method for the determination of nucleic acids was developed. Berberine is very like ethidium bromide (EB), but it is non-poisonous. Determination can be made at any pH between 4 and 10, where the native structure of DNA and RNA is not disrupted. The maximum emission is near 520 nm for excitation at 355 or 450 nm. This method has good sensitivity (0.01 μg ml -1 of ctDNA), high selectivity and a wide linear range (0.05-14.0 μg ml -1 of ctDNA).

  15. The RNA World: Life Before DNA and Protein

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F.

    1993-01-01

    All of the life that is known, all organisms that exist on Earth today or are known to have existed on Earth in the past, are of the same life form: a life form based on DNA and protein. It does not necessarily have to be that way. Why not have two competing life forms on this planet? Why not have biology as we know it and some other biology that occupies its own distinct niche? Yet that is not how evolution has played out. From microbes living on the surface of antarctic ice to tube worms lying near the deep-sea hydrothermal vents, all known organisms on this planet are of the same biology. Looking at the single known biology on Earth, it is clear that this biology could not have simply sprung forth from the primordial soup. The biological system that is the basis for all known. life is far too complicated to have arisen spontaneously. This brings us to the notion that something else something simpler, must have preceded life based on DNA and protein. One suggestion that has gained considerable acceptance over the past decade is that DNA and protein-based life was preceded by RNA-based life in a period referred to as the 'RNA world'. Even an RNA-based life form would have been fairly complicated - not as complicated as our own DNA- and protein-based life form - but far too complicated, according to prevailing scientific thinking, to have arisen spontaneously from the primordial soup. Thus, it has been argued that something else must have preceded RNA-based life, or even that there was a succession of life forms leading from the primordial soup to RNA-based life. The experimental evidence to support this conjecture is not strong because, after all, the origin of life was a historical event that left no direct physical record. However, based on indirect evidence in both the geological record and the phylogenetic record of evolutionary history on earth, it is possible to reconstruct a rough picture of what life was like before DNA and protein.

  16. Crystallization of Macromolecules

    PubMed Central

    Friedmann, David; Messick, Troy; Marmorstein, Ronen

    2014-01-01

    X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

  17. All-atom crystal simulations of DNA and RNA duplexes

    PubMed Central

    Liu, Chunmei; Janowski, Pawel A.; Case, David A.

    2014-01-01

    Background Molecular dynamics simulations can complement experimental measures of structure and dynamics of biomolecules. The quality of such simulations can be tested by comparisons to models refined against experimental crystallographic data. Methods We report simulations of a DNA and RNA duplex in their crystalline environment. The calculations mimic the conditions for PDB entries 1D23 [d(CGATCGATCG)2] and 1RNA [(UUAUAUAUAUAUAA)2], and contain 8 unit cells, each with 4 copies of the Watson-Crick duplex; this yields in aggregate 64 µs of duplex sampling for DNA and 16 µs for RNA. Results The duplex structures conform much more closely to the average structure seen in the crystal than do structures extracted from a solution simulation with the same force field. Sequence-dependent variations in helical parameters, and in groove widths, are largely maintained in the crystal structure, but are smoothed out in solution. However, the integrity of the crystal lattice is slowly degraded in both simulations, with the result that the interfaces between chains become heterogeneous. This problem is more severe for the DNA crystal, which has fewer inter-chain hydrogen bond contacts than does the RNA crystal. Conclusions Crystal simulations using current force fields reproduce many features of observed crystal structures, but suffer from a gradual degradation of the integrity of the crystal lattice. General significance The results offer insights into force-field simulations that tests their ability to preserve weak interactions between chains, which will be of importance also in non-crystalline applications that involve binding and recognition. PMID:25255706

  18. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    PubMed

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. PMID:27117661

  19. DNARNA: What Do Students Think the Arrow Means?

    PubMed Central

    Fisk, J. Nick; Newman, Dina L.

    2014-01-01

    The central dogma of molecular biology, a model that has remained intact for decades, describes the transfer of genetic information from DNA to protein though an RNA intermediate. While recent work has illustrated many exceptions to the central dogma, it is still a common model used to describe and study the relationship between genes and protein products. We investigated understanding of central dogma concepts and found that students are not primed to think about information when presented with the canonical figure of the central dogma. We also uncovered conceptual errors in student interpretation of the meaning of the transcription arrow in the central dogma representation; 36% of students (n = 128; all undergraduate levels) described transcription as a chemical conversion of DNA into RNA or suggested that RNA existed before the process of transcription began. Interviews confirm that students with weak conceptual understanding of information flow find inappropriate meaning in the canonical representation of central dogma. Therefore, we suggest that use of this representation during instruction can be counterproductive unless educators are explicit about the underlying meaning. PMID:26086664

  20. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    DOEpatents

    Von Dreele, Robert B.

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  1. Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

    PubMed Central

    de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

    1988-01-01

    It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment. PMID:2453027

  2. A Course on Macromolecules.

    ERIC Educational Resources Information Center

    Horta, Arturo

    1985-01-01

    Describes a senior-level course that: (1) focuses on the structure and reactions of macromolecules; (2) treats industrial polymers in a unified way; and (3) uses analysis of conformation and conformational statistics as a unifying approach. Also discusses course topics, including polysaccharides, proteins, nucleic acids, and others. (JN)

  3. Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

    PubMed Central

    Roy, Christian K; Olson, Sara; Graveley, Brenton R; Zamore, Phillip D; Moore, Melissa J

    2015-01-01

    Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA–DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1. DOI: http://dx.doi.org/10.7554/eLife.03700.001 PMID:25866926

  4. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

    PubMed Central

    Schiebel, W; Pélissier, T; Riedel, L; Thalmeir, S; Schiebel, R; Kempe, D; Lottspeich, F; Sänger, H L; Wassenegger, M

    1998-01-01

    A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. PMID:9836747

  5. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

    PubMed Central

    Hamperl, Stephan; Cimprich, Karlene A.

    2014-01-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

  6. Structural analysis of hepatitis C RNA genome using DNA microarrays

    PubMed Central

    Martell, María; Briones, Carlos; de Vicente, Aránzazu; Piron, María; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gómez, Jordi

    2004-01-01

    Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5′ non-coding region (5′NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions. PMID:15247323

  7. Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

    PubMed

    Fujiwara, T.; Giesman-Cookmeyer, D.; Ding, B.; Lommel, S. A.; Lucas, W. J.

    1993-12-01

    Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread. PMID:12271056

  8. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

    PubMed Central

    2016-01-01

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design. PMID:25853314

  9. Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

    SciTech Connect

    Mao, Xun; Liu, Guodong; Wang, Shengfu; Lin, Yuehe; Zhang, Aiguo; Zhang, Lurong; Ma, Yunqing

    2008-11-01

    We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

  10. Human age estimation from blood using mRNA, DNA methylation, DNA rearrangement, and telomere length.

    PubMed

    Zubakov, Dmitry; Liu, Fan; Kokmeijer, Iris; Choi, Ying; van Meurs, Joyce B J; van IJcken, Wilfred F J; Uitterlinden, André G; Hofman, Albert; Broer, Linda; van Duijn, Cornelia M; Lewin, Jörn; Kayser, Manfred

    2016-09-01

    Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DNA methylation markers in blood from the same young and old individuals using microarray technologies. The obtained candidate markers were validated in independent samples covering a wide age range using alternative technologies together with previously proposed DNA methylation, sjTREC, and telomere length markers. Cross-validated multiple regression analysis was applied for estimating and validating the age predictive power of various sets of biomarkers within and across different marker types. We found that DNA methylation markers outperformed mRNA, sjTREC, and telomere length in age predictive power. The best performing model included 8 DNA methylation markers derived from 3 CpG islands reaching a high level of accuracy (cross-validated R(2)=0.88, SE±6.97 years, mean absolute deviation 5.07 years). However, our data also suggest that mRNA markers can provide independent age information: a model using a combined set of 5 DNA methylation markers and one mRNA marker could provide

  11. Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

    PubMed

    Zhang, Pinghu; Liu, Fei; Guo, Fang; Zhao, Qiong; Chang, Jinhong; Guo, Ju-Tao

    2016-07-01

    Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted. PMID:27083116

  12. Electrically assisted delivery of macromolecules into the corneal epithelium

    PubMed Central

    Hao, Jinsong; Li, S. Kevin; Liu, Chia-Yang; Kao, Winston W.Y.

    2009-01-01

    Electrically assisted delivery is noninvasive and has been investigated in a number of ocular drug delivery studies. The objectives of this study were to examine the feasibility of electrically assisted delivery of macromolecules such as small interfering RNA (siRNA) into the corneal epithelium, to optimize the iontophoresis and electroporation methods, and to study the mechanisms of corneal iontophoresis for macromolecules. Anodal and cathodal iontophoresis, electroporation and their combinations were the methods examined with mice in vivo. Cyanine 3 (Cy3) glyceraldehyde 3-phosphate dehydrogenease (GAPDH) siRNA and fluorescein isothiocyanate (FITC)-dextran of different molecular weights (4 to 70 kDa) were the macromolecules studied. Microscopy and histology after cryostat sectioning were used to analyze and compare the delivery of the macromolecules to the cornea. Iontophoresis was effective in delivering siRNA and dextran up to 70 kDa into the cornea. The electroporation method studied was less effective than that of iontophoresis. Although both iontophoresis and electroporation alone can deliver the macromolecules into the cornea, these methods alone were not as effective as the combination of iontophoresis and electroporation (iontophoresis followed by electroporation). The significant enhancement of dextran delivery in anodal iontophoresis suggests that electroosmosis can be a significant flux enhancing mechanism during corneal iontophoresis. These results illustrate the feasibility of electrically assisted delivery of macromolecules such as siRNA into the cornea. PMID:19682448

  13. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

    PubMed Central

    Son, Kyung-No; Liang, Zhiguo

    2015-01-01

    ABSTRACT Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds

  14. A Case Study of the Likes and Dislikes of DNA and RNA in Self-Assembly.

    PubMed

    Zuo, Hua; Wu, Siyu; Li, Mo; Li, Yulin; Jiang, Wen; Mao, Chengde

    2015-12-01

    Programmed self-assembly of nucleic acids (DNA and RNA) is an active research area as it promises a general approach for nanoconstruction. Whereas DNA self-assembly has been extensively studied, RNA self-assembly lags much behind. One strategy to boost RNA self-assembly is to adapt the methods of DNA self-assembly for RNA self-assembly because of the chemical and structural similarities of DNA and RNA. However, these two types of molecules are still significantly different. To enable the rational design of RNA self-assembly, a thorough examination of their likes and dislikes in programmed self-assembly is needed. The current work begins to address this task. It was found that similar, two-stranded motifs of RNA and DNA lead to similar, but clearly different nanostructures. PMID:26457993

  15. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    SciTech Connect

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  16. An RNA fraction in chromatin of L-929 cells associated with DNA

    PubMed Central

    Monahan, John J.; Hall, Ross H.

    1974-01-01

    The DNA of L-929 cell nuclei was examined for the presence of an RNA fraction hybridized to the DNA that could possibly serve a role in gene regulation. A low molecular weight ([unk]4.5S) RNA fraction was observed as an RNA-DNA complex in chromatin preparations subfractionated on a Cs2SO4 density gradient. This RNA does not appear to be covalently attached to the DNA. Neither does it appear to be attached to the DNA via a protein component. The RNA is resistant to ribonuclease H but is slowly attached by ribonuclease A + Tl. A role for, as well as the nature of the attachment of this RNA to DNA is suggested. Images PMID:10793701

  17. Nucleocytoplasmic transport of macromolecules.

    PubMed Central

    Corbett, A H; Silver, P A

    1997-01-01

    Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae. PMID:9184010

  18. Oncogenes in human testicular cancer: DNA and RNA studies.

    PubMed Central

    Peltomäki, P.; Alfthan, O.; de la Chapelle, A.

    1991-01-01

    Oncogene dosage and expression were studied in 16 testicular neoplasms, 14 of germ cell and two of non-germ cell origin. In comparison with normal DNA, tumour DNA of a total of eight patients (seven with germ cell neoplasm and one with testicular lymphoma) showed increased dosages of KRAS2, PDGFA, EGFR, MET and PDGFB. The most frequent (occurring in six tumours) and prominent (up to 3-4-fold) increases were detected in the dosages of KRAS2 (on chromosome 12p) and PDGFA (chromosome 7p), relative to a reference locus from chromosome 2. Importantly, there was a similar increase in 12p dosage in general in these tumours, suggesting the presence of the characteristic isochromosome 12p marker. On the contrary, possible 7p polysomy (assessed by molecular methods) did not explain the PDGFA (or EGFR) changes in all cases. NRAS, MYCN, CSFIR, MYB, MYC, ABL, HRASI, TP53, and ERBB2 did not reveal any consistent alterations in tumour DNA. In RNA dot blot assays the expression of KRAS2, PDGFA, EGFR, or MYC was generally not increased in the tumour samples when compared to that in normal testicular tissue of the same patients although there was interindividual variation in mRNA levels. It thus appears that while oncogene dosage changes occur in a proportion of testis cancers, they are often part of changes in large chromosomal regions or whole arms and are seldom accompanied by altered expression. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1829952

  19. Long-Range Charge Transport in Adenine-Stacked RNA:DNA Hybrids.

    PubMed

    Li, Yuanhui; Artés, Juan M; Hihath, Joshua

    2016-01-27

    An extremely important biological component, RNA:DNA can also be used to design nanoscale structures such as molecular wires. The conductance of single adenine-stacked RNA:DNA hybrids is rapidly and reproducibly measured using the break junction approach. The conductance decreases slightly over a large range of molecular lengths, suggesting that RNA:DNA can be used as an oligonucleotide wire. PMID:26596516

  20. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    PubMed

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric

    2016-02-24

    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context. PMID:26808196

  1. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers.

    PubMed

    Jolly, Pawan; Estrela, Pedro; Ladomery, Michael

    2016-06-30

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  2. Programmed self-assembly of DNA/RNA for biomedical applications

    NASA Astrophysics Data System (ADS)

    Wang, Pengfei

    Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

  3. DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

    PubMed Central

    Coulombe, Benoit; Burton, Zachary F.

    1999-01-01

    A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. PMID:10357858

  4. Decoding DNA, RNA and peptides with quantum tunnelling

    NASA Astrophysics Data System (ADS)

    di Ventra, Massimiliano; Taniguchi, Masateru

    2016-02-01

    Drugs and treatments could be precisely tailored to an individual patient by extracting their cellular- and molecular-level information. For this approach to be feasible on a global scale, however, information on complete genomes (DNA), transcriptomes (RNA) and proteomes (all proteins) needs to be obtained quickly and at low cost. Quantum mechanical phenomena could potentially be of value here, because the biological information needs to be decoded at an atomic level and quantum tunnelling has recently been shown to be able to differentiate single nucleobases and amino acids in short sequences. Here, we review the different approaches to using quantum tunnelling for sequencing, highlighting the theoretical background to the method and the experimental capabilities demonstrated to date. We also explore the potential advantages of the approach and the technical challenges that must be addressed to deliver practical quantum sequencing devices.

  5. Decoding DNA, RNA and peptides with quantum tunnelling.

    PubMed

    Di Ventra, Massimiliano; Taniguchi, Masateru

    2016-02-01

    Drugs and treatments could be precisely tailored to an individual patient by extracting their cellular- and molecular-level information. For this approach to be feasible on a global scale, however, information on complete genomes (DNA), transcriptomes (RNA) and proteomes (all proteins) needs to be obtained quickly and at low cost. Quantum mechanical phenomena could potentially be of value here, because the biological information needs to be decoded at an atomic level and quantum tunnelling has recently been shown to be able to differentiate single nucleobases and amino acids in short sequences. Here, we review the different approaches to using quantum tunnelling for sequencing, highlighting the theoretical background to the method and the experimental capabilities demonstrated to date. We also explore the potential advantages of the approach and the technical challenges that must be addressed to deliver practical quantum sequencing devices. PMID:26839257

  6. Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

    PubMed

    Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

    2014-01-29

    We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression. PMID:24392825

  7. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    PubMed Central

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. PMID:24476820

  8. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    NASA Astrophysics Data System (ADS)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  9. Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

    PubMed

    Kilianski, Andy; Roth, Pierce A; Liem, Alvin T; Hill, Jessica M; Willis, Kristen L; Rossmaier, Rebecca D; Marinich, Andrew V; Maughan, Michele N; Karavis, Mark A; Kuhn, Jens H; Honko, Anna N; Rosenzweig, C Nicole

    2016-08-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  10. A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    PubMed Central

    Wan, Guohui; Hu, Xiaoxiao; Liu, Yunhua; Han, Cecil; Sood, Anil K; Calin, George A; Zhang, Xinna; Lu, Xiongbin

    2013-01-01

    A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti-cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA-JADE, that is induced after DNA damage in an ataxia-telangiectasia mutated (ATM)-dependent manner. LncRNA-JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA-JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA-JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA-JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA-JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA-JADE may contribute to breast tumorigenesis. PMID:24097061

  11. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  12. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity.

    PubMed

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M

    2015-12-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA-RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA-RNA and RNA-RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA-RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  13. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    NASA Astrophysics Data System (ADS)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

  14. Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

    PubMed Central

    Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

    2014-01-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  15. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

    PubMed

    Chan, Yujia A; Aristizabal, Maria J; Lu, Phoebe Y T; Luo, Zongli; Hamza, Akil; Kobor, Michael S; Stirling, Peter C; Hieter, Philip

    2014-04-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  16. The Roads to and from the RNA World

    NASA Technical Reports Server (NTRS)

    Dworkin, Jason P.; Lazcano, Antonio; Miller, Stanley L.

    2003-01-01

    The historical existence of the RNA world, in which early life used RNA for both genetic information and catalytic ability, is widely accepted. However, there has been little discussion of whether protein synthesis arose before DNA or what preceded the RNA world (i.e. the pre-RNA world). We outline arguments of what route life may have taken out of the RNA world: whether DNA or protein followed. The metabolic arguments favor the possibility that RNA genomes preceded the use of DNA as the informational macromolecule. However, the opposite can also be argued based on the enhanced stability, reactivity, and solubility of 2-deoxyribose as compared to ribose. The possibility that DNA may have come before RNA is discussed, although it is a less parsimonious explanation than DNA following RNA.

  17. 4'-Epi-DNA: A DNA Mimic Containing 4'-hydroxymethyl-α-l-Xylo-Thymidine with Compact Backbone like RNA.

    PubMed

    Bagmare, Seema; Puranik, Vedavati G; Fernandes, Moneesha; Kumar, Vaijayanti A

    2016-09-01

    Synthesis of C4'-epi-DNA containing 3'→ 5″ linkages is reported for the first time. Crystal structure study of the monomer indicated that though the dihedral angle O3'-C3'-C4'-C5″ in this case would be like in RNA, the sugar conformation would remain like that in DNA. The study of the effect of this backbone configuration in DNA with respect to its binding to cDNA and RNA is reported in this note. PMID:27556783

  18. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    NASA Technical Reports Server (NTRS)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  19. Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends

    PubMed Central

    Vvedenskaya, Irina O.; Goldman, Seth R.; Nickels, Bryce E.

    2015-01-01

    Summary We provide a detailed protocol for preparing cDNA libraries suitable for high throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and post-transcriptional processing of RNAs with single base pair resolution on a genome-wide level. PMID:25665566

  20. Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

    PubMed Central

    Peña-Llopis, Samuel; Brugarolas, James

    2014-01-01

    Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

  1. Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

    PubMed Central

    Massanella, Marta; Gianella, Sara; Lada, Steven M.; Richman, Douglas D.; Strain, Matthew C.

    2016-01-01

    Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR). For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol “Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma” (Vargas-Meneses et al., 2015).

  2. Discovery of Nuclear DNA-like RNA (dRNA, hnRNA) and Ribonucleoproteins Particles Containing hnRNA.

    PubMed

    Georgiev, G P

    2016-01-01

    On August 9-11, 2014, Cold Spring Harbor (USA) hosted a special symposium dedicated to the discovery of messenger or informational RNA and the main events in the subsequent studies of its synthesis, regulation of synthesis, maturation, and transport. The existence of mRNA in bacteria was first suggested in 1961 by Jacob and Monod, based on genetic studies [1]. The same year, Brenner et al. confirmed the hypothesis [2]. Our laboratory played a key role in the discovery of messenger RNA in eukaryotes, as well as in the discovery of the nuclear ribonucleoproteins that contain it and in the elucidation of their structural organization. Therefore, I was invited to represent Russia at the Symposium and deliver a speech on these topics. However, my visa had only been issued after the end of the Symposium, and, therefore, the presentation was delivered by my former colleague G.N. Yenikolopov, who works at Cold Spring Harbor Laboratory. The transcript of the lecture is presented below. PMID:27099780

  3. Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

    PubMed

    Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols. PMID:26796702

  4. A rapid procedure to detect in situ DNA/RNA hybrids

    SciTech Connect

    Reddy, A.R.; Sofer, W.

    1981-12-15

    A new method was developed for detecting DNA/RNA hydrids formed in situ using anti-DNA/RNA antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesized in vitro from cloned Drosophila histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.

  5. A comparison of RNA with DNA in template-directed synthesis

    NASA Technical Reports Server (NTRS)

    Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

  6. Modified method for combined DNA and RNA isolation from peanut and other oil seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  7. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

    SciTech Connect

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley; Ke, Ailong

    2011-07-25

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.

  8. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor.

    PubMed

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley; Ke, Ailong

    2011-05-01

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA. PMID:21471452

  9. An Attempt to Detect siRNA-Mediated Genomic DNA Modification by Artificially Induced Mismatch siRNA in Arabidopsis

    PubMed Central

    Miyagawa, Yosuke; Ogawa, Jun; Iwata, Yuji; Koizumi, Nozomu; Mishiba, Kei-ichiro

    2013-01-01

    Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the ‘RNA cache’ hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the ‘RNA cache’ hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically. PMID:24278423

  10. Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

    PubMed Central

    Kuramae, Eiko E.; Verbruggen, Erik; Hillekens, Remy; de Hollander, Mattias; Röling, Wilfred F. M.; van der Heijden, Marcel G. A.; Kowalchuk, George A.

    2013-01-01

    We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time. PMID:23875012

  11. Ocular delivery of macromolecules

    PubMed Central

    Kim, Yoo-Chun; Chiang, Bryce; Wu, Xianggen; Prausnitz, Mark R.

    2014-01-01

    Biopharmaceuticals are making increasing impact on medicine, including treatment of indications in the eye. Macromolecular drugs are typically given by physician-administered invasive delivery methods, because non--invasive ocular delivery methods, such as eye drops, and systemic delivery, have low bioavailability and/or poor ocular targeting. There is a need to improve delivery of biopharmaceuticals to enable less-invasive delivery routes, less-frequent dosing through controlled-release drug delivery and improved drug targeting within the eye to increase efficacy and reduce side effects. This review discusses the barriers to drug delivery via various ophthalmic routes of administration in the context of macromolecule delivery and discusses efforts to develop controlled-release systems for delivery of biopharmaceuticals to the eye. The growing number of macromolecular therapies in the eye needs improved drug delivery methods that increase drug efficacy, safety and patient compliance. PMID:24998941

  12. Transcriptional regulation mechanism mediated by miRNA-DNA•DNA triplex structure stabilized by Argonaute.

    PubMed

    Toscano-Garibay, Julia D; Aquino-Jarquin, Guillermo

    2014-11-01

    Transcription regulation depends on interactions between repressor or activator proteins with promoter sequences, while post-transcriptional regulation typically relies on microRNA (miRNA) interaction with sequences in 5' and 3'-Untranslated regions (UTRs) of messenger RNA (mRNA). However, several pieces of evidence suggest that miRNA:Argonaute (AGO) complexes may also suppress transcription through RNA interference (RNAi) components and epigenetic mechanisms. However, recent observations suggest that miRNA-induced transcriptional silencing could be exerted by an unknown mechanism independent of chromatin modifiers. The RNA-DNA•DNA triplex structure has emerged as an important RNA tertiary motif in which successive non-canonical base pairs form between a DNA-DNA duplex and a third strand. Frequently, promoters have Purine (PU)-rich tracts, and some Triplex-forming oligonucleotides (TFOs) targeting these regulatory regions have been shown to inhibit transcription selectively. Here, we summarize observations suggesting that miRNAs exert regulation over promoter regions through miRNA-DNA•DNA triplex structure formation stabilized by AGO proteins which represents a plausible model of RNA-mediated Transcriptional gene silencing (TGS). PMID:25086339

  13. Interacting RNA polymerase motors on a DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis.

    PubMed

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions. PMID:18351890

  14. Viral evasion of intracellular DNA and RNA sensing.

    PubMed

    Chan, Ying Kai; Gack, Michaela U

    2016-06-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals. PMID:27174148

  15. DNA, RNA, and Protein Extraction: The Past and The Present

    PubMed Central

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination. PMID:20011662

  16. Construction of infectious cDNA clones for RNA viruses: Turnip crinkle virus.

    PubMed

    Ryabov, Eugene V

    2008-01-01

    Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones. PMID:18370276

  17. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin.

    PubMed

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-01-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecule, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G • U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G • U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation. PMID:26461456

  18. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    NASA Astrophysics Data System (ADS)

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-10-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

  19. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    PubMed

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. PMID:25881991

  20. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  1. Single Molecule Photobleaching (SMPB) Technology for Counting of RNA, DNA, Protein and Other Molecules in Nanoparticles and Biological Complexes by TIRF Instrumentation

    PubMed Central

    Zhang, Hui; Guo, Peixuan

    2014-01-01

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The Single Molecule Photobleaching (SMPB) technology for direct counting developed by our team (Shu et al, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. PMID:24440482

  2. MicroRNA expression and its association with DNA repair in preimplantation embryos

    PubMed Central

    TULAY, Pinar; SENGUPTA, Sioban B.

    2016-01-01

    Active DNA repair pathways are crucial for preserving genomic integrity and are likely among the complex mechanisms involved in the normal development of preimplantation embryos. MicroRNAs (miRNA), short non-coding RNAs, are key regulators of gene expression through the post-transcriptional and post-translational modification of mRNA. The association of miRNA expression with infertility or polycystic ovarian syndrome has been widely investigated; however, there are limited data regarding the importance of miRNA regulation in DNA repair during preimplantation embryo development. In this article, we review normal miRNA biogenesis and consequences of aberrant miRNA expression in the regulation of DNA repair in gametes and preimplantation embryos. PMID:26853522

  3. Non-coding RNA generated following lariat-debranching mediates targeting of AID to DNA

    PubMed Central

    Zheng, Simin; Vuong, Bao Q.; Vaidyanathan, Bharat; Lin, Jia-Yu; Huang, Feng-Ting; Chaudhuri, Jayanta

    2015-01-01

    SUMMARY Transcription through immunoglobulin switch (S) regions is essential for class switch recombination (CSR) but no molecular function of the transcripts has been described. Likewise, recruitment of activation-induced cytidine deaminase (AID) to S regions is critical for CSR; however, the underlying mechanism has not been fully elucidated. Here, we demonstrate that intronic switch RNA acts in trans to target AID to S region DNA. AID binds directly to switch RNA through G-quadruplexes formed by the RNA molecules. Disruption of this interaction by mutation of a key residue in the putative RNA-binding domain of AID impairs recruitment of AID to S region DNA, thereby abolishing CSR. Additionally, inhibition of RNA lariat processing leads to loss of AID localization to S regions and compromises CSR; both defects can be rescued by exogenous expression of switch transcripts in a sequence-specific manner. These studies uncover an RNA-mediated mechanism of targeting AID to DNA. PMID:25957684

  4. Interspersion of sequences in avian myeloblastosis virus rna that rapidly hybridize with leukemic chicken cell DNA.

    PubMed Central

    Drohan, W N; Shoyab, M; Wall, R; Baluda, M A

    1975-01-01

    Liquid hybridization of progressively smaller fragments (35S, 27S, 15.5S, 12.5S, and 8S) of poly(A)-selected avian myeloblastosis virus RNA with excess DNA from leukemic chicken myeloblasts revealed that all sizes of RNA contained sequences complementary to both slowly and rapidly hybridizing cellular DNA sequences. Apparently, the RNA sequences which hybridize rapidly with excesses of cellular DNA are not restricted to any one region of the avian myeloblastosis virus 35S RNA. Instead, they appear to be randomly distributed over the entire 35S avian myeloblastosis virus RNA molecule with some positioned within 200 nucleotides of the poly(A) tract at the 3' end of the RNA. PMID:163372

  5. Determination of DNA and RNA Methylation in Circulating Tumor Cells by Mass Spectrometry.

    PubMed

    Huang, Wei; Qi, Chu-Bo; Lv, Song-Wei; Xie, Min; Feng, Yu-Qi; Huang, Wei-Hua; Yuan, Bi-Feng

    2016-01-19

    DNA methylation (5-methylcytosine, 5-mC) is the best characterized epigenetic mark that has regulatory roles in diverse biological processes. Recent investigation of RNA modifications also raises the possible functions of RNA adenine and cytosine methylations on gene regulation in the form of "RNA epigenetics." Previous studies demonstrated global DNA hypomethylation in tumor tissues compared to healthy controls. However, DNA and RNA methylation in circulating tumor cells (CTCs) that are derived from tumors are still a mystery due to the lack of proper analytical methods. In this respect, here we established an effective CTCs capture system conjugated with a combined strategy of sample preparation for the captured CTCs lysis, nucleic acids digestion, and nucleosides extraction in one tube. The resulting nucleosides were then further analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). With the developed method, we are able to detect DNA and RNA methylation (5-methyl-2'-deoxycytidine, 5-methylcytidine, and N(6)-methyladenosine) in a single cell. We then further successfully determined DNA and RNA methylation in CTCs from lung cancer patients. Our results demonstrated, for the first time, a significant decrease of DNA methylation (5-methyl-2'-deoxycytidine) and increase of RNA adenine and cytosine methylations (N(6)-methyladenosine and 5-methylcytidine) in CTCs compared with whole blood cells. The discovery of DNA hypomethylation and RNA hypermethylation in CTCs in the current study together with previous reports of global DNA hypomethylation in tumor tissues suggest that nucleic acid modifications play important roles in the formation and development of cancer cells. This work constitutes the first step for the investigation of DNA and RNA methylation in CTCs, which may facilitate uncovering the metastasis mechanism of cancers in the future. PMID:26707930

  6. Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

    PubMed Central

    Szabat, Marta; Pedzinski, Tomasz; Czapik, Tomasz; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes. PMID:26579720

  7. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    PubMed Central

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-01-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted. Images PMID:1696290

  8. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

    PubMed Central

    Pai, Dave A.; Kaplan, Craig D.; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C.; Engelke, David R.

    2014-01-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template. PMID:24614752

  9. Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays

    PubMed Central

    Shakhmin, Anton; Jones, John-Paul; Bychinskaya, Inessa; Zibinsky, Mikhail; Oertell, Keriann; Goodman, Myron F.; Prakash, G.K. Surya

    2015-01-01

    Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA polymerase β. PMID:26279588

  10. Molecular underpinnings of Aprataxin RNA/DNA deadenylase function and dysfunction in neurological disease.

    PubMed

    Schellenberg, Matthew J; Tumbale, Percy P; Williams, R Scott

    2015-03-01

    Eukaryotic DNA ligases seal DNA breaks in the final step of DNA replication and repair transactions via a three-step reaction mechanism that can abort if DNA ligases encounter modified DNA termini, such as the products and repair intermediates of DNA oxidation, alkylation, or the aberrant incorporation of ribonucleotides into genomic DNA. Such abortive DNA ligation reactions act as molecular checkpoint for DNA damage and create 5'-adenylated nucleic acid termini in the context of DNA and RNA-DNA substrates in DNA single strand break repair (SSBR) and ribonucleotide excision repair (RER). Aprataxin (APTX), a protein altered in the heritable neurological disorder Ataxia with Oculomotor Apraxia 1 (AOA1), acts as a DNA ligase "proofreader" to directly reverse AMP-modified nucleic acid termini in DNA- and RNA-DNA damage responses. Herein, we survey APTX function and the emerging cell biological, structural and biochemical data that has established a molecular foundation for understanding the APTX mediated deadenylation reaction, and is providing insights into the molecular bases of APTX deficiency in AOA1. PMID:25637650

  11. Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

    PubMed Central

    Thilly, William G; Gostjeva, Elena V; Koledova, Vera V; Zukerberg, Lawrence R; Chung, Daniel; Fomina, Janna N; Darroudi, Firouz; Stollar, B David

    2014-01-01

    Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development. PMID:24418910

  12. Legume genomics: understanding biology through DNA and RNA sequencing

    PubMed Central

    O'Rourke, Jamie A.; Bolon, Yung-Tsi; Bucciarelli, Bruna; Vance, Carroll P.

    2014-01-01

    Background The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. Scope and Conclusions This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes. PMID:24769535

  13. A Simple RNA-DNA Scaffold Templates the Assembly of Monofunctional Virus-Like Particles.

    PubMed

    Garmann, Rees F; Sportsman, Richard; Beren, Christian; Manoharan, Vinothan N; Knobler, Charles M; Gelbart, William M

    2015-06-24

    Using the components of a particularly well-studied plant virus, cowpea chlorotic mottle virus (CCMV), we demonstrate the synthesis of virus-like particles (VLPs) with one end of the packaged RNA extending out of the capsid and into the surrounding solution. This construct breaks the otherwise perfect symmetry of the capsid and provides a straightforward route for monofunctionalizing VLPs using the principles of DNA nanotechnology. It also allows physical manipulation of the packaged RNA, a previously inaccessible part of the viral architecture. Our synthesis does not involve covalent chemistry of any kind; rather, we trigger capsid assembly on a scaffold of viral RNA that is hybridized at one end to a complementary DNA strand. Interaction of CCMV capsid protein with this RNA-DNA template leads to selective packaging of the RNA portion into a well-formed capsid but leaves the hybridized portion poking out of the capsid through a small hole. We show that the nucleic acid protruding from the capsid is capable of binding free DNA strands and DNA-functionalized colloidal particles. Separately, we show that the RNA-DNA scaffold can be used to nucleate virus formation on a DNA-functionalized surface. We believe this self-assembly strategy can be adapted to viruses other than CCMV. PMID:26043403

  14. Hydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages.

    PubMed

    Andrews-Pfannkoch, Cynthia; Fadrosh, Douglas W; Thorpe, Joyce; Williamson, Shannon J

    2010-08-01

    Metagenomics can be used to determine the diversity of complex, often unculturable, viral communities with various nucleic acid compositions. Here, we report the use of hydroxyapatite chromatography to efficiently fractionate double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), dsRNA, and ssRNA genomes from known bacteriophages. Linker-amplified shotgun libraries were constructed to generate sequencing reads from each hydroxyapatite fraction. Greater than 90% of the reads displayed significant similarity to the expected genomes at the nucleotide level. These methods were applied to marine viruses collected from the Chesapeake Bay and the Dry Tortugas National Park. Isolated nucleic acids were fractionated using hydroxyapatite chromatography followed by linker-amplified shotgun library construction and sequencing. Taxonomic analysis demonstrated that the majority of environmental sequences, regardless of their source nucleic acid, were most similar to dsDNA viruses, reflecting the bias of viral metagenomic sequence databases. PMID:20543058

  15. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  16. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

    PubMed Central

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

  17. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization.

    PubMed

    Heissig, Philipp; Klein, Philipp M; Hadwiger, Philipp; Wagner, Ernst

    2016-01-01

    siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4-10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3' end of the siRNA passenger strand was beneficial over the 5' end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol-shielded polyplex to downregulate a luciferase gene in folate receptor-positive cells. PMID:26928236

  18. Replication of hepatitis delta virus RNA in mice after intramuscular injection of plasmid DNA.

    PubMed Central

    Polo, J M; Lim, B; Govindarajan, S; Lai, M M

    1995-01-01

    To establish a readily manipulable small-animal system for the study of human hepatitis delta virus (HDV) replication in vivo, plasmid DNAs containing head-to-tail cDNA dimers of HDV were inoculated intramuscularly into mice. Genomic-sense HDV RNA was detected in the injected muscle within 1 week and increased to substantial levels by week 7 postinjection. The intramuscular accumulation of HDV RNA was determined to be the direct result of viral RNA replication by three lines of evidence: (i) injected tissues also accumulated antigenomic-sense HDV RNA, (ii) plasmid DNA that synthesized primary transcripts of antigenomic sense also led to the accumulation of genomic-sense HDV RNA, and (iii) injection of a cDNA dimer defective in antigenomic RNA cleavage failed to produce detectable HDV RNA in muscle. Immunohistochemical analysis of injected muscle demonstrated the presence and nuclear localization of hepatitis delta antigen in myocytes. Finally, sera from DNA-injected mice contained antibodies specific for delta antigen, indicating the induction of an immunological response to the intracellularly expressed antigen. These findings demonstrated the ability of HDV RNA to replicate in skeletal muscle and provide a useful system for the study of HDV replication, delta antigen processing, and its presentation to the immune system in vivo. Furthermore, this system offers an efficiently replicating RNA as a potential vehicle for in vivo gene transfer. PMID:7609095

  19. Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes

    PubMed Central

    Bai, Li-Ping; Hagihara, Masaki; Nakatani, Kazuhiko; Jiang, Zhi-Hong

    2014-01-01

    A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K+-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na+-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K+-form human telomeric DNA G-quadruplex over the Na+-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands. PMID:25341562

  20. Detection of messenger RNA from the isoleucine--valine operons of Salmonella typhimurium by heterologous DNA-RNA hybridization: involvement of transfer RNA in transcriptional repression.

    PubMed

    Childs, G; Sonnenberg, F; Freundlich, M

    1977-03-01

    A hybridization assay using Escherichia coli K-12 DNA isolated from the specialized transducing bacteriophage gammaCI857St68h80 dilv was used to examine the rate of synthesis of the messenger RNA's (mRNA) derived from the isoleucine-valine (ilv) gene cluster of Salmonella typhimurium. In all cases examined, changes in ilv enzyme levels could be correlated with changes in the rate of synthesis of ilv mRNA. Several well characterized regulatory mutants of S. typhimurium had rates of synthesis of ilv mRNA 3 to 8-fold higher than the repressed wild-type strain. The increased rates of ilv mRNA synthesis found in a hisT strain as well as in isoleucyl-and leucyl-tRNA SYNTHETASE MUTANTS, STRONGLY SUGGESTS A ROLE FOR BRANCHED-CHAIN AMINOACYL-TRNA's in transcriptional control. PMID:327261

  1. Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor.

    PubMed

    Tanaka, Terumichi; Hori, Yoshiaki; Kikuchi, Yo

    2002-10-01

    Previously, we found that a small (approx. 20-mer) DNA hybridizing to the 5'-leader region of a tRNA precursor enhances the cleavage efficiency in bacterial ribonuclease P reaction. We named this technique the 'guide DNA technique'. Detailed analyses showed that the length of the guide DNA, concentration of the guide DNA and the hybridizing position affected the cleavage efficiency: for an effective cleavage reaction, guide DNA should be designed to hybridize to the region on the cleavage site, should be 20 bases or more in length and should be of high concentration. The presence of a 5'-flanking region in the DNA did not affect the cleavage reaction. The guide DNA technique is a useful tool for effective preparation of mature tRNA molecules in vitro. PMID:12241548

  2. Replication of Mengovirus I. Effect on Synthesis of Macromolecules by Host Cell

    PubMed Central

    Plagemann, Peter G. W.; Swim, H. Earle

    1966-01-01

    Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 91:2317–2326. 1966.—The replication of mengovirus was studied in two strains of Novikoff (rat) hepatoma cells propagated in vitro. The replicative cycle in both strains required 6.5 to 7 hr. Infection resulted in a marked depression of ribonucleic acid (RNA) and protein synthesis by strain N1S1-63. Inhibition of RNA synthesis was reflected by a decrease in the deoxyribonucleic acid (DNA)-dependent RNA polymerase activity of isolated nuclei. Mengovirus had no effect on either protein or RNA synthesis or on the DNA-dependent RNA polymerase activity of a second strain, N1S1-67. The time course of viral-induced synthesis of RNA by cells was studied in cells treated with actinomycin D. It was first detectable between 2.5 and 3 hr after infection and continued until 6.5 to 7 hr. The formation of mature virus was estimated biochemically by measuring the amount of RNA synthesized as a result of viral infection which was resistant to degradation by ribonuclease in the presence of deoxycholate. Approximately 70% of the deoxycholate-ribonuclease-resistant RNA was located in mature virus, and the remainder was double-stranded. The formation of mature virus began about 45 min after viral-directed (actinomycin-resistant) synthesis of RNA was detectable in the cell, and only about 18 to 20% of the total RNA synthesized was incorporated into virus. Release of virus from cells began about 1 hr after maturation was first detectable. Release of virus from cells was accompanied by a loss of a large proportion of their cytoplasmic RNA and protein. PMID:4287585

  3. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    EPA Science Inventory

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
    Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  4. Evolution from DNA to RNA Recognition by the bI3 LAGLIDADG Maturase

    SciTech Connect

    Longo,A.; Leonard, C.; Bassi, G.; Berndt, D.; Krahn, J.; Tanaka Hall, T.; Weeks, K.

    2005-01-01

    LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 Angstroms from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.

  5. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  6. Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA.

    PubMed

    Kwok, Albert; McCarthy, David; Hart, Stephen L; Tagalakis, Aristides D

    2016-05-01

    The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved. PMID:26684657

  7. RNA-activated DNA cleavage by the Type III-B CRISPR-Cas effector complex.

    PubMed

    Estrella, Michael A; Kuo, Fang-Ting; Bailey, Scott

    2016-02-15

    The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine-aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems. PMID:26848046

  8. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.

    PubMed

    Ashkenazy, Haim; Abadi, Shiran; Martz, Eric; Chay, Ofer; Mayrose, Itay; Pupko, Tal; Ben-Tal, Nir

    2016-07-01

    The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (http://consurf.tau.ac.il), established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree. PMID:27166375

  9. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules

    PubMed Central

    Ashkenazy, Haim; Abadi, Shiran; Martz, Eric; Chay, Ofer; Mayrose, Itay; Pupko, Tal; Ben-Tal, Nir

    2016-01-01

    The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (http://consurf.tau.ac.il), established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree. PMID:27166375

  10. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    PubMed

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes. PMID:26418169

  11. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

    PubMed Central

    Kassavetis, G A; Riggs, D L; Negri, R; Nguyen, L H; Geiduschek, E P

    1989-01-01

    Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes. Images PMID:2668737

  12. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

    1995-04-11

    A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

  13. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

    1995-01-01

    Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

  14. ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We are reviewing here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the double-stranded RNA and Z-DNA binding domains. PMID:21728134

  15. An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

    PubMed Central

    Zhao, Wei; Jardine, Paul J.

    2015-01-01

    ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful

  16. UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes

    PubMed Central

    Tremblay, Maxime; Charton, Romain; Wittner, Manuel; Levasseur, Geneviève; Griesenbeck, Joachim; Conconi, Antonio

    2014-01-01

    The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5′-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin. PMID:24097442

  17. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity

    PubMed Central

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M.

    2015-01-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA–RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA–RNA and RNA–RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA–RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  18. Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

    PubMed Central

    Lagunavicius, Arunas; Merkiene, Egle; Kiveryte, Zivile; Savaneviciute, Agne; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2009-01-01

    We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3′→5′ RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3′-end. PMID:19244362

  19. Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

    PubMed

    Seio, Kohji; Tokugawa, Munefumi; Tsunoda, Hirosuke; Ohkubo, Akihiro; Arisaka, Fumio; Sekine, Mitsuo

    2013-12-15

    The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration. PMID:24183539

  20. Structure of the RNA claw of the DNA packaging motor of bacteriophage Φ29.

    PubMed

    Harjes, Elena; Kitamura, Aya; Zhao, Wei; Morais, Marc C; Jardine, Paul J; Grimes, Shelley; Matsuo, Hiroshi

    2012-10-01

    Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation. PMID:22879380

  1. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  2. SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

    PubMed Central

    Turner, Kevin B.; Yi-Brunozzi, Hye Young; Brinson, Robert G.; Marino, John P.; Fabris, Daniele; Le Grice, Stuart F.J.

    2009-01-01

    Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (Mr = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed. PMID:19535461

  3. Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants.

    PubMed Central

    Wang, M B; Wesley, S V; Finnegan, E J; Smith, N A; Waterhouse, P M

    2001-01-01

    Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules. PMID:11214177

  4. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    PubMed

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  5. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    PubMed Central

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  6. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases

    PubMed Central

    Iyer, Lakshminarayan M; Koonin, Eugene V; Aravind, L

    2003-01-01

    Background The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. Results Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the β' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi β-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP β' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases

  7. Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

    PubMed Central

    Sun, Yan; Borbat, Peter P.; Grigoryants, Vladimir M.; Myers, William K.; Freed, Jack H.; Scholes, Charles P.

    2015-01-01

    Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ∼35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present. PMID:25692594

  8. Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes.

    PubMed

    Shah, Pratik; Cho, Seok Keun; Thulstrup, Peter Waaben; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

    2014-01-31

    MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded. PMID:24393838

  9. Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes

    NASA Astrophysics Data System (ADS)

    Shah, Pratik; Cho, Seok Keun; Waaben Thulstrup, Peter; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

    2014-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ˜21 nt to ˜25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

  10. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  11. Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.

    PubMed

    Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M

    2015-01-01

    STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. PMID:25284026

  12. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces

    PubMed Central

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-01-01

    Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

  13. Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

    PubMed Central

    Kibler-Herzog, L; Zon, G; Uznanski, B; Whittier, G; Wilson, W D

    1991-01-01

    The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations. PMID:1711677

  14. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

    PubMed

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-09-01

    Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

  15. MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures.

    PubMed

    Mondal, Tanmoy; Subhash, Santhilal; Vaid, Roshan; Enroth, Stefan; Uday, Sireesha; Reinius, Björn; Mitra, Sanhita; Mohammed, Arif; James, Alva Rani; Hoberg, Emily; Moustakas, Aristidis; Gyllensten, Ulf; Jones, Steven J M; Gustafsson, Claes M; Sims, Andrew H; Westerlund, Fredrik; Gorab, Eduardo; Kanduri, Chandrasekhar

    2015-01-01

    Long noncoding RNAs (lncRNAs) regulate gene expression by association with chromatin, but how they target chromatin remains poorly understood. We have used chromatin RNA immunoprecipitation-coupled high-throughput sequencing to identify 276 lncRNAs enriched in repressive chromatin from breast cancer cells. Using one of the chromatin-interacting lncRNAs, MEG3, we explore the mechanisms by which lncRNAs target chromatin. Here we show that MEG3 and EZH2 share common target genes, including the TGF-β pathway genes. Genome-wide mapping of MEG3 binding sites reveals that MEG3 modulates the activity of TGF-β genes by binding to distal regulatory elements. MEG3 binding sites have GA-rich sequences, which guide MEG3 to the chromatin through RNA-DNA triplex formation. We have found that RNA-DNA triplex structures are widespread and are present over the MEG3 binding sites associated with the TGF-β pathway genes. Our findings suggest that RNA-DNA triplex formation could be a general characteristic of target gene recognition by the chromatin-interacting lncRNAs. PMID:26205790

  16. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function

    PubMed Central

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-01-01

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition–fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates “bifacial polymer nucleic acids” (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure–function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  17. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    PubMed

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  18. In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.

    PubMed

    Shah, Pratik; Thulstrup, Peter Waaben; Cho, Seok Keun; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Bjerrum, Morten Jannik; Yang, Seong Wook

    2014-05-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method. PMID:24616905

  19. Viral nanomotors for packaging of dsDNA and dsRNA

    PubMed Central

    Guo, Peixuan; Lee, Tae Jin

    2007-01-01

    While capsid proteins are assembled around single-stranded genomic DNA or RNA in rod-shaped viruses, the lengthy double-stranded genome of other viruses is packaged forcefully within a preformed protein shell. This entropically unfavourable DNA or RNA packaging is accomplished by an ATP-driven viral nanomotor, which is mainly composed of two components, the oligomerized channel and the packaging enzymes. This intriguing DNA or RNA packaging process has provoked interest among virologists, bacteriologists, biochemists, biophysicists, chemists, structural biologists and computational scientists alike, especially those interested in nanotechnology, nanomedicine, AAA+ family proteins, energy conversion, cell membrane transport, DNA or RNA replication and antiviral therapy. This review mainly focuses on the motors of double-stranded DNA viruses, but double-stranded RNA viral motors are also discussed due to interesting similarities. The novel and ingenious configuration of these nanomotors has inspired the development of biomimetics for nanodevices. Advances in structural and functional studies have increased our understanding of the molecular basis of biological movement to the point where we can begin thinking about possible applications of the viral DNA packaging motor in nanotechnology and medical applications. PMID:17501915

  20. Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

    PubMed Central

    Luke, Jeremy; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2009-01-01

    To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1 gm/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination. PMID:19559109

  1. Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

    PubMed

    Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo; Gu, Jianyou; Suwa, Yoshiaki; Pavlov, Youri I; Tahirov, Tahir H

    2016-05-01

    The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs. PMID:26975377

  2. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  3. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

    PubMed

    Karmakar, Saswata; Harcourt, Emily M; Hewings, David S; Scherer, Florian; Lovejoy, Alexander F; Kurtz, David M; Ehrenschwender, Thomas; Barandun, Luzi J; Roost, Caroline; Alizadeh, Ash A; Kool, Eric T

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

  4. DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

    PubMed Central

    Du, Yaqing; Topp, Christopher N.; Dawe, R. Kelly

    2010-01-01

    Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically). It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics. PMID:20140237

  5. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA.

    PubMed

    Fonfara, Ines; Richter, Hagen; Bratovič, Majda; Le Rhun, Anaïs; Charpentier, Emmanuelle

    2016-04-28

    CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described. PMID:27096362

  6. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

  7. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  8. Macromolecules Relevant to Stone Formation

    NASA Astrophysics Data System (ADS)

    Ryall, Rosemary L.; Cook, Alison F.; Thurgood, Lauren A.; Grover, Phulwinder K.

    2007-04-01

    Despite years of research, no single macromolecule in kidney calculi or in urine has yet been shown to fulfill a specific function in stone pathogenesis. In this paper we briefly review papers investigating the urinary excretion of individual macromolecules, their effects on calcium oxalate (CaOx) crystallization and attachment of crystals to renal epithelial cells, and the influence of lithogenic conditions on their renal expression in cultured cells and animal models. Using prothrombin fragment 1 (PTF1) and human serum albumin as examples, we show the types of patterns resulting from the binding of a fluorescently tagged protein to a specific CaOx monohydrate (COM) crystal face and its incorporation into the crystal structure. Molecular modeling is also used to illustrate how PTF1 can align with the atomic array on a COM crystal surface. We conclude that although many macromolecules are, by strict definition, relevant to stone formation, very few are probably truly influential.

  9. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  10. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

    PubMed Central

    Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin; Hartono, Stella R; Chédin, Frédéric

    2015-01-01

    Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE. DOI: http://dx.doi.org/10.7554/eLife.08007.001 PMID:26182405

  11. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  12. UvrD facilitates DNA repair by pulling RNA polymerase backwards.

    PubMed

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-16

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes. PMID:24402227

  13. Simultaneous detection of RNA and DNA targets based on multiplex isothermal amplification.

    PubMed

    Dobnik, David; Morisset, Dany; Lenarčič, Rok; Ravnikar, Maja

    2014-04-01

    The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously. PMID:24625323

  14. Simultaneous DNA and RNA Mapping of Somatic Mitochondrial Mutations across Diverse Human Cancers

    PubMed Central

    Stewart, James B.; Alaei-Mahabadi, Babak; Sabarinathan, Radhakrishnan; Samuelsson, Tore; Gorodkin, Jan; Gustafsson, Claes M.; Larsson, Erik

    2015-01-01

    Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3′ to 5′ direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems. PMID:26125550

  15. Configurational diffusion of coal macromolecules

    SciTech Connect

    Guin, J.A.; Curtis, C.W.; Tarrer, A.R.

    1990-01-01

    As shown in last quarter's report on the configurational diffusion of coal macromolecules, the hindered diffusion data for both TPP and coal macromolecules were significantly different from the theoretical correlations. In order to evaluate the factors which could lead to this difference an error analysis was conducted, and the detailed results reported herein. Generally, we did not find any errors which could account for the deviation from the theory, and thus we conclude that this deviation is real and can be ascribed to some factor not considered by the hindered diffusion theory, i.e., attractive or repulsive forces. 2 refs., 4 figs., 4 tabs.

  16. DNA repair by RNA: Templated, or not templated, that is the question.

    PubMed

    Meers, Chance; Keskin, Havva; Storici, Francesca

    2016-08-01

    Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair. PMID:27237587

  17. DNA repair by RNA: Templated, or not templated, that is the question

    PubMed Central

    Meers, Chance; Keskin, Havva; Storici, Francesca

    2016-01-01

    Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair. PMID:27237587

  18. A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants.

    PubMed

    Martinez, German; Castellano, Mayte; Tortosa, Maria; Pallas, Vicente; Gomez, Gustavo

    2014-02-01

    Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the 'Hop stunt viroid' accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle. PMID:24178032

  19. Unzipping of A-Form DNA-RNA, A-Form DNA-PNA, and B-Form DNA-DNA in the α-Hemolysin Nanopore.

    PubMed

    Perera, Rukshan T; Fleming, Aaron M; Peterson, Amberlyn M; Heemstra, Jennifer M; Burrows, Cynthia J; White, Henry S

    2016-01-19

    Unzipping of double-stranded nucleic acids by an electric field applied across a wild-type α-hemolysin (αHL) nanopore provides structural information about different duplex forms. In this work, comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and the unzipping duration between the two helical forms. We observed that the B-form duplex blocks the channel 1.9 ± 0.2 pA more and unzips ∼15-fold more slowly than an A-form duplex at 120 mV. We developed a model to describe the dependence of duplex unzipping on structure. We demonstrate that the wider A-form duplex (d = 2.4 nm) is unable to enter the vestibule opening of αHL on the cis side, leading to unzipping outside of the nanopore with higher residual current and faster unzipping times. In contrast, the smaller B-form duplexes (d = 2.0 nm) enter the vestibule of αHL, resulting in decreased current blockages and slower unzipping. We investigated the effects of varying the length of the single-stranded overhang, and studied A-form DNA-PNA duplexes to provide additional support for the proposed model. This study identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA. PMID:26789754

  20. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    PubMed

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  1. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review)

    PubMed Central

    JIMÉNEZ-WENCES, HILDA; PERALTA-ZARAGOZA, OSCAR; FERNÁNDEZ-TILAPA, GLORIA

    2014-01-01

    Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53. PMID:24737381

  2. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review).

    PubMed

    Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

    2014-06-01

    Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53. PMID:24737381

  3. Density functional study of the phosphate diester hydrolysis of RNA in RNA/DNA hybrid by RNase HI

    NASA Astrophysics Data System (ADS)

    Kita, Makoto; Nakamura, Haruki; Takano, Yu

    2014-02-01

    Ribonuclease HI (RNase HI) catalyses the non-specific hydrolysis of RNA in an RNA/DNA hybrid. This enzyme is found in almost all organisms and involved in replication initiation and DNA topology restoration. A similar fold has been observed in other enzymes such as DNA transposases. In particular, RNases HI has emerged as important therapeutic targets because the enzymatic activity is absolutely required for proliferation of HIV and other retroviruses. The X-ray crystallographic structures of RNase HI revealed that the Mg2+ ion is essential for the enzymatic reaction and that Asp and Glu coordinate to the Mg2+ ion. There are, however, controversies about the catalytic mechanism of RNase HI, the number of Mg2+ ions, the proton transfer pathway, and the protonation states of the active site residues. In the present study, we have explored the hydrolysis of the phosphate diester group of RNA by RNase HI using density functional theory to elucidate how many Mg2+ ions are required for the catalysis of RNase HI. Our computation demonstrates that both one- and two-metal models show the stepwise hydrolysis pathway via a pentacovalent intermediate. However, the activation barrier of the two-metal model is lower than that of the one-metal model by 11.7 kcal/mol.

  4. RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle of Oreochromis niloticus fed with elevated levels of palm oil

    NASA Astrophysics Data System (ADS)

    Ayisi, Christian Larbi; Zhao, Jinliang

    2016-02-01

    Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected ( P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.

  5. In vivo DNA/RNA adduction of 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BaP) in the liver of rainbow trout (Oncorhynchus mykiss)

    SciTech Connect

    Schnitz, A.R.; O'Connor, J.M. )

    1992-07-01

    Juvenile rainbow trout were exposed via two intramuscular injections to either 14C-DMBA for 24 hr or 14C-BaP for 48 hr, after which the livers were removed for DNA extraction and analysis. In the fish exposed to 14C-BaP, 0.2 ng was bound to the DNA, representing 0.5% of the total liver PAH-derived radioactivity and 2.38% of the administered dose. Liver DNA and RNA were found to contain 0.5% of the administered dose, respectively. Liver analysis of rainbow trout exposed to 14C-DMBA demonstrated that 0.4 ng and 0.3 ng were bound to the DNA and RNA, respectively. This represents 1.0% and 0.6% of the liver DMBA burden, respectively. The DNA adduct concentrations formed were comparable to both in vitro and in vivo experiments with both mammals and fishes, indicating that relatively small, environmentally realistic' doses of PAH have the ability to bind significantly to critical cellular macromolecules of young fish in vivo.

  6. Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome

    PubMed Central

    Hou, Linlin; Klug, Gabriele; Evguenieva-Hackenberg, Elena

    2014-01-01

    The archaeal exosome is a phosphorolytic 3′–5′ exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation. PMID:25326320

  7. Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA.

    PubMed

    Escarmis, C; Domingo, E; Warner, R C

    1975-08-21

    The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15 degrees C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37 degrees C. This difference is responsible for the apparent inhibition of a21 dna transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enxyme is three times greater than that for the complex with the holoenzyme. PMID:1100115

  8. The Chemical Synthesis of DNA/RNA: Our Gift to Science

    PubMed Central

    Caruthers, Marvin H.

    2013-01-01

    It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs. PMID:23223445

  9. Effect of seven days of spaceflight on hindlimb muscle protein, RNA and DNA in adult rats

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1985-01-01

    Effects of seven days of spaceflight on skeletal muscle (soleus, gastrocnemius, EDL) content of protein, RNA and DNA were determined in adult rats. Whereas total protein contents were reduced in parallel with muscle weights, myofibrillar protein appeared to be more affected. There were no significant changes in absolute DNA contents, but a significant (P less than 0.05) increase in DNA concentration (microgram/milligram) in soleus muscles from flight rats. Absolute RNA contents were significantly (P less than 0.025) decreased in the soleus and gastrocnemius muscles of flight rats, with RNA concentrations reduced 15-30 percent. These results agree with previous ground-based observations on the suspended rat with unloaded hindlimbs and support continued use of this model.

  10. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    PubMed

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. PMID:27237373

  11. Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control.

    PubMed

    Vohhodina, Jekaterina; Harkin, D Paul; Savage, Kienan I

    2016-09-01

    Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. WIREs RNA 2016, 7:604-619. doi: 10.1002/wrna.1353 For further resources related to this article, please visit the WIREs website. PMID:27126972

  12. Abnormal rapid non-linear RNA production induced by T7 RNA polymerase in the absence of an exogenous DNA template

    NASA Astrophysics Data System (ADS)

    Kakimoto, Y.; Fujinuma, A.; Fujita, S.; Kikuchi, Y.; Umekage, S.

    2015-02-01

    Although recombinant T7 RNA polymerase is commonly used for in vitro RNA synthesis, several reports have pointed out that T7 RNA polymerase can also induce RNA-directed RNA polymerization or replication. In addition, here we show a new aberrant transcription when using T7 RNA polymerase. This polymerization was observed in the presence of both ribonucleotides and a purchasable T7 RNA polymerase, Thermo T7 RNA polymerase, as well as in the absence of an exogenous DNA template. This cryptic RNA production was detectable after several hours of incubation and was inhibited by adding DNase I. These findings suggested that some contaminated DNA along with the Thermo stable T7 RNA polymerase could be used as template DNA. However, to our surprise, RNA production showed a rapid non-linear increase. This finding strongly indicated that a self-replication cycle emerged from the RNA-directed polymerization or replication by T7 RNA polymerase, triggering the abnormal explosive increase.

  13. Macromolecules in Undergraduate Physical Chemistry.

    ERIC Educational Resources Information Center

    Mattice, Wayne L.

    1981-01-01

    Suggests the topic of macromolecules and synthetic polymers be included in undergraduate courses. Two macromolecular systems (polyethylene in a state unperturbated by long-range interactions and a polypeptide undergoing a helix-coil transition) are described which are suitable for inclusion in the statistical mechanics section of physical…

  14. RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

    NASA Technical Reports Server (NTRS)

    Ordoukhanian, Phillip; Joyce, Gerald F.

    2002-01-01

    In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

  15. Not just "a clever way to detect whether DNA really made RNA": The invention of DNA-RNA hybridization and its outcome.

    PubMed

    Fisher, Susie

    2015-10-01

    The invention of DNA-RNA hybridization in 1960 by Ben Hall and Sol Spiegelman had a powerful impact on the theory and discourse of molecular biology. Yet, despite its importance, the story of this invention has barely been told. Hybridization allowed biologists to bridge the theoretical realm and the material world of organisms, to correlate a hypothetical concept of biological information transfer with a mechanism capable of making an RNA copy of DNA. During the early 1960s, Spiegelman and coworkers employed hybridization to investigate the origin of RNAs found in cells. They operationally defined messenger RNA and elucidated several aspects of genome organization. For Spiegelman, this was the culmination of his longstanding interest in the mechanism of enzyme/protein synthesis; for Hall, it was the beginning of a successful career in genetics. Other scientists immediately recognized the power of the technique and introduced improvements. In 1965, Gillespie and Spiegelman combined several modifications and described a procedure for hybridization that became standard. Since the 1970s, it has become an essential tool in biology and in biotechnology, and a core component in molecular techniques such as DNA microarrays. Notwithstanding its current success, the inventors' names have disappeared from the literature. This curiosity is discussed. PMID:26209888

  16. Rolling circle amplification detection of RNA and DNA

    DOEpatents

    Christian, Allen T.; Pattee, Melissa S.; Attix, Cristina M.; Tucker, James D.

    2004-08-31

    Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

  17. Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase.

    PubMed

    Nabel, Christopher S; Lee, Jae W; Wang, Laura C; Kohli, Rahul M

    2013-08-27

    Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID's functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID's selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2'-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID's reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2'-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID's closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2'-fluoro-RNA substrates, AID's deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID's DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

  18. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

    PubMed

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-01-01

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

  19. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute

    PubMed Central

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-01-01

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

  20. The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions

    PubMed Central

    Kljashtorny, Vladislav; Nikonov, Stanislav; Ovchinnikov, Lev; Lyabin, Dmitry; Vodovar, Nicolas; Curmi, Patrick; Manivet, Philippe

    2015-01-01

    The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids. PMID:26147853

  1. Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies.

    PubMed

    Rombouts, K; Braeckmans, K; Remaut, K

    2016-02-17

    Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs. PMID:26670733

  2. RNA Polymerase Collision versus DNA Structural Distortion: Twists and Turns Can Cause Break Failure.

    PubMed

    Pannunzio, Nicholas R; Lieber, Michael R

    2016-05-01

    The twisting of DNA due to the movement of RNA polymerases is the basis of numerous classic experiments in molecular biology. Recent mouse genetic models indicate that chromosomal breakage is common at sites of transcriptional turbulence. Two key studies on this point mapped breakpoints to sites of either convergent or divergent transcription but arrived at different conclusions as to which is more detrimental and why. The issue hinges on whether DNA strand separation is the basis for the chromosomal instability or collision of RNA polymerases. PMID:27153532

  3. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    PubMed

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. PMID:25579785

  4. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

  5. A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

    PubMed Central

    Chak, Li-Ling; Mohammed, Jaaved; Lai, Eric C.; Tucker-Kellogg, Greg

    2015-01-01

    Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the pre-rRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5′ and 3′ ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha–Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms. PMID:25605965

  6. INFLUENCE OF MACROMOLECULES ON CHEMICAL TRANSPORT

    EPA Science Inventory

    Macromolecules in the pore fluid influence the mobility of hydrophobic compounds through soils. his study evaluated the significance of macromolecules in facilitating chemical transport under laboratory conditions. Partition coefficients between 14C-labeled hexachlorobenzene and ...

  7. Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition.

    PubMed

    Kazlauskiene, Migle; Tamulaitis, Gintautas; Kostiuk, Georgij; Venclovas, Česlovas; Siksnys, Virginijus

    2016-04-21

    Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA. PMID:27105119

  8. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

    SciTech Connect

    Ausin, Israel; Greenberg, Maxim V.C.; Simanshu, Dhirendra K.; Hale, Christopher J.; Vashisht, Ajay A.; Simon, Stacey A.; Lee, Tzuu-fen; Feng, Suhua; Española, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2012-10-23

    At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

  9. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    PubMed

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  10. Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III.

    PubMed

    Wenzel, Michael; Wunderlich, Michael; Besch, Robert; Poeck, Hendrik; Willms, Simone; Schwantes, Astrid; Kremer, Melanie; Sutter, Gerd; Endres, Stefan; Schmidt, Andreas; Rothenfusser, Simon

    2012-01-01

    A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome. PMID:22140256

  11. Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

    PubMed Central

    Bardeesy, N; Pelletier, J

    1998-01-01

    The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein. PMID:9512553

  12. mRNA and DNA selection via protein multimerization: YB-1 as a case study

    PubMed Central

    Kretov, Dmitry A.; Curmi, Patrick A.; Hamon, Loic; Abrakhi, Sanae; Desforges, Bénédicte; Ovchinnikov, Lev P.; Pastré, David

    2015-01-01

    Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance. PMID:26271991

  13. Base Pair Fraying in Molecular Dynamics Simulations of DNA and RNA.

    PubMed

    Zgarbová, Marie; Otyepka, Michal; Šponer, Jiří; Lankaš, Filip; Jurečka, Petr

    2014-08-12

    Terminal base pairs of DNA and RNA molecules in solution are known to undergo frequent transient opening events (fraying). Accurate modeling of this process is important because of its involvement in nucleic acid end recognition and enzymatic catalysis. In this article, we describe fraying in molecular dynamics simulations with the ff99bsc0, ff99bsc0χOL3, and ff99bsc0χOL4 force fields, both for DNA and RNA molecules. Comparison with the experiment showed that while some features of fraying are consistent with the available data, others indicate potential problems with the force field description. In particular, multiple noncanonical structures are formed at the ends of the DNA and RNA duplexes. Among them are tWC/sugar edge pair, C-H edge/Watson-Crick pair, and stacked geometries, in which the terminal bases are stacked above each other. These structures usually appear within the first tens to hundreds of nanoseconds and substantially limit the usefulness of the remaining part of the simulation due to geometry distortions that are transferred to several neighboring base pairs ("end effects"). We show that stability of the noncanonical structures in ff99bsc0 may be partly linked to inaccurate glycosidic (χ) torsion potentials that overstabilize the syn region and allow for rapid anti to syn transitions. The RNA refined glycosidic torsion potential χOL3 provides an improved description and substantially more stable MD simulations of RNA molecules. In the case of DNA, the χOL4 correction gives only partial improvement. None of the tested force fields provide a satisfactory description of the terminal regions, indicating that further improvement is needed to achieve realistic modeling of fraying in DNA and RNA molecules. PMID:26588288

  14. Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

    PubMed Central

    Tbakhi, A.; Totos, G.; Hauser-Kronberger, C.; Pettay, J.; Baunoch, D.; Hacker, G. W.; Tubbs, R. R.

    1998-01-01

    Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions. Images Figure 1 Figure 2 Figure 3 PMID:9422521

  15. Heat shock protein-mediated cell penetration and cytosolic delivery of macromolecules by a telomerase-derived peptide vaccine.

    PubMed

    Lee, Seoung-Ae; Kim, Bo-Ram; Kim, Bu-Kyung; Kim, Dong-Won; Shon, Won-Jun; Lee, Na-Rae; Inn, Kyung-Soo; Kim, Bum-Joon

    2013-10-01

    A reverse-transcriptase-subunit of telomerase (hTERT) derived peptide, GV1001, has been developed as a vaccine against various cancers. Here, we report an unexpected function of GV1001 as a cell-penetrating peptide (CPP). GV1001 was delivered into a variety of cells including various cancer cell lines and primary blood cells. Moreover, the delivered GV1001 was predominantly located in the cytoplasm of the cells, while a significantly higher proportion of TAT peptide was localized in the nucleus. Macromolecules such as proteins, DNA and siRNA, which were linked to GV1001 by direct covalent conjugation or non-covalent complexation through poly-lysine, were successfully delivered into cells, indicating that GV1001 can be used as a carrier for macromolecules. Expression of the delivered DNA, and lowered expression of the target gene by the delivered siRNA, suggest the potential therapeutic use of GV1001. Pull-down analysis identified Heat Shock Protein 90 (HSP90) and 70 (HSP70) as GV1001 interacting proteins. Treatment of Anti-HSP90 and HSP70 antibodies lowered the internalization of GV1001, indicating that the interaction is critical for the efficient internalization of GV1001. Collectively, the results of this study suggest the pharmaceutical potential of GV1001, already proven safe in clinical trials, as a carrier for the delivery of macromolecular therapeutics into cells, in addition to its own anti-cancer activity. PMID:23827187

  16. Non-Coding RNA: Sequence-Specific Guide for Chromatin Modification and DNA Damage Signaling

    PubMed Central

    Francia, Sofia

    2015-01-01

    Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair. PMID:26617633

  17. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms

    PubMed Central

    Swift, Robert V.; Amaro, Rommie E.

    2009-01-01

    Background Members of the nucleotidyltransferase superfamily known as DNA and RNA ligases carry out the enzymatic process of polynucleotide ligation. These guardians of genomic integrity share a three-step ligation mechanism, as well as common core structural elements. Both DNA and RNA ligases have experienced a surge of recent interest as chemotherapeutic targets for the treatment of a range of diseases, including bacterial infection, cancer, and the diseases caused by the protozoan parasites known as trypanosomes. Objective In this review, we will focus on efforts targeting pathogenic microorganisms; specifically, bacterial NAD+-dependent DNA ligases, which are promising broad-spectrum antibiotic targets, and ATP-dependent RNA editing ligases from Trypanosoma brucei, the species responsible for the devastating neurodegenerative disease, African sleeping sickness. Conclusion High quality crystal structures of both NAD+-dependent DNA ligase and the Trypanosoma brucei RNA editing ligase have facilitated the development of a number of promising leads. For both targets, further progress will require surmounting permeability issues and improving selectivity and affinity. PMID:20354588

  18. Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA.

    PubMed

    Yamano, Takashi; Nishimasu, Hiroshi; Zetsche, Bernd; Hirano, Hisato; Slaymaker, Ian M; Li, Yinqing; Fedorova, Iana; Nakane, Takanori; Makarova, Kira S; Koonin, Eugene V; Ishitani, Ryuichiro; Zhang, Feng; Nureki, Osamu

    2016-05-01

    Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox. PMID:27114038

  19. De novo reconstruction of plant RNA and DNA virus genomes from viral siRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  20. Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

    PubMed

    Fu, J; Gerstein, M; David, P R; Gnatt, A L; Bushnell, D A; Edwards, A M; Kornberg, R D

    1998-07-17

    X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein. PMID:9665838

  1. Differential DNA and RNA sequence discrimination by PNA having charged side chains.

    PubMed

    De Costa, N Tilani S; Heemstra, Jennifer M

    2014-05-15

    PNA sequences modified with charged side chains were evaluated for base-pairing sequence selectivity under physiological conditions. PNA having negatively charged aspartic acid side chains shows higher selectivity with RNA, while PNA having positively charged lysine side chains shows higher selectivity with DNA. These observations provide insight into the binding selectivity of modified PNA in antisense and antigene applications. PMID:24731279

  2. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    PubMed

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  3. Sensitive SERS detection of miRNA via enzyme-free DNA machine signal amplification.

    PubMed

    Li, Xiaoxiao; Ye, Sujuan; Luo, Xiliang

    2016-08-11

    In this work, an enzyme-free signal amplified detection platform is described for miRNA detection with a DNA fueled molecular machine. Coupling SERS technology with multiple amplification modes, this flexible biosensing system exhibits high sensitivity and specificity. PMID:27469084

  4. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  5. Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer

    PubMed Central

    Bersani, Francesca; Lee, Eunjung; Kharchenko, Peter V.; Xu, Andrew W.; Liu, Mingzhu; Xega, Kristina; MacKenzie, Olivia C.; Brannigan, Brian W.; Wittner, Ben S.; Jung, Hyunchul; Ramaswamy, Sridhar; Park, Peter J.; Maheswaran, Shyamala; Ting, David T.; Haber, Daniel A.

    2015-01-01

    Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture. PMID:26575630

  6. Endogenous Small RNA Mediates Meiotic Silencing of a Novel DNA Transposon

    PubMed Central

    Wang, Yizhou; Smith, Kristina M.; Taylor, John W.; Freitag, Michael; Stajich, Jason E.

    2015-01-01

    Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory “wild-type” strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference−silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element. PMID:26109355

  7. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    NASA Astrophysics Data System (ADS)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  8. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

    PubMed

    Henderson, Ian R; Zhang, Xiaoyu; Lu, Cheng; Johnson, Lianna; Meyers, Blake C; Green, Pamela J; Jacobsen, Steven E

    2006-06-01

    Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs. PMID:16699516

  9. Molecular approaches for forensic cell type identification: On mRNA, miRNA, DNA methylation and microbial markers.

    PubMed

    Sijen, Titia

    2015-09-01

    Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well. PMID:25488609

  10. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    SciTech Connect

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    2012-12-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  11. MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression

    PubMed Central

    Wang, Yemin; Huang, Jen-Wei; Li, Ming; Cavenee, Webster K.; Mitchell, Patrick S.; Zhou, Xiaofeng; Tewari, Muneesh; Furnari, Frank B.; Taniguchi, Toshiyasu

    2011-01-01

    Precise regulation of DNA damage response is crucial for cellular survival after DNA damage, and its abrogation often results in genomic instability in cancer. Phosphorylated histone H2AX (γH2AX) forms nuclear foci at sites of DNA damage and facilitates DNA damage response and repair. MicroRNAs are short, non-protein-encoding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation of and/or degrading mRNA. How microRNAs modulate DNA damage response is largely unknown. In this study, we developed a cell-based screening assay utilizing ionizing radiation-induced γH2AX foci formation in a human osteosarcoma cell line, U2OS, as the readout. By screening a library of human microRNA mimics, we identified several microRNAs that inhibited γH2AX foci formation. Among them, miR-138 directly targeted the histone H2AX 3′-UTR, reduced histone H2AX expression and induced chromosomal instability after DNA damage. Overexpression of miR-138 inhibited homologous recombination and enhanced cellular sensitivity to multiple DNA damaging agents (cisplatin, camptothecin, and ionizing radiation). Reintroduction of histone H2AX in miR-138 overexpressing cells attenuated miR-138-mediated sensitization to cisplatin and camptothecin. Our study suggests that miR-138 is an important regulator of genomic stability and a potential therapeutic agent to improve the efficacy of radiotherapy and chemotherapy with DNA damaging agents. PMID:21693595

  12. Compositional changes in RNA, DNA and proteins for bacterial adaptation to higher and lower temperatures.

    PubMed

    Nakashima, Hiroshi; Fukuchi, Satoshi; Nishikawa, Ken

    2003-04-01

    It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed. PMID:12761299

  13. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

    PubMed Central

    Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A; Gwerder, Myriam; Gutsche, Katrin; Altmeyer, Matthias; Jungmichel, Stephanie; Toledo, Luis I; Fink, Daniel; Rask, Maj-Britt; Grøfte, Merete; Lukas, Claudia; Nielsen, Michael L; Smerdon, Stephen J; Lukas, Jiri; Stucki, Manuel

    2016-01-01

    Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in-trans signaling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identified TCOF1-Treacle, a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle-dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in-trans in the presence of distant chromosome breaks. PMID:25064736

  14. Opposing Effects of Multivalent Ions on the Flexibility of DNA and RNA.

    PubMed

    Drozdetski, Aleksander V; Tolokh, Igor S; Pollack, Lois; Baker, Nathan; Onufriev, Alexey V

    2016-07-01

    Increasing the concentration of counterions (salt) is known to reduce the bending persistence length of DNA. Here we use atomistic molecular dynamics simulations to predict that multivalent counterions have the opposite effect on double-stranded RNA, increasing its bending rigidity by at least 30%. This counterintuitive effect is observed for various tri- and tetravalent ions alike, and is robust to methodological details and the RNA sequence. In contrast to DNA, multivalent counterions bind inside the RNA major groove, causing significant contraction of the molecule along its helical axis-as a result, its further deformation due to bending becomes energetically more expensive compared to bending without bound multivalent ions. Thus, the relationship between mechanical properties of a charged polymer and its ionic atmosphere may be richer than previously thought. PMID:27447528

  15. Regulating infidelity: RNA-mediated recruitment of AID to DNA during class switch recombination.

    PubMed

    DiMenna, Lauren J; Chaudhuri, Jayanta

    2016-03-01

    The mechanism by which the DNA deaminase activation-induced cytidine deaminase (AID) is specifically recruited to repetitive switch region DNA during class switch recombination is still poorly understood. Work over the past decade has revealed a strong link between transcription and RNA polymerase-associated factors in AID recruitment, yet none of these processes satisfactorily explain how AID specificity is affected. Here, we review a recent finding wherein AID is guided to switch regions not by a protein factor but by an RNA moiety, and especially one associated with a noncoding RNA that has been long thought of as being inert. This work explains the long-standing requirement of splicing of noncoding transcripts during class switching, and has implications in both B cell-mediated immunity as well as the underlying pathological syndromes associated with the recombination reaction. PMID:26799454

  16. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    2007-04-06

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

  17. Opposing Effects of Multivalent Ions on the Flexibility of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Drozdetski, Aleksander V.; Tolokh, Igor S.; Pollack, Lois; Baker, Nathan; Onufriev, Alexey V.

    2016-07-01

    Increasing the concentration of counterions (salt) is known to reduce the bending persistence length of DNA. Here we use atomistic molecular dynamics simulations to predict that multivalent counterions have the opposite effect on double-stranded RNA, increasing its bending rigidity by at least 30%. This counterintuitive effect is observed for various tri- and tetravalent ions alike, and is robust to methodological details and the RNA sequence. In contrast to DNA, multivalent counterions bind inside the RNA major groove, causing significant contraction of the molecule along its helical axis—as a result, its further deformation due to bending becomes energetically more expensive compared to bending without bound multivalent ions. Thus, the relationship between mechanical properties of a charged polymer and its ionic atmosphere may be richer than previously thought.

  18. Stability of triple helices containing RNA and DNA strands: experimental and molecular modeling studies.

    PubMed Central

    Escudé, C; François, J C; Sun, J S; Ott, G; Sprinzl, M; Garestier, T; Hélène, C

    1993-01-01

    UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes. Images PMID:7506827

  19. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  20. Comparative Dynamics and Sequence Dependence of DNA and RNA Binding to Single Walled Carbon Nanotubes

    PubMed Central

    Landry, Markita P.; Vuković, Lela; Kruss, Sebastian; Bisker, Gili; Landry, Alexandra M.; Islam, Shahrin; Jain, Rishabh; Schulten, Klaus; Strano, Michael S.

    2015-01-01

    Noncovalent polymer-single walled carbon nanotube (SWCNT) conjugates have gained recent interest due to their prevalent use as electrochemical and optical sensors, SWCNT-based therapeutics, and for SWCNT separation. However, little is known about the effects of polymer-SWCNT molecular interactions on functional properties of these conjugates. In this work, we show that SWCNT complexed with related polynucleotide polymers (DNA, RNA) have dramatically different fluorescence stability. Surprisingly, we find a difference of nearly 2500-fold in fluorescence emission between the most fluorescently stable DNA-SWCNT complex, C30 DNA-SWCNT, compared to the least fluorescently stable complex, (AT)7A-(GU)7G DNA-RNA hybrid-SWCNT. We further reveal the existence of three regimes in which SWCNT fluorescence varies nonmonotonically with SWCNT concentration. We utilize molecular dynamics simulations to elucidate the conformation and atomic details of SWCNT-corona phase interactions. Our results show that variations in polynucleotide sequence or sugar backbone can lead to large changes in the conformational stability of the polymer SWCNT corona and the SWCNT optical response. Finally, we demonstrate the effect of the coronae on the response of a recently developed dopamine nanosensor, based on (GT)15 DNA- and (GU)15 RNA-SWCNT complexes. Our results clarify several features of the sequence dependence of corona phases produced by polynucleotides adsorbed to single walled carbon nanotubes, and the implications for molecular recognition in such phases. PMID:26005509

  1. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  2. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    PubMed Central

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  3. First Principles Dynamics of Photoexcited DNA and RNA Bases

    SciTech Connect

    Hudock, Hanneli R.; Levine, Benjamin G.; Thompson, Alexis L.; Martinez, Todd J.

    2007-12-26

    The reaction dynamics of excited electronic states in nucleic acid bases is a key process in DNA photodamage. Recent ultrafast spectroscopy experiments have shown multi-component decays of excited uracil and thymine, tentatively assigned to nonadiabatic transitions involving multiple electronic states. Using both quantum chemistry and first principles quantum molecular dynamics methods we show that a true minimum on the bright S{sub 2} electronic state is responsible for the first step which occurs on a femtosecond timescale. Thus the observed femtosecond decay does not correspond to surface crossing as previously thought. We suggest that subsequent barrier crossing to the minimal energy S{sub 2}/S{sub 1} conical intersection is responsible for the picosecond decay.

  4. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  5. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  6. The DNA Habitat and its RNA Inhabitants: At the Dawn of RNA Sociology

    PubMed Central

    Villarreal, Luis P; Witzany, Guenther

    2013-01-01

    Most molecular biological concepts derive from physical chemical assumptions about the genetic code that are basically more than 40 years old. Additionally, systems biology, another quantitative approach, investigates the sum of interrelations to obtain a more holistic picture of nucleotide sequence order. Recent empirical data on genetic code compositions and rearrangements by mobile genetic elements and noncoding RNAs, together with results of virus research and their role in evolution, does not really fit into these concepts and compel a reexamination. In this review, we try to find an alternate hypothesis. It seems plausible now that if we look at the abundance of regulatory RNAs and persistent viruses in host genomes, we will find more and more evidence that the key players that edit the genetic codes of host genomes are consortia of RNA agents and viruses that drive evolutionary novelty and regulation of cellular processes in all steps of development. This agent-based approach may lead to a qualitative RNA sociology that investigates and identifies relevant behavioral motifs of cooperative RNA consortia. In addition to molecular biological perspectives, this may lead to a better understanding of genetic code evolution and dynamics. PMID:26217106

  7. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    PubMed

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods. PMID:25629590

  8. DNA and RNA sequencing by nanoscale reading through programmable electrophoresis and nanoelectrode-gated tunneling and dielectric detection

    DOEpatents

    Lee, James W.; Thundat, Thomas G.

    2005-06-14

    An apparatus and method for performing nucleic acid (DNA and/or RNA) sequencing on a single molecule. The genetic sequence information is obtained by probing through a DNA or RNA molecule base by base at nanometer scale as though looking through a strip of movie film. This DNA sequencing nanotechnology has the theoretical capability of performing DNA sequencing at a maximal rate of about 1,000,000 bases per second. This enhanced performance is made possible by a series of innovations including: novel applications of a fine-tuned nanometer gap for passage of a single DNA or RNA molecule; thin layer microfluidics for sample loading and delivery; and programmable electric fields for precise control of DNA or RNA movement. Detection methods include nanoelectrode-gated tunneling current measurements, dielectric molecular characterization, and atomic force microscopy/electrostatic force microscopy (AFM/EFM) probing for nanoscale reading of the nucleic acid sequences.

  9. Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

    PubMed Central

    Strom, Shane; Shiskova, Evgenia; Hahm, Yaeeun

    2015-01-01

    Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl2 or 1 M KCl. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47–2.06 kcal/mol more favorable for the RNA bulge loops. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride. PMID:26022248

  10. Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.

    PubMed

    Boros, Gábor; Karikó, Katalin; Muramatsu, Hiromi; Miko, Edit; Emri, Eszter; Hegedűs, Csaba; Emri, Gabriella; Remenyik, Éva

    2016-01-01

    In vitro-synthesized mRNA containing nucleoside modifications has great therapeutical potential to transiently express proteins with physiological importance. One such protein is photolyase which rapidly removes UV-induced DNA damages, but this enzyme is absent in humans. Here, we apply a novel mRNA-based platform to achieve functional nonhuman photolyase production in cultured human keratinocytes. Transfection of nucleoside-modified mRNA encoding photolyase leads to accelerated repair of DNA photolesions in human keratinocytes. PMID:27236802

  11. The non-coding B2 RNA binds to the DNA cleft and active-site region of RNA polymerase II.

    PubMed

    Ponicsan, Steven L; Houel, Stephane; Old, William M; Ahn, Natalie G; Goodrich, James A; Kugel, Jennifer F

    2013-10-01

    The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA. PMID:23416138

  12. Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model

    PubMed Central

    Pfingsten, Jennifer S.; Goodrich, Karen J.; Taabazuing, Cornelius; Ouenzar, Faissal; Chartrand, Pascal; Cech, Thomas R.

    2012-01-01

    SUMMARY In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes its telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt non-homologous end-joining (NHEJ), telomeric gene silencing and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PMID:22365814

  13. Macromolecules

    PubMed Central

    Mark, H.

    1964-01-01

    Natural polymers such as wood, cotton, wool, starch, and rubber have been known and used for a long time even though their composition and structure were unknown. At the beginning of this century systematic efforts were started to analyze these materials, elucidate their molecular pattern, and fit them into the framework of organic chemistry. It was found that they all are characterized by the presence of chain-like molecules with very high molecular weights ranging from 10000 to several millions. It was also found that these chains can be oriented by mechanical deformations and assume a laterally ordered crystal-like supermolecular arrangement. As soon as these facts become known as systematic search for synthetic counterparts of natural polymers was started which led to the laboratory preparation and large scale production of many synthetic materials which cover a wide range of properties and applications and which are becoming an increasingly important factor in science and industry. With growing understanding and know-how it also became possible to synthesize polymers with biochemically interesting properties such as the polyelectrolytes and other biopolymers. PMID:14104084

  14. Stacking interactions in RNA and DNA: Roll-slide energy hyperspace for ten unique dinucleotide steps.

    PubMed

    Mukherjee, Sanchita; Kailasam, Senthilkumar; Bansal, Manju; Bhattacharyya, Dhananjay

    2015-03-01

    Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by ωB97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. PMID:25257334

  15. PATTERNS OF RNA SYNTHESIS IN T5-INFECTED CELLS I. AS STUDIED BY THE TECHNIQUE OF DNA-RNA HYBRIDIZATION-COMPETITION*

    PubMed Central

    Moyer, Richard W.; Buchanan, John M.

    1969-01-01

    The RNA-labeling patterns obtained after T5 infection of Escherichia coli F agree with the patterns of protein labeling published by McCorquodale and Buchanan.1 Three distinct classes of RNA formed sequentially during the period of viral development can be recognized by the DNA-RNA hybridization-competition technique. Class I RNA is formed within 5 minutes after the beginning of viral metabolism and corresponds to the RNA synthesized in response to infection with the 8 per cent segment of T5 DNA. Protein synthesis directed by this 8 per cent segment is required in some capacity for the cessation of class I synthesis and the beginning of the synthesis of class II at 4 to 5 min after infection. Class III RNA synthesis begins between 9 and 12 minutes. Its appearance is prevented when chloramphenicol is added immediately after complete expression of class I functions. PMID:4916923

  16. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  17. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  18. Junk DNA and the long non-coding RNA twist in cancer genetics

    PubMed Central

    Ling, Hui; Vincent, Kimberly; Pichler, Martin; Fodde, Riccardo; Berindan-Neagoe, Ioana; Slack, Frank J.; Calin, George A

    2015-01-01

    The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions, and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function, and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual’s susceptibility to cancer. PMID:25619839

  19. RNA involvement in T4 DNA synthesis in toluene-treated cells.

    PubMed

    Dicou, E

    1980-01-01

    In T4-infected cells made permeable with toluene, pulses with [(alpha-32P deoxyribonucleoside triphosphates demonstrated covalent linkage of RNA to DNA of the Okazaki fragments. Analysis of the transfer of the 32P label to the 2'(3') ribonucleoside monophosphates indicated that the 3'-end of the RNA primer is heterogeneous. The most frequently encountered ribonucleotide was rCMP, but also transfer to rUMP, rAMP and rGMP occurred at different frequencies. In contrast, no heterogeneity was observed for the deoxyribonucleoside at the RNA-DNA junction. Of all the [to-32P] deoxyribonucleoside triphosphates tested, transfer of the 32P label to 2'(3') rNMPs was predominant when [alpha32P] dGTP was the substrate, indicating that the deoxyribonucleoside most frequently encountered at the RNA-DNA linkage is dG. These observations suggest that the starts for the Okazaki fragments may occur at unique sites of the T4 genome. PMID:17941178

  20. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

    PubMed

    He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

    2015-08-01

    Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery. PMID:25937003

  1. Junk DNA and the long non-coding RNA twist in cancer genetics.

    PubMed

    Ling, H; Vincent, K; Pichler, M; Fodde, R; Berindan-Neagoe, I; Slack, F J; Calin, G A

    2015-09-24

    The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single-nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual's susceptibility to cancer. PMID:25619839

  2. The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

    PubMed Central

    Domingo-Calap, Pilar; Cuevas, José M.; Sanjuán, Rafael

    2009-01-01

    Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses. PMID:19956760

  3. The arabidopsis polyamine transporter LHRI/AtPUT3 modulates heat responsive gene expression by regulating mRNA stability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyamines (PA) involve in the gene regulation by interacting with various anionic macromolecules such as DNA, RNA and proteins and modulating their structure and function. Previous studies have showed that changing in polyamine biosynthesis alters plant response to different abiotic stresses. Here,...

  4. An RNA aptamer that interferes with the DNA binding of the HSF transcription activator

    PubMed Central

    Zhao, Xiaoching; Shi, Hua; Sevilimedu, Aarti; Liachko, Nicole; Nelson, Hillary C. M.; Lis, John T.

    2006-01-01

    Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem–loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-κB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF. PMID:16893958

  5. Crystal structure of Cpf1 in complex with guide RNA and target DNA

    PubMed Central

    Yamano, Takashi; Nishimasu, Hiroshi; Zetsche, Bernd; Hirano, Hisato; Slaymaker, Ian M.; Li, Yinqing; Fedorova, Iana; Nakane, Takanori; Makarova, Kira S.; Koonin, Eugene V.; Ishitani, Ryuichiro; Zhang, Feng; Nureki, Osamu

    2016-01-01

    Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA, at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA–DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for the cleavage of the non-target and target strands, respectively, and jointly generate staggered DNA double-strand breaks. AsCpf1 recognizes the 5′-TTTN-3′ protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1, and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox. PMID:27114038

  6. SL1 RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian human coprolites.

    PubMed

    Iñiguez, Alena Mayo; Reinhard, Karl; Carvalho Gonçalves, Marcelo Luiz; Ferreira, Luiz Fernando; Araújo, Adauto; Paulo Vicente, Ana Carolina

    2006-11-01

    Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further

  7. Increased DNA and RNA damage by oxidation in patients with bipolar I disorder.

    PubMed

    Jacoby, A S; Vinberg, M; Poulsen, H E; Kessing, L V; Munkholm, K

    2016-01-01

    The mechanisms underlying bipolar disorder (BD) and the associated medical burden are unclear. Damage generated by oxidation of nucleosides may be implicated in BD pathophysiology; however, evidence from in vivo studies is limited and the extent of state-related alterations is unclear. This prospective study investigated for we believe the first time the damage generated by oxidation of DNA and RNA strictly in patients with type I BD in a manic or mixed state and subsequent episodes and remission compared with healthy control subjects. Urinary excretion of 8-oxo-deoxyguanosine (8-oxodG) and 8-oxo-guanosine (8-oxoGuo), valid markers of whole-body DNA and RNA damage by oxidation, respectively, was measured in 54 patients with BD I and in 35 healthy control subjects using a modified ultraperformance liquid chromatography and mass spectrometry assay. Repeated measurements were evaluated in various affective phases during a 6- to 12-month period and compared with repeated measurements in healthy control subjects. Independent of lifestyle and demographic variables, a 34% (P<0.0001) increase in RNA damage by oxidation across all affective states, including euthymia, was found in patients with BD I compared with healthy control subjects. Increases in DNA and RNA oxidation of 18% (P<0.0001) and 8% (P=0.02), respectively, were found in manic/hypomanic states compared with euthymia, and levels of 8-oxodG decreased 15% (P<0.0001) from a manic or mixed episode to remission. The results indicate a role for DNA and RNA damage by oxidation in BD pathophysiology and a potential for urinary 8-oxodG and 8-oxoGuo to function as biological markers of diagnosis, state and treatment response in BD. PMID:27505230

  8. RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

    PubMed

    Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

    2007-09-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

  9. Differential DNA Methylation of MicroRNA Genes in Temporal Cortex from Alzheimer's Disease Individuals

    PubMed Central

    Villela, Darine; Ramalho, Rodrigo F.; Silva, Aderbal R. T.; Brentani, Helena; Suemoto, Claudia K.; Pasqualucci, Carlos Augusto; Grinberg, Lea T.; Krepischi, Ana C. V.; Rosenberg, Carla

    2016-01-01

    This study investigated for the first time the genomewide DNA methylation changes of noncoding RNA genes in the temporal cortex samples from individuals with Alzheimer's disease (AD). The methylome of 10 AD individuals and 10 age-matched controls were obtained using Illumina 450 K methylation array. A total of 2,095 among the 15,258 interrogated noncoding RNA CpG sites presented differential methylation, 161 of which were associated with miRNA genes. In particular, 10 miRNA CpG sites that were found to be hypermethylated in AD compared to control brains represent transcripts that have been previously associated with the disease. This miRNA set is predicted to target 33 coding genes from the neuregulin receptor complex (ErbB) signaling pathway, which is required for the neurons myelination process. For 6 of these miRNA genes (MIR9-1, MIR9-3, MIR181C, MIR124-1, MIR146B, and MIR451), the hypermethylation pattern is in agreement with previous results from literature that shows downregulation of miR-9, miR-181c, miR-124, miR-146b, and miR-451 in the AD brain. Our data implicate dysregulation of miRNA methylation as contributor to the pathogenesis of AD. PMID:27213057

  10. Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART.

    PubMed

    Saracino, Annalisa; Gianotti, Nicola; Marangi, Marianna; Cibelli, Donatella C; Galli, Andrea; Punzi, Grazia; Monno, Laura; Lazzarin, Adriano; Angarano, Gioacchino

    2008-10-01

    The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 +/- 2.76) than in DNA (2.88 +/- 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had >or=1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. PMID:18712823

  11. The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.

    PubMed

    Molodtsov, Vadim; Anikin, Michael; McAllister, William T

    2014-09-01

    Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway. PMID:24976131

  12. Blind Predictions of DNA and RNA Tweezers Experiments with Force and Torque

    PubMed Central

    Chou, Fang-Chieh; Lipfert, Jan; Das, Rhiju

    2014-01-01

    Single-molecule tweezers measurements of double-stranded nucleic acids (dsDNA and dsRNA) provide unprecedented opportunities to dissect how these fundamental molecules respond to forces and torques analogous to those applied by topoisomerases, viral capsids, and other biological partners. However, tweezers data are still most commonly interpreted post facto in the framework of simple analytical models. Testing falsifiable predictions of state-of-the-art nucleic acid models would be more illuminating but has not been performed. Here we describe a blind challenge in which numerical predictions of nucleic acid mechanical properties were compared to experimental data obtained recently for dsRNA under applied force and torque. The predictions were enabled by the HelixMC package, first presented in this paper. HelixMC advances crystallography-derived base-pair level models (BPLMs) to simulate kilobase-length dsDNAs and dsRNAs under external forces and torques, including their global linking numbers. These calculations recovered the experimental bending persistence length of dsRNA within the error of the simulations and accurately predicted that dsRNA's “spring-like” conformation would give a two-fold decrease of stretch modulus relative to dsDNA. Further blind predictions of helix torsional properties, however, exposed inaccuracies in current BPLM theory, including three-fold discrepancies in torsional persistence length at the high force limit and the incorrect sign of dsRNA link-extension (twist-stretch) coupling. Beyond these experiments, HelixMC predicted that ‘nucleosome-excluding’ poly(A)/poly(T) is at least two-fold stiffer than random-sequence dsDNA in bending, stretching, and torsional behaviors; Z-DNA to be at least three-fold stiffer than random-sequence dsDNA, with a near-zero link-extension coupling; and non-negligible effects from base pair step correlations. We propose that experimentally testing these predictions should be powerful next steps for

  13. Blind predictions of DNA and RNA tweezers experiments with force and torque.

    PubMed

    Chou, Fang-Chieh; Lipfert, Jan; Das, Rhiju

    2014-08-01

    Single-molecule tweezers measurements of double-stranded nucleic acids (dsDNA and dsRNA) provide unprecedented opportunities to dissect how these fundamental molecules respond to forces and torques analogous to those applied by topoisomerases, viral capsids, and other biological partners. However, tweezers data are still most commonly interpreted post facto in the framework of simple analytical models. Testing falsifiable predictions of state-of-the-art nucleic acid models would be more illuminating but has not been performed. Here we describe a blind challenge in which numerical predictions of nucleic acid mechanical properties were compared to experimental data obtained recently for dsRNA under applied force and torque. The predictions were enabled by the HelixMC package, first presented in this paper. HelixMC advances crystallography-derived base-pair level models (BPLMs) to simulate kilobase-length dsDNAs and dsRNAs under external forces and torques, including their global linking numbers. These calculations recovered the experimental bending persistence length of dsRNA within the error of the simulations and accurately predicted that dsRNA's "spring-like" conformation would give a two-fold decrease of stretch modulus relative to dsDNA. Further blind predictions of helix torsional properties, however, exposed inaccuracies in current BPLM theory, including three-fold discrepancies in torsional persistence length at the high force limit and the incorrect sign of dsRNA link-extension (twist-stretch) coupling. Beyond these experiments, HelixMC predicted that 'nucleosome-excluding' poly(A)/poly(T) is at least two-fold stiffer than random-sequence dsDNA in bending, stretching, and torsional behaviors; Z-DNA to be at least three-fold stiffer than random-sequence dsDNA, with a near-zero link-extension coupling; and non-negligible effects from base pair step correlations. We propose that experimentally testing these predictions should be powerful next steps for

  14. Catalytic Activity of a Binary Informational Macromolecule

    NASA Technical Reports Server (NTRS)

    Reader, John S.; Joyce, Gerald F.

    2003-01-01

    RNA molecules are thought to have played a prominent role in the early history of life on Earth based on their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, due to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unie'. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyze the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalyzed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.

  15. Cloning and physical mapping of DNA complementary to potato leafroll virus RNA

    SciTech Connect

    Smith, O.P.

    1987-01-01

    Potato leafroll virus (PLRV) was aphid-transmitted from potato (Solanum tuberosum cultivar Russett Burbank) to ground cherry (Physalis floridana), where it was maintained by serial aphid transmission. Serological and plant differential tests indicated that the isolate was not contaminated with beet western yellows virus. Purified PLRV RNA was poly(A)-tailed in vitro and used as a template for reverse transcriptase, primed with oligo(dT). Alkaline gel electrophoresis of /sup 32/P-labeled first-strand complementary DNA (cDNA) indicated a major size range of 0.1 to 3.5 kilobases (kb). A small percentage of transcripts corresponded to full length PLRV RNA. Following RNase H and DNA polymerase I-mediated second strand synthesis, double-stranded cDNA was cloned into the Pst I site of the plasmid pUC9 using oligo (dC)-oligo(dG) tailing methodology. Escherichia coli JM109 transformants were screened with first-strand /sup 32/P-cDNA in colony hybridization experiments to confirm that recombinants contained PLRV-specific sequences.

  16. Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

    SciTech Connect

    Gleghorn, M.; Davydova, E; Rothman-Denes, L; Murakami, K

    2008-01-01

    Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

  17. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    SciTech Connect

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S.; Arbuthnot, Patrick

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  18. Bacterial and archaeal communities in long-term contaminated surface and subsurface soil evaluated through coextracted RNA and DNA.

    PubMed

    Mikkonen, Anu; Santalahti, Minna; Lappi, Kaisa; Pulkkinen, Anni-Mari; Montonen, Leone; Suominen, Leena

    2014-10-01

    Soil RNA and DNA were coextracted along a contamination gradient at a landfarming field with aged crude oil contamination to investigate pollution-dependent differences in 16S rRNA and rRNA gene pools. Microbial biomass correlated with nucleic acid yields as well as bacterial community change, indicating that the same factors controlled community size and structure. In surface soil, bacterial community evenness, estimated through length heterogeneity PCR (LH-PCR) fingerprinting, appeared higher for RNA-based than for DNA-based communities. The RNA-based community profiles resembled the DNA-based communities of soil with a lower contamination level. Cloning-based identification of bacterial hydrocarbon-degrading taxa in the RNA pool, representing the viable community with high protein synthesis potential, indicated that decontamination processes still continue. Analyses of archaea revealed that only Thaumarchaeota were present in the aerobic samples, whereas more diverse communities were found in the compacted subsurface soil with more crude oil. For subsurface bacteria, hydrocarbon concentration explained neither the community structure nor the difference between RNA-based and DNA-based communities. However, rRNA of bacterial taxa associated with syntrophic and sulphate-reducing alkane degradation was detected. Although the same prokaryotic taxa were identified in DNA and RNA, comparison of the two nucleic acid pools can aid in the assessment of past and future restoration success. PMID:24986450

  19. The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

    SciTech Connect

    Reich, Charles F.; Pisetsky, David S.

    2009-03-10

    Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for {beta}-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

  20. HYPOXIA-INDUCED GROWTH LIMITATION OF JUVENILE FISHES IN AN ESTUARINE NURSERY: ASSESSMENT OF SMALL-SCALE TEMPORAL DYNAMICS USING RNA:DNA

    EPA Science Inventory

    The ratio of RNA to DNA (RNA:DNA) in white muscle tissue of juvenile summer flounder (Paralichthys dentatus) and weakfish (Cynoscion regalis) was used as a proxy for recent growth rate in an estuarine nursery. Variability in RNA:DNA was examined relative to temporal changes in te...

  1. Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus.

    PubMed Central

    Dhundale, A; Furuichi, T; Inouye, M; Inouye, S

    1988-01-01

    A deletion mutation of the gene (msd-msr) for the branched RNA-linked msDNA of Myxococcus xanthus was constructed by replacing the chromosomal 0.7-kilobase (kb) SmaI-XhoI fragment encompassing msd-msr with a 1.4-kb fragment carrying a gene for kanamycin resistance. It was found that this deletion strain (delta msSX) could not produce msDNA, although it still contained another species of msDNA, mrDNA (msDNA, reduced size). No apparent differences between delta msSX and the wild-type strain were observed in terms of cell growth, morphogenesis, fruiting-body formation, or motility. Both a deletion mutation at the region 100 base pairs upstream of msd and an insertion mutation at a site 500 base pairs upstream of msd showed a significant reduction of msDNA production, indicating that there is a cis- or trans-acting positive element in this region. When the 3.5-kb BamHI fragment carrying msd-msr from Stigmatella aurantiaca was inserted into the M. xanthus chromosome, the S. aurantiaca msDNA was found to be produced in M. xanthus. Images PMID:2461359

  2. Heat shock induces a loss of rRNA-encoding DNA repeats in Brassica nigra.

    PubMed Central

    Waters, E R; Schaal, B A

    1996-01-01

    Stress-induced mutations may play an important role in the evolution of plants. Plants do not sequester a germ line, and thus any stress-induced mutations could be passed on to future generations. We report a study of the effects of heat shock on genomic components of Brassica nigra Brassicaceae. Plants were submitted to heat stress, and the copy number of two nuclear-encoded single-copy genes, rRNA-encoding DNA (rDNA) and a chloroplast DNA gene, was determined and compared to a nonstressed control group. We determined whether genomic changes were inherited by examining copy number in the selfed progeny of control and heat-treated individuals. No effects of heat shock on copy number of the single-copy nuclear genes or on chloroplast DNA are found. However, heat shock did cause a statistically significant reduction in rDNA copies inherited by the F1 generation. In addition, we propose a DNA damage-reppair hypothesis to explain the reduction in rDNA caused by heat shock. Images Fig. 1 PMID:8643652

  3. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    PubMed

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-01

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii. PMID:27352216

  4. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  5. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B

    PubMed Central

    Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  6. Transcription-coupled Hypernegative Supercoiling of Plasmid DNA by T7 RNA Polymerase in Escherichia coli Topoisomerase I Deficient Strains

    PubMed Central

    Samul, Rebecca; Leng, Fenfei

    2007-01-01

    Summary Transcription by RNA polymerase can stimulate negative DNA supercoiling in Escherichia coli topA strains. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription in which positive DNA supercoils are generated in front of a translocating RNA polymerase and negative supercoils behind it. However, since a specific system is lacking to study the factors governing this biologically important process, the parameters regulating transcription-coupled DNA supercoiling (TCDS) in Escherichia coli still remain elusive. In this paper, we describe our efforts to study TCDS in Escherichia coli using a newly developed system. This system consists of a topA strain, VS111(DE3) or DM800(DE3), in which a λDE3 prophage containing a T7 RNA polymerase gene under control of the lacUV5 promoter has been integrated into the cell chromosome, along with a set of plasmids producing RNA transcripts of various lengths by T7 RNA polymerase. Using this system, we found that transcription by T7 RNA polymerase strikingly induced formation of hypernegatively supercoiled plasmid DNA. We also discovered, for the first time, that TCDS was dependent on the length of RNA transcripts in vivo, precisely predicted by the “twin-supercoiled-domain” model of transcription. Furthermore, our results demonstrated that hypernegative supercoiling of plasmid DNA by T7 RNA polymerase did not require anchoring of DNA to the bacterial cytoplasmic membrane. These results indicate that a transcribing RNA polymerase alone is sufficient to cause change of local DNA superhelicity, which can have a powerful impact on the conformation and function of critical DNA sequence elements, such as promoters and DNA replication origins. PMID:17980389

  7. Multifunctional iron oxide nanoparticles for diagnostics, therapy and macromolecule delivery.

    PubMed

    Yen, Swee Kuan; Padmanabhan, Parasuraman; Selvan, Subramanian Tamil

    2013-01-01

    In recent years, multifunctional nanoparticles (NPs) consisting of either metal (e.g. Au), or magnetic NP (e.g. iron oxide) with other fluorescent components such as quantum dots (QDs) or organic dyes have been emerging as versatile candidate systems for cancer diagnosis, therapy, and macromolecule delivery such as micro ribonucleic acid (microRNA). This review intends to highlight the recent advances in the synthesis and application of multifunctional NPs (mainly iron oxide) in theranostics, an area used to combine therapeutics and diagnostics. The recent applications of NPs in miRNA delivery are also reviewed. PMID:24396508

  8. Multifunctional Iron Oxide Nanoparticles for Diagnostics, Therapy and Macromolecule Delivery

    PubMed Central

    Yen, Swee Kuan; Padmanabhan, Parasuraman; Selvan, Subramanian Tamil

    2013-01-01

    In recent years, multifunctional nanoparticles (NPs) consisting of either metal (e.g. Au), or magnetic NP (e.g. iron oxide) with other fluorescent components such as quantum dots (QDs) or organic dyes have been emerging as versatile candidate systems for cancer diagnosis, therapy, and macromolecule delivery such as micro ribonucleic acid (microRNA). This review intends to highlight the recent advances in the synthesis and application of multifunctional NPs (mainly iron oxide) in theranostics, an area used to combine therapeutics and diagnostics. The recent applications of NPs in miRNA delivery are also reviewed. PMID:24396508

  9. Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

    PubMed Central

    Haag, Jeremy R.; Pontes, Olga; Pikaard, Craig S.

    2009-01-01

    Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active. PMID:19119310

  10. Differentiating the Protein Coding and Noncoding RNA Segments of DNA Using Shannon Entropy

    NASA Astrophysics Data System (ADS)

    Mazaheri, P.; Shirazi, A. H.; Saeedi, N.; Reza Jafari, G.; Sahimi, Muhammad

    The complexity of DNA sequences is evaluated in order to differentiate between protein-coding and noncoding RNA segments. The method is based on computing the Shannon entropy of the sequences. By comparing the entropy of the original sequence with that of its shuffled one, we identify the source of the difference between the two segments and their relative contributions to the sequence. To demonstrate the method, the DNA sequences of the bacterium Clostridium difficile 630 (G + C = 29.1%) and Bdellovibrio bacteriovorus (G + C = 50.6%) are analyzed, which are representatives of bacteria with unbalanced and balanced nucleotide content, respectively. It is shown that in both bacteria, regardless of nucleotide content, ΔrS — the relative difference of the two entropies — is significantly greater in protein-coding regions, when compared with noncoding RNA segments.

  11. Stopped in its tracks: the RNA polymerase molecular motor as a robust sensor of DNA damage.

    PubMed

    Howan, K; Monnet, J; Fan, J; Strick, T R

    2014-08-01

    DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C). PMID:24685770

  12. Configurational diffusion of coal macromolecules

    SciTech Connect

    Guin, J.A.; Curtis, C.W.; Tarrer, A.R.; Kim, S.; Hwang, D.; Chen, C.C.; Chiou, Z.

    1991-01-01

    The objective of our research was to obtain fundamental information regarding the functional dependence of the diffusion coefficient of coal molecules on the ratio of molecule to pore diameter. That is, the objective of our study was to examine the effect of molecule size and configuration on hindered diffusion of coal macromolecules through as porous medium. To best accomplish this task, we circumvented the complexities of an actual porous catalyst by using a well defined porous matrix with uniform capillaric pores, i.e., a track-etched membrane. In this way, useful information was obtained regarding the relationship of molecular size and configuration on the diffusion rate of coal derived macromolecules through a pore structure with known geometry. Similar studies were performed using a pellet formed of porous alumina, to provide a link between the idealized membranes and the actual complex pore structure of real catalyst extrudates. The fundamental information from our study will be useful toward the tailoring of catalysts to minimize diffusional influences and thereby increase coal conversion and selectivity for desirable products. (VC)

  13. Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA.

    PubMed Central

    Jones, P C; Hayward, G S; Roizman, B

    1977-01-01

    Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection. Images PMID:189068

  14. Effects of trace elements and pesticides on dephosphorylation of RNA and DNA added to soils

    SciTech Connect

    Frankenberger, W.T. Jr.; Johanson, J.B.; Lund L.J.

    1986-01-01

    This study was carried out to assess the effects of 14 trace elements, 12 herbicides, and two fungicides on dephosphorylation of yeast ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) added to soils (Xerollic Calciorthids and Typic Haploxeralfs). The cumulative amount of ortho phosphate (Pi) released from nucleic acids increased linearly with time of incubation (up to 72 h), decreased with profile depth, and was highly influenced by soil pH. When trace elements were applied and compared by using 2.5 mmol kg/sup -1/ of soil, the average inhibition in dephosphorylation of RNA and DNA in two soils ranged from 17% with Co(II) to 52% with Cu(II). The most effective inhibitors of nucleic acid dephosphorylation were Ag(I), Cu(I), Cd(II), Cu(II), Mn(II), Ni(II), and Pb(II) (avg inhibition greater than or equal to 35%). Other elements that inhibited dephosphorylation of RNA and DNA added to soils included Ba(II), Co(II), Hg(II), Zn(II), Ti(IV), V(IV), and W(VI). When the pesticides were compared by using 5 mg of active ingredient kg/sup -1/ of soil, the average inhibition in nucleic acid dephosphorylation ranged from 14% with butylate to 39% with chloramben. The most effective inhibitors (> 25%) were atrazine, naptalam, chloramben, dicamba, trifluralin, and maneb. Other pesticides that inhibited RNA and DNA dephosphorylation in soils included cyanazine, 2,4-D, dinitroamine, EPTC plus R-25788, alachlor, paraquat, butylate, and captan.

  15. RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

    PubMed Central

    Chícharo, Maria Alexandra; Chícharo, Luis

    2008-01-01

    Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. PMID:19325815

  16. RNA/DNA ratio as biomarkers for periphyton and macroinvertebrate growth

    NASA Astrophysics Data System (ADS)

    Mewes, Daniela; Winkelmann, Carola

    2015-04-01

    A biocenosis is a complex assembly of organisms driven and shaped by numerous processes and interactions. Yet, in order to describe the biocenosis of a stream often only state variables, such as algal biomass or invertebrate diversity and abundance, are measured. But these variables fail to provide much needed information on those driving processes. Because processes such as growth of periphyton and invertebrates can hardly be measured directly in the field, the use of biomarkers is a promising approach to quantify biological rates under natural conditions. Periphyton represents the main food source for invertebrate grazers and periphyton growth rate rather than standing stocks alone allows the estimation of the availability of this resource. A linear relationship of RNA/DNA ratios and growth rate has previously been established for single species cultures of algae and bacteria but not for naturally occurring freshwater periphyton assemblages. In this study it could be shown that linear relationships of RNA/DNA ratios and growth rate are also valid for naturally occurring freshwater periphyton assemblages and can be used as biomarkers for periphyton growth rate. Moreover, recent results indicate that the RNA/DNA ratio might also be used as biomarker for invertebrates, because high-quality food was observed to increase the RNA/DNA ratios of the freshwater amphipod Dikerogammarus villosus. These are very promising results with regard to the usefulness and applicability of biomarkers ecosystem analysis in running waters. Additional biomarkers allowing the analysis of further processes and interactions within the food web such as PLFAs (phospholipid-fatty acids), neutral lipids and PUFAs (polyunsaturated fatty acids) are to be tested for their applicability in stream ecosystems.

  17. Ion distributions around left- and right-handed DNA and RNA duplexes: a comparative study.

    PubMed

    Pan, Feng; Roland, Christopher; Sagui, Celeste

    2014-12-16

    The ion atmosphere around nucleic acids is an integral part of their solvated structure. However, detailed aspects of the ionic distribution are difficult to probe experimentally, and comparative studies for different structures of the same sequence are almost non-existent. Here, we have used large-scale molecular dynamics simulations to perform a comparative study of the ion distribution around (5'-CGCGCGCGCGCG-3')2 dodecamers in solution in B-DNA, A-RNA, Z-DNA and Z-RNA forms. The CG sequence is very sensitive to ionic strength and it allows the comparison with the rare but important left-handed forms. The ions investigated include Na(+), K(+) and Mg(2 +), with various concentrations of their chloride salts. Our results quantitatively describe the characteristics of the ionic distributions for different structures at varying ionic strengths, tracing these differences to nucleic acid structure and ion type. Several binding pockets with rather long ion residence times are described, both for the monovalent ions and for the hexahydrated Mg[(H2O)6](2+) ion. The conformations of these binding pockets include direct binding through desolvated ion bridges in the GpC steps in B-DNA and A-RNA; direct binding to backbone oxygens; binding of Mg[(H2O)6](2+) to distant phosphates, resulting in acute bending of A-RNA; tight 'ion traps' in Z-RNA between C-O2 and the C-O2' atoms in GpC steps; and others. PMID:25428372

  18. Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

    PubMed Central

    Marczynski, G T; Schultz, P W; Jaehning, J A

    1989-01-01

    We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested. Images PMID:2677667

  19. ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects

    PubMed Central

    Qiu, Haiyan; Lee, Sebum; Shang, Yulei; Wang, Wen-Yuan; Au, Kin Fai; Kamiya, Sherry; Barmada, Sami J.; Finkbeiner, Steven; Lui, Hansen; Carlton, Caitlin E.; Tang, Amy A.; Oldham, Michael C.; Wang, Hejia; Shorter, James; Filiano, Anthony J.; Roberson, Erik D.; Tourtellotte, Warren G.; Chen, Bin; Tsai, Li-Huei; Huang, Eric J.

    2014-01-01

    Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C–associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions. PMID:24509083

  20. Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

    SciTech Connect

    Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

    1985-07-01

    To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

  1. Effects of 12-O-tetradecanoylphorbol-13-acetate on the incorporation of labelled precursors into RNA, DNA and protein in epidermis, dermis and subcutis from precancerous mouse skin with reference to enhanced tumorigenesis

    SciTech Connect

    Bhisey, R.A.; Ramchandani, A.G.; Sirsat, S.M.

    1984-02-01

    The effects of a single application of 1.8 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) on precursor incorporation into RNA, DNA and protein in the epidermis, dermis and subcutis from 3-methylcholanthrene (MCA) injected precancerous mouse skin were studied at various time points between 3 and 96 h. In the precancerous tissues, the rates of incorporation of (/sup 3/H)uridine into RNA did not alter appreciably from those in the control tissues; while the rates of (/sup 3/H)methylthymidine incorporation into DNA were elevated with peaks appearing between 6 and 12 h, at 24 h and at 72 h in epidermis, dermis and subcutis. The rate of incorporation of (/sup 14/C)leucine into protein was markedly elevated in all the three tissues which showed 3-4 sharp peaks. The maximum stimulation ranged between 14 and 20 times that of the control. A single application of TPA to the precancerous mouse skin induced early stimulation of precursor incorporation into all the three macromolecules in epidermis, dermis and subcutis. The increased stimulation was maintained for 36-72 h. The patterns of incorporation of (/sup 3/H)methylthymidine into DNA gave rise to 2-3 peaks of elevated uptake in each tissue up to 36-48 h. A lowered rate of DNA synthesis between 48 and 60 h was followed by a peak at 72 h. In each group, epidermal mitotic activity correlated well with spurts of precursor incorporation into cellular DNA. The observations indicate that TPA recruits more cells into the DNA synthetic phase and accelerates selective growth of preneoplastic cells during tumor progression.

  2. Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation.

    PubMed

    Chow, Michael Y T; Lam, Jenny K W

    2015-01-01

    Nucleic acid therapeutics has huge potential for the treatment of a wide range of diseases including respiratory diseases. Plasmid DNA (pDNA) and small interfering RNA (siRNA) are the two most widely investigated nucleic acids for therapeutic development. However, efficient and safe delivery of nucleic acids is still a major hurdle in translating nucleic acid therapy into clinical practice. For the treatment of respiratory diseases, administration via inhalation is the most direct and effective way to deliver therapeutic nucleic acids to the lungs. Although liquid aerosol formulation is investigated in most of the studies, it is not desirable in terms of maintaining the stability of nucleic acid especially during long-term storage. This problem could be circumvented by formulating the therapeutic nucleic acids into dry powder for inhalation, and should be considered as the future direction of developing inhalable nucleic acids. In this review, the three major particle engineering methods investigated for the preparation of inhalable pDNA and siRNA formulations, including spray drying (SD), spray freeze drying (SFD) and supercritical fluid (SFC) drying, are discussed and compared. Moreover, common assessment methods and the challenges of evaluating the biological activities of inhalable nucleic acid powders are also reviewed. PMID:26290202

  3. Adsorption of DNA/RNA nucleobases on hexagonal boron nitride sheet: an ab initio study.

    PubMed

    Lin, Qing; Zou, Xiaolong; Zhou, Gang; Liu, Rui; Wu, Jian; Li, Jia; Duan, Wenhui

    2011-07-14

    Our ab initio calculations indicate that the interaction of deoxyribonucleic/ribonucleic acid (DNA/RNA) nucleobases [guanine (G), adenine (A), thymine (T), cytosine (C), and uracil (U)] with the hexagonal boron nitride (h-BN) sheet, a polar but chemically inert surface, is governed by mutual polarization. Unlike the case of graphene, all nucleobases exhibit the same stacking arrangement on the h-BN sheet due to polarization effects: the anions (N and O atoms) of nucleobases prefer to stay on top of cations (B) of the substrate as far as possible, regardless of the biological properties of nucleobases. The adsorption energies, ranging from 0.5 eV to 0.69 eV, increase in the order of U, C, T, A and G, which can be attributed to different side groups or atoms of nucleobases. The fundamental nature of DNA/RNA nucleobases and h-BN sheet remains unchanged upon adsorption, suggesting that the h-BN sheet is a promising template for DNA/RNA-related research, such as self-assembly. PMID:21637870

  4. Reduction of RNA and DNA in Methylococcus capsulatus by endogenous nucleases.

    PubMed

    Larsen, J; Joergensen, L

    1996-03-01

    A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90 degrees C. Cells were then incubated at 60 degrees C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight loss in connection with an 80% reduction of the nucleic acid content was 20%-25%, giving a final product with a raw protein content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO4 and ZnSO4. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO4. Reduction of RNA was not increased by any of the minerals tested. PMID:8920188

  5. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method.

    PubMed

    Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  6. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

    PubMed Central

    Laguerre, Aurélien; Wong, Judy M. Y.; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  7. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    PubMed Central

    2014-01-01

    Background Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. Conclusions A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities. PMID:25183040

  8. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    SciTech Connect

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  9. RNA exosome regulates AID DNA mutator activity in the B cell genome

    PubMed Central

    Pefanis, Evangelos; Basu, Uttiya

    2015-01-01

    The immunoglobulin diversification processes of somatic hypermutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome-wide studies have uncovered pervasive AID activity throughout the B cell genome. It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co-transcriptional RNA degradation mechanisms. Here we review aspects and consequences of eukaryotic transcription that lead to early termination, RNA exosome recruitment, and ultimately targeting of AID mutagenic activity. PMID:26073986

  10. Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR.

    PubMed

    Ferracin, Manuela; Salamon, Irene; Lupini, Laura; Miotto, Elena; Sabbioni, Silvia; Negrini, Massimo

    2016-01-01

    Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective. PMID:27403944

  11. Connecting the dots of RNA-directed DNA methylation in Arabidopsis thaliana.

    PubMed

    Costa-Nunes, Pedro; Vitins, Alexa; Pontes, Olga

    2014-06-01

    Noncoding RNAs are the rising stars of genome regulation and are crucial to an organism's metabolism, development, and defense. One of their most notable functions is its ability to direct epigenetic modifications through small RNA molecules to specific genomic regions, ensuring transcriptional regulation, proper genome organization, and maintenance of genome integrity. Here, we review the current knowledge of the spatial organization of the Arabidopsis thaliana RNA-directed DNA methylation pathway within the cell nucleus, which, while known to be essential for the proper establishment of epigenetic modifications, remains poorly understood. We will also discuss possible future cytological approaches that have the potential of unveiling functional insights into how small RNA-directed epigenetics is regulated through the spatiotemporal regulation of its major components within the cell. PMID:24846724

  12. Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses

    PubMed Central

    Chiappinelli, Katherine B.; Strissel, Pamela L.; Desrichard, Alexis; Li, Huili; Henke, Christine; Akman, Benjamin; Hein, Alexander; Rote, Neal S.; Cope, Leslie M.; Snyder, Alexandra; Makarov, Vladimir; Buhu, Sadna; Slamon, Dennis J.; Wolchok, Jedd D.; Pardoll, Drew M.; Beckmann, Matthias W.; Zahnow, Cynthia A.; Mergoub, Taha; Chan, Timothy A.; Baylin, Stephen B.; Strick, Reiner

    2015-01-01

    Summary We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a Type I Interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response twofold, and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model. PMID:26317466

  13. Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

    PubMed

    Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

    2006-08-18

    DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes. PMID:16859707

  14. Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA-Programmed Nanoparticle Disassembly.

    PubMed

    He, Xuewen; Zeng, Tao; Li, Zhi; Wang, Ganglin; Ma, Nan

    2016-02-24

    Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low-abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal-to-background ratio of conventional molecular imaging probes. Herein, we report a class of DNA-templated gold nanoparticle (GNP)-quantum dot (QD) assembly-based probes for catalytic imaging of cancer-related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA-programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non-catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells. PMID:26694689

  15. Seasonal Succession Leads to Habitat-Dependent Differentiation in Ribosomal RNA:DNA Ratios among Freshwater Lake Bacteria

    PubMed Central

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-01-01

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors. PMID:27199936

  16. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    DOE PAGESBeta

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

  17. mRNA and DNA Detection of Human Papillomaviruses in Women of All Ages Attending Two Colposcopy Clinics

    PubMed Central

    Spathis, Aris; Kottaridi, Christine; Chranioti, Aikaterini; Meristoudis, Christos; Chrelias, Charalambos; Panayiotides, Ioannis G.; Paraskevaidis, Evangelos; Karakitsos, Petros

    2012-01-01

    Objective HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. Methods 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. Results HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests’ positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. Conclusions Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results. PMID:23166611

  18. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

    SciTech Connect

    Kim, Sunyoung; Baltimore, D. Massachusetts Institute of Technology, Cambridge ); Byrn, R.; Groopman, J. )

    1989-09-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4{sup +} lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.

  19. Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification.

    PubMed

    Chetverina, Helena V; Falaleeva, Marina V; Chetverin, Alexander B

    2004-11-15

    A universal procedure that permits the whole human blood to be tested for the presence of single molecules of DNA and RNA targets is described. The procedure includes a novel protocol for the isolation of total nucleic acids from the guanidinium thiocyanate lysate of unfractionated blood in which, prior to phenol/chloroform extraction, the sample is deproteinized by precipitation with isopropanol. The procedure results in a nearly 100% yield of DNA and RNA, preserves the integrity of RNA, and removes any polymerase chain reaction (PCR) inhibitors. Following reverse transcription (RT), target molecules are counted after having been amplified as molecular colonies by carrying out PCR in a polyacrylamide gel. The entire procedure was checked by assaying viral DNA and RNA in 100-microl aliquots of the whole blood and was found to be capable of detecting 100% molecules of DNA target and 50% molecules of RNA target. Unexpectedly, nucleic acids at relatively high concentrations (1 ng/microl) were found to selectively inhibit the RT activity of Thermus thermophilus DNA polymerase without affecting its DNA-dependent polymerization activity. It follows that the popular single-enzyme RT-PCR format, in which this DNA polymerase serves for both RT and PCR, is not appropriate for assaying rare RNA targets. PMID:15494145

  20. A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA

    PubMed Central

    Subramaniam, Siddharth; Vohra, Iqbal; Iyer, Aishwarya; Nair, Naveen K; Mittra, Indraneel

    2016-01-01

    Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical relationship between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical relationship is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu. PMID:27134724

  1. Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

    PubMed Central

    Xia, Gang; Chen, Liangjing; Sera, Takashi; Fa, Ming; Schultz, Peter G.; Romesberg, Floyd E.

    2002-01-01

    The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. PMID:12011423

  2. DNA/RNA chimera templates improve the emission intensity and target the accessibility of silver nanocluster-based sensors for human microRNA detection.

    PubMed

    Shah, Pratik; Choi, Suk Won; Kim, Ho-jin; Cho, Seok Keun; Thulstrup, Peter Waaben; Bjerrum, Morten Jannik; Bhang, Yong-Joo; Ahn, Jong Cheol; Yang, Seong Wook

    2015-05-21

    In recent years microRNAs (miRNAs) have been established as important biomarkers in a variety of diseases including cancer, diabetes, cardiovascular disease, aging, Alzheimer's disease, asthma, autoimmune disease and liver diseases. As a consequence, a variety of monitoring methods for miRNAs have been developed, including a fast and simple method for miRNA detection by exploitation of the unique photoluminescence of DNA-templated silver nanoclusters (DNA/AgNCs). To increase the versatility of the AgNC-based method, we have adopted DNA/RNA chimera templates for AgNC-based probes, allowing response from several human miRNAs which are hardly detectable with DNA-based probes. Here, we demonstrate in detail the power of DNA/RNA chimera/AgNC probes in detecting two human miRNAs, let-7a and miR-200c. The DNA/RNA chimera-based probes are highly efficient to determine the level of miRNAs in several human cell lines. PMID:25759134

  3. Technological development of structural DNA/RNA-based RNAi systems and their applications.

    PubMed

    Jeong, Eun Hye; Kim, Hyejin; Jang, Bora; Cho, Hyesoo; Ryu, Jaehee; Kim, Boyeon; Park, Youngkuk; Kim, Jieun; Lee, Jong Bum; Lee, Hyukjin

    2016-09-01

    RNA interference (RNAi)-based gene therapy has drawn tremendous attention due to its highly specific gene regulation by selective degradation of any target mRNA. There have been multiple reports regarding the development of various cationic materials for efficient siRNA delivery, however, many studies still suffer from the conventional delivery problems such as suboptimal transfection performance, a lack of tissue specificity, and potential cytotoxicity. Despite the huge therapeutic potential of siRNAs, conventional gene carriers have failed to guarantee successful gene silencing in vivo, thus not warranting clinical trials. The relatively short double-stranded structure of siRNAs has resulted in uncompromising delivery formulations, as well as low transfection efficiency, compared with the conventional nucleic acid drugs such as plasmid DNAs. Recent developments in structural siRNA and RNAi nanotechnology have enabled more refined and reliable in vivo gene silencing with multiple advantages over naked siRNAs. This review focuses on recent progress in the development of structural DNA/RNA-based RNAi systems and their potential therapeutic applications. In addition, an extensive list of prior reports on various RNAi systems is provided and categorized by their distinctive molecular characters. PMID:26494399

  4. Strong Inverse Correlation Between MicroRNA-125b and Human Papillomavirus DNA in Productive Infection

    PubMed Central

    Nuovo, Gerard J.; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F.; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D.; Croce, Carlo

    2014-01-01

    Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, — 99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis. PMID:20736742

  5. cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the indianmeal moth, Plodia interpunctella.

    PubMed

    Zhu, Y C; Oppert, B; Kramer, K J; McGaughey, W H; Dowdy, A K

    2000-02-01

    Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two

  6. MACROMOLECULES FACILITATE THE TRANSPORT OF TRACE ORGANICS

    EPA Science Inventory

    Macromolecules in the pore fluid of a soil may influence the mobility of hydrophobic compounds by their partitioning to the macromolecule, which moves with, or even faster than, the water. The mobility is described mathematically by a chemical transport model. The significance of...

  7. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-01

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended. PMID:23796243

  8. The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

    NASA Technical Reports Server (NTRS)

    Dworkin, Jason P.; Miller, Stanley L.

    1995-01-01

    If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could

  9. De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.

    PubMed

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R; Kasschau, Kristin; Dolja, Valerian V; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M

    2014-01-01

    Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

  10. De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs

    PubMed Central

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R.; Kasschau, Kristin; Dolja, Valerian V.; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M.

    2014-01-01

    Virus-infected plants accumulate abundant, 21–24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this ‘siRNA omics’ approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

  11. Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition.

    PubMed

    Gao, Pu; Yang, Hui; Rajashankar, Kanagalaghatta R; Huang, Zhiwei; Patel, Dinshaw J

    2016-08-01

    CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRISPR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9. PMID:27444870

  12. Human papillomavirus DNA and mRNA prevalence and association with cervical cytological abnormalities in the Irish HIV population.

    PubMed

    Loy, Aisling; McInerney, Jamie; Pilkington, Loretto; Keegan, Helen; Delamere, Sandra; Martin, Cara M; Sheils, Orla; O'Leary, John J; Mulcahy, Fiona

    2015-10-01

    The complex interplay between HIV and human papillomavirus and its link to cervical dysplasia is poorly understood. This is the first study to assess the prevalence of oncogenic human papillomavirus mRNA in HIV-positive women, its relationship to HIV and its potential use in the triage of cervical cancer screening in HIV-positive women. In this cross-sectional study, we included 321 HIV-positive women. In all, 28.7% had abnormal cervical cytology, 51.1% were human papillomavirus DNA-positive and 21.8% tested positive for human papillomavirus mRNA. Women with a CD4 count of <200 × 10(6)/L were more likely to test positive for human papillomavirus DNA and mRNA. Virally suppressed women were less likely to be human papillomavirus DNA-positive; however, the same did not hold true for human papillomavirus mRNA. We found the human papillomavirus mRNA screening to be more specific when screening for low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion than human papillomavirus DNA at 84.53% compared to 57.36%. However, the sensitivity was less at 51.59% versus 91.07% for human papillomavirus DNA. It may be possible in the future to use human papillomavirus mRNA/DNA testing within a triage algorithm for the screening and management of cervical cancer in the HIV-positive patient. PMID:25258395

  13. A Three-Helix Junction Is the Interface between Two Functional Domains of Prohead RNA in ϕ29 DNA Packaging

    PubMed Central

    Zhao, Wei; Saha, Mitul; Ke, Ailong; Morais, Marc C.; Jardine, Paul J.

    2012-01-01

    The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage ϕ29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA. PMID:22896620

  14. Hg(2+) detection using a phosphorothioate RNA probe adsorbed on graphene oxide and a comparison with thymine-rich DNA.

    PubMed

    Huang, Po-Jung Jimmy; van Ballegooie, Courtney; Liu, Juewen

    2016-06-01

    Mercury is a highly toxic heavy metal and many DNA-based biosensors have been recently developed for Hg(2+) detection in water. Among them, thymine-rich DNA is the most commonly used for designing Hg(2+) sensors. However, the thymine-Hg(2+) interaction is strongly affected by the buffer conditions. We recently reported a molecular beacon containing phosphorothioate (PS)-modified RNA linkages that can be cleaved by Hg(2+). In this work, the fluorescence quenching and DNA adsorption properties of nano-sized graphene oxide (NGO) were used to develop a new sensor using the PS-RNA chemistry. Three DNA probes, containing one, three and five PS-RNA linkages, respectively, were tested. Finally, a fluorophore-labeled poly-A DNA with five PS-RNA linkages was selected and adsorbed by NGO. In the presence of Hg(2+), the fluorophore was released from NGO due to the cleavage reaction, resulting in a fluorescence enhancement. This sensor is highly selective for Hg(2+) with a detection limit of 8.5 nM Hg(2+). For comparison, a fluorophore-labeled poly-T DNA was also tested, which responded to Hg(2+) more slowly and was inhibited by high NaCl concentrations, while the PS-RNA probe was more tolerant to different buffer conditions. This work indicates a new method for interfacing DNA with NGO for Hg(2+) detection. PMID:26580137

  15. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts.

    PubMed

    Liu, Y; Santi, D V

    2000-07-18

    A family of RNA m(5)C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MTase, Fmu, as an initial probe. The RNA m(5)C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m(5)C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m(5)U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m(5)C MTases remains unknown. PMID:10899996

  16. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

    PubMed Central

    Liu, Yaoquan; Santi, Daniel V.

    2000-01-01

    A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown. PMID:10899996

  17. Micelle-like Nanoparticles as Carriers for DNA and siRNA

    PubMed Central

    Navarro, Gemma; Pan, Jiayi; Torchilin, Vladimir P.

    2015-01-01

    Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor in vivo stability, gene molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, and low toxicity may contribute to the successful application of siRNA- and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimulus-sensitive functions to respond specifically to pathological or externally applied “triggers” (e.g., temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acid combinations. PMID:25557580

  18. Major degradable polycations as carriers for DNA and siRNA.

    PubMed

    Islam, Mohammad Ariful; Park, Tae-Eun; Singh, Bijay; Maharjan, Sushila; Firdous, Jannatul; Cho, Myung-Haing; Kang, Sang-Kee; Yun, Cheol-Heui; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-10

    Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA). PMID:24942341

  19. Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA.

    PubMed

    Bishop, Corey J; Tzeng, Stephany Y; Green, Jordan J

    2015-01-01

    Gold nanoparticles have utility for in vitro, ex vivo and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable the simultaneous co-delivery of DNA and short interfering RNA (siRNA). To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells. PMID:25246314

  20. Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA

    NASA Astrophysics Data System (ADS)

    Reichl, Maren; Herzog, Mario; Greiss, Ferdinand; Wolff, Manuel; Braun, Dieter

    2015-06-01

    Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012), 10.1073/pnas.1215764109], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis.

  1. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    PubMed Central

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  2. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

    Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  3. Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

    PubMed

    Barsky, D; Colvin, M E; Zon, G; Gryaznov, S M

    1997-02-15

    Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone. PMID:9016634

  4. Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

    PubMed Central

    Barsky, D; Colvin, M E; Zon, G; Gryaznov, S M

    1997-01-01

    Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone. PMID:9016634

  5. Analysis of dsDNA and RNA viromes in methanogenic digesters reveals novel viral genetic diversity.

    PubMed

    Calusinska, Magdalena; Marynowska, Martyna; Goux, Xavier; Lentzen, Esther; Delfosse, Philippe

    2016-04-01

    Although viruses are not the key players of the anaerobic digestion process, they may affect the dynamics of bacterial and archaeal populations involved in biogas production. Until now viruses have received very little attention in this specific habitat; therefore, as a first step towards their characterization, we optimized a virus filtration protocol from anaerobic sludge. Afterwards, to assess dsDNA and RNA viral diversity in sludge samples from nine different reactors fed either with waste water, agricultural residues or solid municipal waste plus agro-food residues, we performed metagenomic analyses. As a result we showed that, while the dsDNA viromes (21 assigned families in total) were dominated by dsDNA phages of the order Caudovirales, RNA viruses (14 assigned families in total) were less diverse and were for the main part plant-infecting viruses. Interestingly, less than 2% of annotated contigs were assigned as putative human and animal pathogens. Our study greatly extends the existing view of viral genetic diversity in methanogenic reactors and shows that these viral assemblages are distinct not only among the reactor types but also from nearly 30 other environments already studied, including the human gut, fermented food, deep sea sediments and other aquatic habitats. PMID:26568175

  6. Human origin recognition complex binds preferentially to G-quadruplex-preferable RNA and single-stranded DNA.

    PubMed

    Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

    2013-10-18

    Origin recognition complex (ORC), consisting of six subunits ORC1-6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs. PMID:24003239

  7. Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA*

    PubMed Central

    Hoshina, Shoko; Yura, Kei; Teranishi, Honami; Kiyasu, Noriko; Tominaga, Ayumi; Kadoma, Haruka; Nakatsuka, Ayaka; Kunichika, Tomoko; Obuse, Chikashi; Waga, Shou

    2013-01-01

    Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs. PMID:24003239

  8. Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA

    PubMed Central

    Christensen, Ulf B.; Pedersen, Erik B.

    2002-01-01

    We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9°C per modification when the IPN was added as a dangling end and up to 6.7°C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8°C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes. PMID:12433995

  9. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  10. A microfluorometric method for quantifying RNA and DNA in terrestrial insects

    PubMed Central

    Kyle, M.; Watts, T.; Schade, J.; Elser, J.J.

    2003-01-01

    Evidence is accumulating for a mechanistic linkage between body phosphorus content and growth and reproduction of individual organisms, due in part to variation in allocation of resources to ribosomal RNA. Testing this connection requires reliable methods of quantifying the nucleic acid content of individual organisms. Although methods for quantifying nucleic acids are available for a wide array of organisms, adaptation of such methods for study of insects has been neglected. Sensitive stains and high throughput fluorometric measurements are now available that substantially improve past methodologies. Here we present methods for the extraction and quantification of insect RNA and DNA based on the use of N-lauroylsarcosine and sonication for extraction, the nucleases RNase and DNase, and the use of microplate fluorescent assays to quantify nucleic acids as percent of body weight in insects. We illustrate the method using Drosophila and curculionid weevils. PMID:15841218

  11. Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma

    PubMed Central

    Vargas-Meneses, Milenka V.; Massanella, Marta; Ignacio, Caroline C.; Gianella, Sara

    2016-01-01

    Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology. PMID:27376109

  12. Split Spinach Aptamer for Highly Selective Recognition of DNA and RNA at Ambient Temperatures.

    PubMed

    Kikuchi, Nanami; Kolpashchikov, Dmitry M

    2016-09-01

    Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested. In SSA design, two RNA or RNA/DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for low-fluorescent 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) dye, which resulted in up to a 270-fold increase in fluorescence. The major advantage of the SSA over state-of-the art fluorescent probes is high selectivity: it produces only background fluorescence in the presence of a single-base-mismatched analyte, even at room temperature. SSA is therefore a promising tool for label-free analysis of nucleic acids at ambient temperatures. PMID:27305425

  13. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    PubMed

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471. PMID:27275414

  14. Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

    PubMed Central

    Barakat, Tahsin Stefan; Gribnau, Joost

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

  15. Cytophotometric investigation of DNA and RNA content in nuclei of active Strasburger cells in Pinus nigra var. austriaca (Hoess) Badoux.

    PubMed

    Sauter, J J; Ulrich, H

    1977-01-01

    The nuclei of active, sieve cell-associated Strasburger cells in the secondary phloem of Pinus nigra var. austriaca (Hoess) Badoux have been studied for their structure and DNA and RNA content. No difference in size compared to those of ordinary ray cells was found. The nuclear surface is often increased by an ameboid or lobed shape. The amount of highly decondensed chromatin is greatly increased. Cytophotometric measurements of DNA content of both Feulgen and gallocyanine chromalum-stained nuclei showed normal DNA levels and proved absence of endomitotic polyploidization. RNA content, however, was significantly increased as compared to nuclei of young Strasburger cells and of ordinary ray parenchyma cells. PMID:24420510

  16. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  17. The impact of α-hydrazino acids embedded in short fluorescent peptides on peptide interactions with DNA and RNA.

    PubMed

    Suć, Josipa; Tumir, Lidija-Marija; Glavaš-Obrovac, Ljubica; Jukić, Marijana; Piantanida, Ivo; Jerić, Ivanka

    2016-06-01

    A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino < alternating-hydrazino < sequential-hydrazino group. Binding studies of ss-polynucleotides suggest intercalation of phenanthridine between polynucleotide bases, whereby affinity and fluorimetric response decrease with the number of α-hydrazino groups in the peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore. PMID:27161341

  18. Submillimeter wave spectroscopy of biological macromolecules

    NASA Astrophysics Data System (ADS)

    Globus, Tatiana

    2005-03-01

    The recently emergence of submillimeter-wave or terahertz (THz) spectroscopy of biological molecules has demonstrated the capability to detect low-frequency internal molecular vibrations involving the weakest hydrogen bonds of the DNA base pairs and/or non-bonded interactions. These multiple bonds, although having only ˜ 5% of the strength of covalent bonds, stabilize the structure of bio-polymers, by holding the two strands of the DNA double helix together, or polypeptides together in different secondary structure conformations. There will be a review of THz-frequency transmission (absorption) results for biological materials obtained from Fourier Transform Infrared (FTIR) spectroscopy during the last few years^1,2. Multiple resonances, due to low frequency vibrational modes within biological macromolecules, have been unambiguously demonstrated in qualitative agreement with theoretical prediction, thereby confirming the fundamental physical nature of observed resonance features. The discovery of resonance character of interaction between THz radiation and biological materials opens many possible applications for THz spectroscopy technique in biological sensing and biomedicine using multiple resonances as distinctive spectral fingerprints. However, many issues still require investigation. Kinetics of interactions with radiation at THz has not been studied and vibrational lifetimes have not been measured directly as a function of frequency. The strength of resonant modes of bio-molecules in aqueous environment and strong dependence of spectra on molecular orientation need explanation. Vibrational modes have not been assigned to specific motions within molecules. THz spectroscopy of bio-polymers makes it only in first steps. 1. T. Globus, D. Woolard, M. Bykhovskaia, B. Gelmont, L. Werbos, A. Samuels. International Journal of High Speed Electronics and Systems (IJHSES), 13, No. 4, 903-936 (2003). 2. T. Globus, T. Khromova, D. Woolard and B. Gelmont. Proceedings of

  19. Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.

    PubMed

    Aschenbrenner, Joos; Marx, Andreas

    2016-05-01

    Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  20. Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    PubMed Central

    Aschenbrenner, Joos; Marx, Andreas

    2016-01-01

    Methylation of the 2′-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2′-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2′-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  1. DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal.

    PubMed

    Britton, Sébastien; Dernoncourt, Emma; Delteil, Christine; Froment, Carine; Schiltz, Odile; Salles, Bernard; Frit, Philippe; Calsou, Patrick

    2014-08-01

    We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15. PMID:25030905

  2. DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal

    PubMed Central

    Britton, Sébastien; Dernoncourt, Emma; Delteil, Christine; Froment, Carine; Schiltz, Odile; Salles, Bernard; Frit, Philippe; Calsou, Patrick

    2014-01-01

    We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15. PMID:25030905

  3. DUPLEX: A molecular mechanics program in torsion angle space for computing structures of DNA and RNA

    SciTech Connect

    Hingerty, B.E.

    1992-07-01

    DUPLEX produces energy minimized structures of DNA and RNA of any base sequence for single and double strands. The smallest subunits are deoxydinucleoside monophosphates, and up to 12 residues, single or double stranded can be treated. In addition, it can incorporate NMR derived interproton distances an constraints in the minimizations. Both upper and lower bounds for these distances can be specified. The program has been designed to run on a UNICOS Cray supercomputer, but should run, albeit slowly, on a laboratory computer such as a VAX or a workstation.

  4. DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy

    PubMed Central

    Chen, Tao; Sam Wu, Cuichen; Jimenez, Elizabeth; Zhu, Zhi; Dajac, Joshua G.; You, Mingxu; Han, Da

    2013-01-01

    Multifunctional DNA micelles: Molecular beacon micelle flares (MBMFs), based on diacyllipid-molecular beacon conjugate (L-MB) self-assembly, have been developed for combined mRNA detection and gene therapy. The advantages of these micelle flares include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high signal-to-background ratio, excellent target selectivity, and superior biocompatibility. In addition, these probes possess a hydrophobic cavity that can be used for additional hydrophobic agents, holding great promise for constructing an all-in-one nucleic acid probe. PMID:23319350

  5. Simplified Identification of mRNA or DNA in Whole Cells

    NASA Technical Reports Server (NTRS)

    Almeida, Eduardo; Kadambi, Geeta

    2007-01-01

    A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

  6. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

    PubMed Central

    Coombs, N J; Gough, A C; Primrose, J N

    1999-01-01

    Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-100, 15% with classical xylene-based extraction and 60% of sections using the kit. Successful RNA extraction was observed, by beta-actin amplification, in 83.7% sections for samples treated by the thermal cycler and Chelex-100 method. Thermal cycler and Chelex-100 extraction of nucleic acid is reliable, quick and inexpensive. PMID:10454649

  7. Genome-wide analysis of mRNA polysomal profiles with spotted DNA microarrays.

    PubMed

    Melamed, Daniel; Arava, Yoav

    2007-01-01

    The sedimentation of an mRNA in sucrose gradients is highly affected by its ribosomal association. Sedimentation analysis has therefore become routine for studying changes in ribosomal association of mRNAs of interest. DNA microarray technology has been combined with sedimentation analysis to characterize changes in ribosomal association for thousands of mRNAs in parallel. Such analyses revealed mRNAs that are translationally regulated and have provided new insights into the translation process. In this chapter, we describe possible experimental designs for analyzing genome-wide changes in ribosomal association, and discuss some of their advantages and disadvantages. We then provide a detailed protocol for analysis of polysomal fractions using spotted DNA microarrays. PMID:17923236

  8. Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5

    PubMed Central

    Kassube, Susanne A.; Jinek, Martin; Fang, Jie; Tsutakawa, Susan; Nogales, Eva

    2013-01-01

    RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein–coding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo–electron microscopy structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read–through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II–TFIIS interaction. PMID:23748380

  9. A novel approach on fluid dispensing for a DNA/RNA extraction chip package

    NASA Astrophysics Data System (ADS)

    Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

    2008-02-01

    Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

  10. Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination.

    PubMed

    Futai, Kazuki; Sumaoka, Jun; Komiyama, Makoto

    2016-05-01

    By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids. PMID:27057646

  11. MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells.

    PubMed

    Xie, Chen; Chen, Wei; Zhang, Mengdie; Cai, Qiuxian; Xu, Weiyi; Li, Xiaodi; Jiang, Songshan

    2015-07-01

    Despite extensive investigation into the role of let-7 miRNAs in pathological tumor processes, their involvement in the DNA damage response remains unclear. Here we show that most let-7 family members down-regulate MDM4 expression via binding to MDM4 mRNA at a conserved DNA sequence. Expression of exogenous let-7 miRNA mimics decreased MDM4 protein but not mRNA levels. Several DNA damage reagents increased let-7 expression, thereby decreasing MDM4 protein levels in glioma cells. Inhibition of endogenous let-7 with antisense RNAs rescued MDM4 protein levels with or without MG132, a proteasome-dependent degradation inhibitor. An MDM4 mutation identified in a glioma patient was associated with loss of the putative MDM4 target site. Therefore, let-7 binding to MDM4 is implicated in the DNA damage response. PMID:26028311

  12. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    SciTech Connect

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  13. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    PubMed

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  14. RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

    PubMed Central

    Li, Qing; Gent, Jonathan I.; Zynda, Greg; Song, Jawon; Makarevitch, Irina; Hirsch, Cory D.; Hirsch, Candice N.; Dawe, R. Kelly; Madzima, Thelma F.; McGinnis, Karen M.; Lisch, Damon; Schmitz, Robert J.; Vaughn, Matthew W.; Springer, Nathan M.

    2015-01-01

    The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes. PMID:26553984

  15. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

    PubMed

    Morrill, Summer A; Exner, Alexandra E; Babokhov, Michael; Reinfeld, Bradley I; Fuchs, Stephen M

    2016-05-27

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  16. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain*

    PubMed Central

    Morrill, Summer A.; Exner, Alexandra E.; Babokhov, Michael; Reinfeld, Bradley I.

    2016-01-01

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  17. Differential utility of the Bacteroidales DNA and RNA markers in the tiered approach for microbial source tracking in subtropical seawater.

    PubMed

    Liu, Rulong; Cheng, Ken H F; Wong, Klaine; Cheng, Samuel C S; Lau, Stanley C K

    2015-07-01

    Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater. PMID:25652655

  18. Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

    PubMed Central

    Taylor, N R; Kaplan, B E; Swiderski, P; Li, H; Rossi, J J

    1992-01-01

    Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents. Images PMID:1408757

  19. [Investigation of Epstein-Barr virus DNA and RNA in tissues of patients with lymphoma].

    PubMed

    Zeytinoğlu, Ayşin; Hekimgil, Mine; Erensoy, Selda; Aydemir, Söhret; Berber, Sahir; Cağirgan, Seçkin; Soydan, Saliha; Bilgiç, Altinay

    2005-10-01

    Relation between Epstein-Barr virus (EBV) and nasopharyngeal carsinoma, Burkitt's lymphoma, and lymphomas in immunosupressed patients have been shown previously in different studies. The same relationship was also shown in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) by some researchers. The aim of this study was to demonstrate EBV nucleic acids in tissue sections of adult patients with lymphoma. The presence of EBV encoded RNA (EBER) were investigated with in situ hybridization and EBV-DNA with PCR method in 29 formalin-fixed paraffin-embedded tissue sections (19 lymph nodes, the others being gastric, orbital, skin, salivary gland, testicle, small intestinal, tongue root, bone marrow and gingival tissues) of 8 patients with HL and 21 patients with NHL who were followed-up in Haematology Clinics of our university hospital. EBER and EBV-DNA positivity rates were found as follows respectively; 50% (n: 4) and 37.5% (n: 3) of 8 HL patients, and 23.8% (n: 5) and 47.6% (n: 10) of 21 NHL patients. In total evaluation EBER and/or EBV-DNA were positive in 5 of 8 (62.5%) HL, and 12 of 21 (57.1%) NHL tissue sections. There was no significant difference in EBER and EBV-DNA positivity between HL and NHL groups. As a result, our study emphasize a possible EBV related aetiology in HL and NHL. PMID:16544549

  20. Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.

    PubMed

    Wei, Na; Shi, Yi; Truong, Lan N; Fisch, Kathleen M; Xu, Tao; Gardiner, Elisabeth; Fu, Guangsen; Hsu, Yun-Shiuan Olivia; Kishi, Shuji; Su, Andrew I; Wu, Xiaohua; Yang, Xiang-Lei

    2014-10-23

    Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage. PMID:25284223

  1. Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury.

    PubMed

    Ando, Hideyuki; Fukuda, Noboru; Kotani, Motoko; Yokoyama, Shin ichiro; Kunimoto, Satoshi; Matsumoto, Koichi; Saito, Satoshi; Kanmatsuse, Katsuo; Mugishima, Hideo

    2004-01-12

    We designed and synthesized a chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor (TGF)-beta 1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-beta 1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-beta 1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-beta 1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-beta 1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-beta 1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. PMID:14729108

  2. The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains.

    PubMed Central

    Moser, M J; Holley, W R; Chatterjee, A; Mian, I S

    1997-01-01

    Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain. PMID:9396823

  3. Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA.

    PubMed

    Zhang, C; Trottier, M; Guo, P

    1995-03-10

    A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both the 5'- and 3'-termini of the pRNA are proximate and crucial for biological function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging. Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA. As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure. Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure. PMID:7533964

  4. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  5. RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

    PubMed

    Haas, C; Hanson, E; Banemann, R; Bento, A M; Berti, A; Carracedo, Á; Courts, C; De Cock, G; Drobnic, K; Fleming, R; Franchi, C; Gomes, I; Hadzic, G; Harbison, S A; Hjort, B; Hollard, C; Hoff-Olsen, P; Keyser, C; Kondili, A; Maroñas, O; McCallum, N; Miniati, P; Morling, N; Niederstätter, H; Noël, F; Parson, W; Porto, M J; Roeder, A D; Sauer, E; Schneider, P M; Shanthan, G; Sijen, T; Syndercombe Court, D; Turanská, M; van den Berge, M; Vennemann, M; Vidaki, A; Zatkalíková, L; Ballantyne, J

    2015-05-01

    The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology. PMID:25600397

  6. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  7. Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

    PubMed

    Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

    2015-02-01

    Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells. PMID:25579461

  8. Characterization of Damage to Bacteria and Bio-macromolecules Caused by (V)UV Radiation and Particles Generated by a Microscale Atmospheric Pressure Plasma Jet

    NASA Astrophysics Data System (ADS)

    Lackmann, Jan-Wilm; Schneider, Simon; Narberhaus, Franz; Benedikt, Jan; Bandow, Julia E.

    Atmospheric pressure plasma jets effectively inactivate bacteria on ­surfaces including infected tissues. This is due to the combined effects of (V)UV radiation, reactive oxygen and nitrogen species, ions, and high electric fields. A well-characterized microscale atmospheric pressure plasma jet (μ-APPJ) operated with He/O2 gas mixture has been modified so that (V)UV radiation and heavy reactive particles (mainly O3 molecules and O atoms) emitted from the plasma source can be separated effectively. The separation is achieved by an additional lateral He flow, which diverts the heavy particles from the jet axis. The new jet geometry is called X-Jet. Separation of different plasma components allows studying their effects on living cells and bio-macromolecules separately. First, the effectiveness of the separation of different plasma components was demonstrated by treatment of monolayers of vegetative Bacillus subtilis cells. To characterize effects on nucleic acids, dried plasmid DNA and total cellular RNA were treated with the separated plasma components. Dried bovine serum albumin was used to study etching effects of (V)UV radiation and heavy particles on proteins. We found that heavy particles emitted from the X-Jet kill vegetative cells more effectively than the (V)UV radiation from this type of plasma source. All bio-macromolecules investigated, DNA, RNA, and proteins, are affected by plasma treatment. DNA exposed to the (V)UV-channel of the jet seems to be prone to thymine dimer formation not only in vitro but also in vivo as indicated by induction of the photolyase in Escherichia coli, while DNA strand breaks occur under both jet channels. Heavy particles seem more effective in degrading RNA and in etching protein in vitro.

  9. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    PubMed Central

    Halaby, Marie-Jo; Li, Yan; Harris, Benjamin R.; Jiang, Shuxia; Miskimins, W. Keith; Cleary, Margot P.; Yang, Da-Qing

    2015-01-01

    Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5′-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress. PMID:26273641

  10. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  11. A novel microRNA assay with optical detection and enzyme-free DNA circuits

    NASA Astrophysics Data System (ADS)

    Liao, Yuhui; Zhou, Xiaoming

    2014-09-01

    MicroRNAs (miRNAs) participate in the significant processes of life course, can be used as quantificational biomarkers for cellular level researches and related diseases. Conventional methods of miRNAs' quantitative detection are obsessed with low sensitivity, time and labour consuming. Otherwise, the emerging miRNAs detection approaches are mostly exposed to the expensive equipment demands and the professional operation, remains at the stage of laboratory and concept demonstration phase. Herein, we designed a novel miRNAs detection platform that based on enzyme-free DNA circuits and electrochemical luminescence (ECL). MicroRNA21 was chosen as the ideal analyte of this platform. The whole process consists of enzyme-free DNA circuits and ECL signal giving-out steps, achieves advantages of operating in constant temperature condition, without the participation of the enzyme, preferable sensitivity and specificity. Through this approach, the sensitivity achieved at 10pM. It is indicated that this miRNAs detection platform possesses potentials to be an innovation of miRNA detection technologies in routine tests. Benefits of the high penetration of ECL in well-equipped medical establishment, this approach could greatly lessen the obstacles in process of popularization and possess excellent prospects of practical application.

  12. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  13. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae.

    PubMed Central

    Hood, M A; Guckert, J B; White, D C; Deck, F

    1986-01-01

    The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation. Images PMID:2430523

  14. Degradable Polymer-Coated Gold Nanoparticles for Co-Delivery of DNA and siRNA

    PubMed Central

    Bishop, Corey J.; Tzeng, Stephany Y.; Green, Jordan J.

    2014-01-01

    Gold nanoparticles have utility for in vitro, ex vivo, and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable co-delivery of both DNA and short interfering RNA simultaneously. To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200 nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells. PMID:25246314

  15. DNA/RNA binding and anticancer/antimicrobial activities of polymer-copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Lakshmipraba, Jagadeesan; Arunachalam, Sankaralingam; Riyasdeen, Anvarbatcha; Dhivya, Rajakumar; Vignesh, Sivanandham; Akbarsha, Mohammad Abdulkader; James, Rathinam Arthur

    2013-05-01

    Water soluble polymer-copper(II) complexes with various degrees of coordination in the polymer chain were synthesized and characterized by elemental analysis, IR, UV-visible and EPR spectra. The DNA/RNA binding behavior of these polymer-copper(II) complexes was examined by UV-visible absorption, emission and circular dichroism spectroscopic methods, and cyclic voltammetry techniques. The binding of the polymer-copper(II) complexes with DNA/RNA was mainly through intercalation but some amount of electrostatic interaction was also observed. This binding capacity increased with the degree of coordination of the complexes. The polymer-copper(II) complex having the highest degree of coordination was subjected to analysis of cytotoxic and antimicrobial properties. The cytotoxicity study indicated that the polymer-copper(II) complexes affected the viability of MCF-7 mammary carcinoma cells, and the cells responded to the treatment with mostly through apoptosis although a few cells succumbed to necrosis. The antimicrobial screening showed activity against some human pathogens.

  16. Automatic on-chip RNA-DNA hybridization assay with integrated phase change microvalves

    NASA Astrophysics Data System (ADS)

    Weng, Xuan; Jiang, Hai; Wang, Junsheng; Chen, Shu; Cao, Honghe; Li, Dongqing

    2012-07-01

    An RNA-DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

  17. Antibody binding of macromolecular DNA and RNA in the plasma of SLE patients.

    PubMed

    Krapf, F; Herrmann, M; Leitmann, W; Kalden, J R

    1989-03-01

    Plasmapheresis fluids from 20 patients with clinically active SLE, from three patients with Waldenstrom's disease, from three patients with rheumatoid arthritis, two patients with myasthenia gravis and other diseases including active systemic disorders were precipitated using polyethylene glycol 6000 (PEG). By applying ethidium bromide staining, plasma nucleic acids (PNA) could be demonstrated in PEG-precipitates of SLE patients exclusively. Purified immunoglobulins of SLE plasma precipitates were shown to form antigen-antibody complexes with PNA as demonstrated by electronmicroscopy. Further characterization of PNA by agarose gel electrophoresis revealed a molecular weight up to 20 kbp. Cesium chloride buoyant density gradients showed non-homogeneous molecules, excluding pure microbial origin. In spite of RNase digestion, the PNA contained RNA with 30-70% riboguanosine as shown by nucleoside analysis. The high amount of guanosine-rich RNA was further supported by similarities between PNA and polyriboguanylic acid in hyperchrome shifting due to thermic denaturation. HPLC analysis showed a molecular weight of ribonucleic acids of more than 60 b thus excluding mere oligonucleotides. In contrast to B-type dsDNA, PNA from SLE patients were immunogenic. Antibodies against PNA could be induced in rabbits by subcutaneous injection. The antisera thus obtained showed crossreactivity with polyriboguanylic acid and dsDNA preparations. PMID:2467774

  18. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-01-01

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

  19. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    PubMed Central

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

  20. Integrative DNA, RNA, and Protein Evidence Connects TREML4 to Coronary Artery Calcification

    PubMed Central

    Sen, Shurjo K.; Boelte, Kimberly C.; Barb, Jennifer J.; Joehanes, Roby; Zhao, XiaoQing; Cheng, Qi; Adams, Lila; Teer, Jamie K.; Accame, David S.; Chowdhury, Soma; Singh, Larry N.; Kavousi, Maryam; Peyser, Patricia A.; Quigley, Laura; Priel, Debra Long; Lau, Karen; Kuhns, Douglas B.; Yoshimura, Teizo; Johnson, Andrew D.; Hwang, Shih-Jen; Chen, Marcus Y.; Arai, Andrew E.; Green, Eric D.; Mullikin, James C.; Kolodgie, Frank D.; O’Donnell, Christopher J.; Virmani, Renu; Munson, Peter J.; McVicar, Daniel W.; Biesecker, Leslie G.

    2014-01-01

    Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease. PMID:24975946

  1. Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes

    PubMed Central

    Bergues-Pupo, Ana Elisa; Arias-Gonzalez, J. Ricardo; Morón, María Carmen; Fiasconaro, Alessandro; Falo, Fernando

    2015-01-01

    Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\rm {K}^+$\\end{document} central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery. PMID:26170233

  2. Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes.

    PubMed

    Bergues-Pupo, Ana Elisa; Arias-Gonzalez, J Ricardo; Morón, María Carmen; Fiasconaro, Alessandro; Falo, Fernando

    2015-09-01

    Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of [Formula: see text] central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery. PMID:26170233

  3. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    SciTech Connect

    Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

    1987-05-01

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific TSP-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect.

  4. Trm9 Catalyzed tRNA Modifications Link Translation to the DNA Damage Response

    PubMed Central

    Begley, Ulrike; Dyavaiah, Madhu; Patil, Ashish; Rooney, John P.; DiRenzo, Dan; Young, Christine M.; Conklin, Douglas S.; Zitomer, Richard S.; Begley, Thomas J.

    2007-01-01

    Summary Transcriptional and post-translational signals are known mechanisms which promote efficient responses to DNA damage. We have identified Saccharomyces cerevisiae tRNA methyltransferase 9 (Trm9) as an enzyme that prevents cell death via translational enhancement of DNA damage response proteins. Trm9 methylates the uridine wobble base of tRNAARG(UCU) and tRNAGLU(UUC). We used computational and molecular approaches to predict that Trm9 enhances the translation of some transcripts over-represented with specific arginine and glutamic acid codons. We found that translation elongation factor 3 (YEF3) and the ribonucleotide reductase (RNR1 and RNR3) large subunits are over-represented with specific arginine and glutamic acid codons, and demonstrated that Trm9 significantly enhances Yef3, Rnr1, and Rnr3 protein levels. Additionally, we identified 425 genes, which included YEF3, RNR1, and RNR3, with a unique codon usage pattern linked to Trm9. We propose that Trm9-specific tRNA modifications enhance codon-specific translation elongation and promote increased levels of key damage response proteins. PMID:18082610

  5. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner. PMID:27198220

  6. Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis

    PubMed Central

    Liebl, Korbinian; Drsata, Tomas; Lankas, Filip; Lipfert, Jan; Zacharias, Martin

    2015-01-01

    Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over- or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure. PMID:26464435

  7. Systemic oxidative DNA and RNA damage are not increased during early phases of psychosis: A case control study.

    PubMed

    Nordholm, Dorte; Poulsen, Henrik Enghusen; Hjorthøj, Carsten; Randers, Lasse; Nielsen, Mette Ø; Wulff, Sanne; Krakauer, Kristine; Nørbak-Emig, Henrik; Henriksen, Trine; Glenthøj, Birte; Nordentoft, Merete

    2016-07-30

    It has been suggested that patients with schizophrenia develop higher levels of oxidative stress, which may contribute to deteriorating mental illness. In order to examine oxidative stress in the early stages of severe mental illness, we examined the levels of systemic Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, perceived stress and recent life events in patients at ultra high-risk (UHR) of developing psychosis, in antipsychotic naïve patients with first-episode schizophrenia (FES), and in healthy controls. We included 41 UHR patients, 35 FES patients, and 29 healthy controls. There was no difference in the level of DNA/RNA oxidative damage between UHR patients and FES patients compared with healthy controls. We found no association between levels of DNA/RNA oxidative damage and perceived stress/life events. Based on the results, we suggest that DNA and RNA oxidative markers are not increased during the early stages of illness, but further longitudinal studies in first-episode psychosis should be carried out to examine whether DNA and RNA oxidative damage are potential markers of severe illness. PMID:27183105

  8. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  9. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

    PubMed

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

    2015-01-01

    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses. PMID:26782533

  10. Co-delivery of DNA and siRNA via Arginine-Rich PEI-based Polyplexes

    PubMed Central

    Shan, Lu; Morris, Viola B; Labhasetwar, Vinod

    2015-01-01

    In this study, we formulated polyplexes with different compositions for co-delivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their co-delivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, co-delivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100 fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects, because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression. PMID:25591125

  11. Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

    PubMed Central

    Ketzer, Patrick; Kaufmann, Johanna K.; Engelhardt, Sarah; Bossow, Sascha; von Kalle, Christof; Hartig, Jörg S.; Ungerechts, Guy; Nettelbeck, Dirk M.

    2014-01-01

    Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule–triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses. PMID:24449891

  12. DNA/RNA markers for colorectal cancer risk in preserved stool specimens: a pilot study

    PubMed Central

    Kato, Ikuko; Badsha, Kawsar Z; Land, Susan; Nechvatal, Jordan M; Matherly, Larry H; Tarca, Adi L; Majumdar, Adhip P; Basson, Marc D; Ram, Jeffrey L

    2016-01-01

    Aims and background Exfoliated cells in human stool offer excellent opportunities to non-invasively detect molecular markers associated with colorectal tumorigenesis, and to evaluate the effects of exposures to exogenous and endogenous carcinogenic or chemopreventive substances. This pilot study investigated the feasibility of determining DNA methylation and RNA expression simultaneously in stool specimens treated with a single type of nucleic acid preservatives. Methods Stool specimens from 56 volunteers that were preserved up to a week with RNAlater were used in this study. Bisulfite sequencing was used to determine methylation at 27 CpG loci on the estrogen receptor 1 (ESR1) promoter. Taqman assay was used for quantitative reverse transcription polymerase chain reactions to measure cyclooxygenase 2 (COX2) and epidermal growth factor receptor (EGFR) mRNA expression. Subjects’ basic demographic and other selected risk factors for colorectal cancer were captured through questionnaires and correlated with the levels of these markers. Results Less than 10% of the samples failed in individual assays. Overall, 24.0% of the CpG loci on the ESR1 promoter were methylated. COX2 expression and alcohol use were positively correlated; an inverse association was present between EGFR expression and cigarette smoking; and subjects using anti-diabetic medication had higher ESR1 methylation. In addition, higher EGFR expression levels were marginally associated with history of polyps and family history of colorectal cancer. Conclusions The present study demonstrates that simultaneous analyses for DNA and RNA markers are feasible in stool samples treated with a single type of nucleotide preservatives. Among several associations observed, the association between EGFR expression and polyps deserves further investigation as a potential target for colorectal cancer screening. Larger studies are warranted to confirm some of our observations. PMID:20210241

  13. Activation of different split functionalities on re-association of RNA-DNA hybrids.

    PubMed

    Afonin, Kirill A; Viard, Mathias; Martins, Angelica N; Lockett, Stephen J; Maciag, Anna E; Freed, Eric O; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A

    2013-04-01

    Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications. PMID:23542902

  14. Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

    PubMed

    Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit; Al Husini, Nadra; Dhoondia, Zuzer; Ansari, Athar; Pascuzzi, Pete E; Tran, Elizabeth J

    2016-02-01

    Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. PMID:26833086

  15. Applications of Hairpin DNA-Functionalized Gold Nanoparticles for Imaging mRNA in Living Cells.

    PubMed

    Jackson, S R; Wong, A C; Travis, A R; Catrina, I E; Bratu, D P; Wright, D W; Jayagopal, A

    2016-01-01

    Molecular imaging agents are useful for imaging molecular processes in living systems in order to elucidate the function of molecular mediators in health and disease. Here, we demonstrate a technique for the synthesis, characterization, and application of hairpin DNA-functionalized gold nanoparticles (hAuNPs) as fluorescent hybridization probes for imaging mRNA expression and spatiotemporal dynamics in living cells. These imaging probes feature gold colloids linked to fluorophores via engineered oligonucleotides to resemble a molecular beacon in which the gold colloid serves as the fluorescence quencher in a fluorescence resonance energy transfer system. Target-specific hybridization of the hairpin oligonucleotide enables fluorescence de-quenching and subsequent emission with high signal to noise ratios. hAuNPs exhibit high specificity without adverse toxicity or the need for transfection reagents. Furthermore, tunability of hAuNP emission profiles by selection of spectrally distinct fluorophores enables multiplexed mRNA imaging applications. Therefore, hAuNPs are promising tools for imaging gene expression in living cells. As a representative application of this technology, we discuss the design and applications of hAuNP targeted against distinct matrix metalloproteinase enzymes for the multiplexed detection of mRNA expression in live breast cancer cells using flow cytometry and fluorescence microscopy. PMID:27241751

  16. Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor

    PubMed Central

    Shu, Dan; Khisamutdinov, Emil F.; Zhang, Le; Guo, Peixuan

    2014-01-01

    Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies. PMID:24084081

  17. The Origins of the RNA World

    PubMed Central

    Robertson, Michael P; Joyce, Gerald F

    2012-01-01

    The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA. PMID:20739415

  18. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  19. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

    DOE PAGESBeta

    Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J.; Wang, Dong; Kashlev, Mikhail

    2015-01-20

    In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5´-templating base, indicating that it derives from nontemplated synthesismore » according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. The translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.« less

  20. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

    SciTech Connect

    Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J.; Wang, Dong; Kashlev, Mikhail

    2015-01-20

    In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5´-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. The translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

  1. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  2. Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding.

    PubMed

    Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

    2015-02-18

    The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S). PMID:25578965

  3. Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding

    PubMed Central

    Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

    2015-01-01

    The RpoS/σS sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σS-dependent control, that of a repressor. Negative regulation by σS has been proposed to result largely from competition between σS and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σS binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σS protein proficient for EσS complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σS requires its binding to DNA. Although the mechanisms of repression by σS are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by EσS. PMID:25578965

  4. Chromosomal Mapping of Repetitive DNA Sequences in Five Species of Astyanax (Characiformes, Characidae) Reveals Independent Location of U1 and U2 snRNA Sites and Association of U1 snRNA and 5S rDNA.

    PubMed

    Silva, Duilio M Z A; Utsunomia, Ricardo; Pansonato-Alves, José C; Oliveira, Cláudio; Foresti, Fausto

    2015-01-01

    Astyanax is a genus of Characidae fishes currently composed of 155 valid species. Previous cytogenetic studies revealed high chromosomal diversification among them, and several studies have been performed using traditional cytogenetic techniques to investigate karyotypes and chromosomal locations of 18S and 5S rDNA genes. However, only a few studies are currently available about other repetitive sequences. Here, the chromosomal location of small nuclear RNA genes, identified as U1 and U2 snRNA clusters, was established and compared to the distribution of 5S rDNA and histone clusters in 5 Astyanax species (A. paranae, A. fasciatus, A. bockmanni, A. altiparanae, and A. jordani) using FISH. The cytogenetic mapping of U1 and U2 snRNA demonstrated a conserved pattern in the number of sites per genome independent of the location in Astyanax species. The location of the U1 snRNA gene was frequently associated with 5S rDNA sequences, indicating a possible interaction between the distinct repetitive DNA families. Finally, comparisons involving the location of U1 and U2 snRNA clusters in the chromosomes of Astyanax species revealed a very diverse pattern, suggesting that many rearrangements have occurred during the diversification process of this group. PMID:26329975

  5. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

    PubMed Central

    Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

    2015-01-01

    Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5′ → 3′ direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA. PMID:25775526

  6. RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus.

    PubMed

    Buck, Stephen W; Maqani, Nazif; Matecic, Mirela; Hontz, Robert D; Fine, Ryan D; Li, Mingguang; Smith, Jeffrey S

    2016-07-27

    RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing. PMID:27060141

  7. Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA.

    PubMed

    Tonin, Yann; Heckel, Anne-Marie; Vysokikh, Mikhail; Dovydenko, Ilya; Meschaninova, Mariya; Rötig, Agnès; Munnich, Arnold; Venyaminova, Alya; Tarassov, Ivan; Entelis, Nina

    2014-05-01

    Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene. PMID:24692550

  8. Ccr4-not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae.

    PubMed

    Traven, Ana; Hammet, Andrew; Tenis, Nora; Denis, Clyde L; Heierhorst, Jörg

    2005-01-01

    DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Delta phenotypes, including synthetic HU hypersensitivity with dun1Delta, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Delta and caf1Delta DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Delta, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Delta. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner. PMID:15466434

  9. Ccr4-Not Complex mRNA Deadenylase Activity Contributes to DNA Damage Responses in Saccharomyces cerevisiae

    PubMed Central

    Traven, Ana; Hammet, Andrew; Tenis, Nora; Denis, Clyde L.; Heierhorst, Jörg

    2005-01-01

    DNA damage checkpoints regulate gene expression at the transcriptional and post-transcriptional level. Some components of the yeast Ccr4-Not complex, which regulates transcription as well as transcript turnover, have previously been linked to DNA damage responses, but it is unclear if this involves transcriptional or post-transcriptional functions. Here we show that CCR4 and CAF1, which together encode the major cytoplasmic mRNA deadenylase complex, have complex genetic interactions with the checkpoint genes DUN1, MRC1, RAD9, and RAD17 in response to DNA-damaging agents hydroxyurea (HU) and methylmethane sulfonate (MMS). The exonuclease-inactivating ccr4-1 point mutation mimics ccr4Δ phenotypes, including synthetic HU hypersensitivity with dun1Δ, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4Δ and caf1Δ DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5Δ, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1Δ. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner. PMID:15466434

  10. A major role for Tau in neuronal DNA and RNA protection in vivo under physiological and hyperthermic conditions

    PubMed Central

    Violet, Marie; Delattre, Lucie; Tardivel, Meryem; Sultan, Audrey; Chauderlier, Alban; Caillierez, Raphaelle; Talahari, Smail; Nesslany, Fabrice; Lefebvre, Bruno; Bonnefoy, Eliette; Buée, Luc; Galas, Marie-Christine

    2014-01-01

    Nucleic acid protection is a substantial challenge for neurons, which are continuously exposed to oxidative stress in the brain. Neurons require powerful mechanisms to protect DNA and RNA integrity and ensure their functionality and longevity. Beside its well known role in microtubule dynamics, we recently discovered that Tau is also a key nuclear player in the protection of neuronal genomic DNA integrity under reactive oxygen species (ROS)-inducing heat stress (HS) conditions in primary neuronal cultures. In this report, we analyzed the capacity of Tau to protect neuronal DNA integrity in vivo in adult mice under physiological and HS conditions. We designed an in vivo mouse model of hyperthermia/HS to induce a transient increase in ROS production in the brain. Comet and Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate ni