Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Science.gov (United States)

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Programmable autonomous synthesis of single-stranded DNA

Science.gov (United States)

Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

2018-02-01

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

MicroRNA-target binding structures mimic microRNA duplex structures in humans.

Directory of Open Access Journals (Sweden)

Xi Chen

Full Text Available Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient--the microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis

International Nuclear Information System (INIS)

Sawicki, S.G.; Sawicki, D.L.

1986-01-01

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [ 3 H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minis-strand RNA synthesis was three- to fourfold more sensitive to inhibition of cycloheximide than was plus-strand synthesis

Packaging signals in single-stranded RNA viruses: nature?s alternative to a purely electrostatic assembly mechanism

OpenAIRE

Stockley, Peter G.; Twarock, Reidun; Bakker, Saskia E.; Barker, Amy M.; Borodavka, Alexander; Dykeman, Eric; Ford, Robert J.; Pearson, Arwen R.; Phillips, Simon E. V.; Ranson, Neil A.; Tuma, Roman

2013-01-01

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative the...

Influence of RNA Strand Rigidity on Polyion Complex Formation with Block Catiomers.

Science.gov (United States)

Hayashi, Kotaro; Chaya, Hiroyuki; Fukushima, Shigeto; Watanabe, Sumiyo; Takemoto, Hiroyasu; Osada, Kensuke; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

2016-03-01

Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA secondary structures of the bacteriophage phi6 packaging regions.

Science.gov (United States)

Pirttimaa, M J; Bamford, D H

2000-06-01

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

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Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

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.

Computer-Aided Design of RNA Origami Structures.

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Sparvath, Steffen L; Geary, Cody W; Andersen, Ebbe S

2017-01-01

RNA nanostructures can be used as scaffolds to organize, combine, and control molecular functionalities, with great potential for applications in nanomedicine and synthetic biology. The single-stranded RNA origami method allows RNA nanostructures to be folded as they are transcribed by the RNA polymerase. RNA origami structures provide a stable framework that can be decorated with functional RNA elements such as riboswitches, ribozymes, interaction sites, and aptamers for binding small molecules or protein targets. The rich library of RNA structural and functional elements combined with the possibility to attach proteins through aptamer-based binding creates virtually limitless possibilities for constructing advanced RNA-based nanodevices.In this chapter we provide a detailed protocol for the single-stranded RNA origami design method using a simple 2-helix tall structure as an example. The first step involves 3D modeling of a double-crossover between two RNA double helices, followed by decoration with tertiary motifs. The second step deals with the construction of a 2D blueprint describing the secondary structure and sequence constraints that serves as the input for computer programs. In the third step, computer programs are used to design RNA sequences that are compatible with the structure, and the resulting outputs are evaluated and converted into DNA sequences to order.

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

Science.gov (United States)

Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

2010-05-01

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

Novel Positive-Sense, Single-Stranded RNA (+ssRNA) Virus with Di-Cistronic Genome from Intestinal Content of Freshwater Carp (Cyprinus carpio)

Science.gov (United States)

Pankovics, Péter; Simmonds, Peter

2011-01-01

A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature. PMID:22195010

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

Science.gov (United States)

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.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

Science.gov (United States)

Garmann, Rees F; Comas-Garcia, Mauricio; Koay, Melissa S T; Cornelissen, Jeroen J L M; Knobler, Charles M; Gelbart, William M

2014-09-01

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

Science.gov (United States)

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Near-Complete Genome Sequence of a Novel Single-Stranded RNA Virus Discovered in Indoor Air.

Science.gov (United States)

Rosario, Karyna; Fierer, Noah; Breitbart, Mya

2018-03-22

Viral metagenomic analysis of heating, ventilation, and air conditioning (HVAC) filters recovered the near-complete genome sequence of a novel virus, named HVAC-associated R NA v irus 1 (HVAC-RV1). The HVAC-RV1 genome is most similar to those of picorna-like viruses identified in arthropods but encodes a small domain observed only in negative-sense single-stranded RNA viruses. Copyright © 2018 Rosario et al.

Global Analysis of RNA Secondary Structure in Two Metazoans

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Fan Li

2012-01-01

Full Text Available The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA] and unpaired (single-stranded RNA [ssRNA] components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.

On topological RNA interaction structures.

Science.gov (United States)

Qin, Jing; Reidys, Christian M

2013-07-01

Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

RNA secondary structures of the bacteriophage phi6 packaging regions.

OpenAIRE

Pirttimaa, M J; Bamford, D H

2000-01-01

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models ...

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

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Renier Myburgh

2014-01-01

Full Text Available Gene knockdown using micro RNA (miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp, positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

DEFF Research Database (Denmark)

Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

2015-01-01

) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

Science.gov (United States)

Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

2018-05-25

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Functional RNA structures throughout the Hepatitis C Virus genome.

Science.gov (United States)

Adams, Rebecca L; Pirakitikulr, Nathan; Pyle, Anna Marie

2017-06-01

The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

On RNA-RNA interaction structures of fixed topological genus.

Science.gov (United States)

Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

2015-04-01

Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

Evolutionary rate variation and RNA secondary structure prediction

DEFF Research Database (Denmark)

Knudsen, B.; Andersen, E.S.; Damgaard, C.

2004-01-01

Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type...... by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions...

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

Science.gov (United States)

Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

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Nikolay Kovalev

2012-02-01

Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV. To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-RNA, rendering the RNA compatible for initiation of (+-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which is another host factor for TBSV, play non-overlapping functions to enhance (+-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV, a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

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Po-Yuan Yeh

2014-07-01

Full Text Available It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−-strand] complement. However, the cis-acting elements on the positive-strand [(+-strand] sgmRNA required for (−-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV sgmRNA 7 required for the synthesis of its (−-strand counterpart by deletion mutagenesis. The major findings are as follows. (1 Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−-strand sgmRNA complement. (2 Deletions of the 3' untranslated region (UTR bulged stem-loop showed no effect on (−-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−-strand sgmRNA. (3 Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−-strand sgmRNA synthesis. (4 Nucleotide species at the 3'-most position (−1 of sgmRNA 7 is correlated to the efficiency of (−-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−-strand sgmRNA synthesis in BCoV.

Strategies underlying RNA silencing suppression by negative strand RNA viruses

NARCIS (Netherlands)

Hemmes, J.C.

2007-01-01

The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

Packaging signals in single-stranded RNA viruses: nature's alternative to a purely electrostatic assembly mechanism.

Science.gov (United States)

Stockley, Peter G; Twarock, Reidun; Bakker, Saskia E; Barker, Amy M; Borodavka, Alexander; Dykeman, Eric; Ford, Robert J; Pearson, Arwen R; Phillips, Simon E V; Ranson, Neil A; Tuma, Roman

2013-03-01

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA-coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

Double Stranded RNA in Human Seminal Plasma

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Maxim V. Zagoskin

2017-10-01

Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

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Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

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Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

2014-01-01

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

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Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

A G-C-rich palindromic structural motif and a stretch of single-stranded purines are required for optimal packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA.

Science.gov (United States)

Jaballah, Soumeya Ali; Aktar, Suriya J; Ali, Jahabar; Phillip, Pretty Susan; Al Dhaheri, Noura Salem; Jabeen, Aayesha; Rizvi, Tahir A

2010-09-03

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process. Copyright (c) 2010 Elsevier Ltd

Prediction of guide strand of microRNAs from its sequence and secondary structure

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Ahmed Firoz

2009-04-01

Full Text Available Abstract Background MicroRNAs (miRNAs are produced by the sequential processing of a long hairpin RNA transcript by Drosha and Dicer, an RNase III enzymes, and form transitory small RNA duplexes. One strand of the duplex, which incorporates into RNA-induced silencing complex (RISC and silences the gene expression is called guide strand, or miRNA; while the other strand of duplex is degraded and called the passenger strand, or miRNA*. Predicting the guide strand of miRNA is important for better understanding the RNA interference pathways. Results This paper describes support vector machine (SVM models developed for predicting the guide strands of miRNAs. All models were trained and tested on a dataset consisting of 329 miRNA and 329 miRNA* pairs using five fold cross validation technique. Firstly, models were developed using mono-, di-, and tri-nucleotide composition of miRNA strands and achieved the highest accuracies of 0.588, 0.638 and 0.596 respectively. Secondly, models were developed using split nucleotide composition and achieved maximum accuracies of 0.553, 0.641 and 0.602 for mono-, di-, and tri-nucleotide respectively. Thirdly, models were developed using binary pattern and achieved the highest accuracy of 0.708. Furthermore, when integrating the secondary structure features with binary pattern, an accuracy of 0.719 was seen. Finally, hybrid models were developed by combining various features and achieved maximum accuracy of 0.799 with sensitivity 0.781 and specificity 0.818. Moreover, the performance of this model was tested on an independent dataset that achieved an accuracy of 0.80. In addition, we also compared the performance of our method with various siRNA-designing methods on miRNA and siRNA datasets. Conclusion In this study, first time a method has been developed to predict guide miRNA strands, of miRNA duplex. This study demonstrates that guide and passenger strand of miRNA precursors can be distinguished using their

TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

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Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

DEFF Research Database (Denmark)

He, Yangzi

and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

Second-strand cDNA synthesis: classical method

International Nuclear Information System (INIS)

Gubler, U.

1987-01-01

The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

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Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

Crystal Structure of a CRISPR RNA-guided Surveillance Complex Bound to a ssDNA Target

Energy Technology Data Exchange (ETDEWEB)

Mulepati, Sabin [Johns Hopkins Univ., Baltimore, MD (United States); Heroux, Annie; Bailey, Scott [Johns Hopkins Univ., Baltimore, MD (United States)

2014-09-19

In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

Use of Cellular Decapping Activators by Positive-Strand RNA Viruses

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Jennifer Jungfleisch

2016-12-01

Full Text Available Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.

Studies of the effects of ultraviolet radiation on the structural integrities of ribosomal RNA components of the Escherichia coli 50S ribosomal subunit

International Nuclear Information System (INIS)

Gorelic, L.; Parker, D.

1978-01-01

The effects of 254-nm radiation on the structural integrities of free and 50S ribosome-bound 5S and 23S ribosomal ribonucleic acids (rRNA) have been elucidated. Irradiation of aqueous solutions of Escherichia coli 50S ribosomes with 253.7-nm radiation results in the formation of single-strand breaks in double-stranded regions of the 23S rRNA component, but not in rRNA chain scission, and destabilization of the secondary structure of the 23S rRNA toward denaturation. The minimum doses of 253.7-nm radiation required for the first detection of the two effects are 7 x 10 19 quanta for the production of single-strand breaks in double-stranded regions of the 23S rRNA, and 19 quanta for destabilization of the 23S rRNA secondary structure. Free 23S rRNA is resistant toward photoinduced chain breakage at doses of 253.7-nm radiation up to at least 2.3 x 10 20 and is much less sensitive toward destabilization of secondary structure than ribosome-bound 23S rRNA. In contrast to the photosensitivity of 50S ribosome-bound 23S rRNA toward chain breakage, 50S ribosome-bound 5S rRNA is resistant toward chain breakage at doses of 253.7-nm radiation up to at least 2.3 x 10 20 quanta. Ribosome-bound 5S and 23S rRNA are also not photosensitive toward intermolecular 5S/23S rRNA cross-linkage

MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

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Qian Feng

2012-11-01

Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

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Watters, Kyle E; Choudhary, Krishna; Aviran, Sharon; Perry, Keith L

2018-01-01

Abstract In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites. PMID:29294088

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with Nitrogen-15

International Nuclear Information System (INIS)

Gewirth, D.T.; Abo, S.R.; Leontis, N.B.; Moore, P.B.

1987-01-01

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15 N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a normal chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for the authors understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed

Global organization of a positive-strand RNA virus genome.

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Baodong Wu

Full Text Available The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2'-hydroxyl acylation analysed by primer extension (i.e. SHAPE, which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.

Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

Science.gov (United States)

Akbari, Fahimeh; Foroutan, Masumeh

2018-02-14

In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

Science.gov (United States)

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.

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.

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Wei-Yu Liao

Full Text Available The synthesis of the negative-strand [(--strand] complement of the ∼30 kilobase, positive-strand [(+-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (--strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (--strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR to detect and quantitate the synthesis of bovine coronavirus (BCoV defective interfering (DI RNA (- strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts which function in the synthesis of (-- or (+-strand BCoV DI RNA. The major findings are as follows: (i nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (--strand BCoV DI RNA synthesis, (ii nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+-strand BCoV DI RNA synthesis, and (iii the nucleotide species at the 3'-most position (-1 is important, but not critical, for both (-- and (+-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-- and (+-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (--strand RNA synthesis in coronaviruses.

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

International Nuclear Information System (INIS)

Dong Hongping; Zhang Bo; Shi Peiyong

2008-01-01

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5 binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis

A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast.

Science.gov (United States)

Yang, Yong; Gordenin, Dmitry A; Resnick, Michael A

2010-08-05

Localized hyper-mutability (LHM) can be important in evolution, immunity, and genetic diseases. We previously reported that single-strand DNA (ssDNA) can be an important source of damage-induced LHM in yeast. Here, we establish that the generation of LHM by methyl methanesulfonate (MMS) during repair of a chromosomal double-strand break (DSB) can result in over 0.2 mutations/kb, which is approximately 20,000-fold higher than the MMS-induced mutation density without a DSB. The MMS-induced mutations associated with DSB repair were primarily due to substitutions via translesion DNA synthesis at damaged cytosines, even though there are nearly 10 times more MMS-induced lesions at other bases. Based on this mutation bias, the promutagenic lesion dominating LHM is likely 3-methylcytosine, which is single-strand specific. Thus, the dramatic increase in mutagenesis at a DSB is concluded to result primarily from the generation of non-repairable lesions in ssDNA associated with DSB repair along with efficient induction of highly mutagenic ssDNA-specific lesions. These findings with MMS-induced LHM have broad biological implications for unrepaired damage generated in ssDNA and possibly ssRNA. Published by Elsevier B.V.

Brickworx builds recurrent RNA and DNA structural motifs into medium- and low-resolution electron-density maps

Energy Technology Data Exchange (ETDEWEB)

Chojnowski, Grzegorz, E-mail: gchojnowski@genesilico.pl [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Waleń, Tomasz [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); University of Warsaw, Banacha 2, 02-097 Warsaw (Poland); Piątkowski, Paweł; Potrzebowski, Wojciech [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Bujnicki, Janusz M. [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Adam Mickiewicz University, Umultowska 89, 61-614 Poznan (Poland)

2015-03-01

A computer program that builds crystal structure models of nucleic acid molecules is presented. Brickworx is a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs including double-stranded helices. In a first step, the program searches for electron-density peaks that may correspond to phosphate groups; it may also take into account phosphate-group positions provided by the user. Subsequently, comparing the three-dimensional patterns of the P atoms with a database of nucleic acid fragments, it finds the matching positions of the double-stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure is RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real space to find the most likely conformations, including a fit of the sequence to the electron-density map. The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx.

Brickworx builds recurrent RNA and DNA structural motifs into medium- and low-resolution electron-density maps

International Nuclear Information System (INIS)

Chojnowski, Grzegorz; Waleń, Tomasz; Piątkowski, Paweł; Potrzebowski, Wojciech; Bujnicki, Janusz M.

2015-01-01

A computer program that builds crystal structure models of nucleic acid molecules is presented. Brickworx is a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs including double-stranded helices. In a first step, the program searches for electron-density peaks that may correspond to phosphate groups; it may also take into account phosphate-group positions provided by the user. Subsequently, comparing the three-dimensional patterns of the P atoms with a database of nucleic acid fragments, it finds the matching positions of the double-stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure is RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real space to find the most likely conformations, including a fit of the sequence to the electron-density map. The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

Science.gov (United States)

Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

2015-09-15

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

KAUST Repository

Butt, Haroon

2017-08-24

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

Science.gov (United States)

Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

2008-07-01

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate si

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

KAUST Repository

Butt, Haroon; Eid, Ayman; Ali, Zahir; Atia, Mohamed A. M.; Mokhtar, Morad M.; Hassan, Norhan; Lee, Ciaran M.; Bao, Gang; Mahfouz, Magdy M.

2017-01-01

used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate

Influence of DNA conformation on radiation-induced single-strand breaks

International Nuclear Information System (INIS)

Barone, F.; Belli, M.; Mazzei, F.

1994-01-01

We performed experiments on two DNA fragments of about 300 bp having different conformation to test whether radiation-induced single-strand breakage is dependent on DNA conformation. Breakage analysis was carried out by denaturing polyacrylamide gel electrophoresis, which allows determination of the broken site at single nucleotide resolution. We found uniform cutting patterns in B-form regions. On the contrary, X- or γ-irradiation of curved fragments of kinetoplast DNA showed that the distribution of single-strand breaks was not uniform along the fragment, as the cleavage pattern was modulated in phase with the runs of A-T pairs. This modulation likely reflected the reduced accessibility of the sites which on hydroxyl-radical attack give rise to strand breaks. The cleavage pattern was phased with the runs of A-T pairs. Moreover, the overall yield of strand breaks was considerably lower in curved DNA fragments than in those with extended straight regions. The conformation effect found here indicates that the cleavage pattern reflects the fine structural features of DNA. (orig./MG)

Negative-strand RNA viruses: the plant-infecting counterparts.

Science.gov (United States)

Kormelink, Richard; Garcia, Maria Laura; Goodin, Michael; Sasaya, Takahide; Haenni, Anne-Lise

2011-12-01

While a large number of negative-strand (-)RNA viruses infect animals and humans, a relative small number have plants as their primary host. Some of these have been classified within families together with animal/human infecting viruses due to similarities in particle morphology and genome organization, while others have just recently been/or are still classified in floating genera. In most cases, at least two striking differences can still be discerned between the animal/human-infecting viruses and their plant-infecting counterparts which for the latter relate to their adaptation to plants as hosts. The first one is the capacity to modify plasmodesmata to facilitate systemic spread of infectious viral entities throughout the plant host. The second one is the capacity to counteract RNA interference (RNAi, also referred to as RNA silencing), the innate antiviral defence system of plants and insects. In this review an overview will be presented on the negative-strand RNA plant viruses classified within the families Bunyaviridae, Rhabdoviridae, Ophioviridae and floating genera Tenuivirus and Varicosavirus. Genetic differences with the animal-infecting counterparts and their evolutionary descendants will be described in light of the above processes. Copyright © 2011 Elsevier B.V. All rights reserved.

Single-fluorophore monitoring of DNA hybridization for investigating the effect of secondary structure on the nucleation step.

Science.gov (United States)

Jo, Joon-Jung; Kim, Min-Ji; Son, Jung-Tae; Kim, Jandi; Shin, Jong-Shik

2009-07-17

Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.

STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE SINGLE-STRAND ORIGIN OF REPLICATION FROM THE LACTOCOCCAL PLASMID PWVO1

NARCIS (Netherlands)

SEEGERS, JFML; ZHAO, AC; MEIJER, WJJ; KHAN, SA; VENEMA, G; BRON, S

1995-01-01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on

Conjugation and Evaluation of Triazole?Linked Single Guide RNA for CRISPR?Cas9 Gene Editing

OpenAIRE

He, Kaizhang; Chou, Eldon T.; Begay, Shawn; Anderson, Emily M.; van?Brabant?Smith, Anja

2016-01-01

Abstract The CRISPR?Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site?specific double?stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne?azide cycloaddition to generate a triazole?linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified s...

Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Energy Technology Data Exchange (ETDEWEB)

Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas; Patel, Dinshaw J.

2015-09-08

RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3'-end.

Science.gov (United States)

Merkiene, Egle; Gaidamaviciute, Edita; Riauba, Laurynas; Janulaitis, Arvydas; Lagunavicius, Arunas

2010-08-01

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3' --> 5' single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3'-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3'-end and customize this technique for the inner RNA sequence analysis.

General enumeration of RNA secondary structures based on new ...

African Journals Online (AJOL)

akpobome

coding, transferring and retrieving genetic information, and in directing cell metabolism. The nucleic acid includes DNA and RNA molecule. RNA molecule is a single-stranded nucleic acid of four different kinds of nucleotides. The four nucleotides only differ by one part, called bases. Hence, one usually identifies nucleotides.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

Directory of Open Access Journals (Sweden)

Rebecca Johnson

2007-11-01

Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template*

OpenAIRE

Yuan, Quan; McHenry, Charles S.

2009-01-01

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of γ-complex to support the reaction in the absence of τ. However, if γ-complex is p...

RNA-SSPT: RNA Secondary Structure Prediction Tools.

Science.gov (United States)

Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

2013-01-01

The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

Negative-strand RNA viruses: The plant-infecting counterparts

NARCIS (Netherlands)

Kormelink, R.J.M.; Garcia, M.L.; Goodin, M.; Sasaya, T.; Haenni, A.L.

2011-01-01

While a large number of negative-strand (-)RNA viruses infect animals and humans, a relative small number have plants as their primary host. Some of these have been classified within families together with animal/human infecting viruses due to similarities in particle morphology and genome

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.

Science.gov (United States)

Park, June Hyun; Shin, Chanseok

2015-10-30

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Study of a steel strand tension sensor with difference single bypass excitation structure based on the magneto-elastic effect

International Nuclear Information System (INIS)

Tang Dedong; Huang Shanglian; Chen Weimin; Jiang Jianshan

2008-01-01

With many steel strands used in various important machines and architectural structures, health monitoring of strand tension becomes more and more important to ensure the equipment or structures' safety. Contrasted with the method of vibration frequency and strain gages, the method of measuring the steel strand tension based on the magneto-elastic effect is more capable of meeting the requirements of health monitoring. Yet the structure of the sensor is mainly a sleeve structure, and the steel strand to be measured serves as the core of primary and secondary solenoids. This structure is very difficult to fix and maintain. On the other hand, a change of temperature will strongly affect measurement results, and experiments prove that temperature error compensation by using a temperature compensation curve is not effective enough. Therefore in this paper the principle of a cable tension sensor based on the magneto-elastic effect is expounded, the theory of temperature influence is explored, a difference structure by single bypass excitation is devised, its magnetic loop is analyzed, an experiment is designed, and experiments on temperature compensation and pulling tension are carried out. The experiment results indicated that the structure of the sensor is feasible, temperature errors can be compensated for automatically, after which temperature errors become less than 0.012 MPa °C −1 , and repeating errors of tension are less than 0.15%, which meet the measurement requirements

Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

Energy Technology Data Exchange (ETDEWEB)

Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

2015-11-15

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

International Nuclear Information System (INIS)

Cheng, Xiaofei; Deng, Ping; Cui, Hongguang; Wang, Aiming

2015-01-01

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.

Science.gov (United States)

Mutisya, Daniel; Hardcastle, Travis; Cheruiyot, Samwel K; Pallan, Pradeep S; Kennedy, Scott D; Egli, Martin; Kelley, Melissa L; Smith, Anja van Brabant; Rozners, Eriks

2017-08-21

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA-DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

Science.gov (United States)

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-02-10

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

Science.gov (United States)

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-01-01

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans.

Science.gov (United States)

Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Lu, Rui

2013-10-01

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

International Nuclear Information System (INIS)

Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

1986-01-01

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . Cs 2 SO 4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs

Characteristics and Prediction of RNA Structure

Directory of Open Access Journals (Sweden)

Hengwu Li

2014-01-01

Full Text Available RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, and L, where L is the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding.

Spectroelectrochemical detection of microRNA-155 based on functional RNA immobilization onto ITO/GNP nanopattern.

Science.gov (United States)

Mohammadniaei, Mohsen; Yoon, Jinho; Lee, Taek; Choi, Jeong-Woo

2018-05-20

We fabricated a microRNA biosensor using the combination of surface enhanced Raman spectroscopy (SERS) and electrochemical (EC) techniques. For the first time, the weaknesses of each techniques for microRNA detection was compensated by the other ones to give rise to the specific and wide-range detection of miR-155. A single stranded 3' methylene blue (MB) and 5' thiol-modified RNA (MB-ssRNA-SH) was designed to detect the target miR-155 and immobilized onto the gold nanoparticle-modified ITO (ITO/GNP). Upon the invasion of target strand, the double-stranded RNA transformed rapidly to an upright structure resulting in a notable decrease in SERS and redox signals of the MB. For the first time, by combination of SERS and EC techniques in a single platform we extended the dynamic range of both techniques from 10 pM to 450 nM (SERS: 10 pM-5 nM and EC: 5 nM-450 nM). As well, the SERS technique improved the detection limit of the EC method from 100 pM to 100 fM, while the EC method covered single-mismatch detection which was the SERS deficiency. The fabricated single-step biosensor possessing a good capability of miRNA detection in human serum, could be employed throughout the broad ranges of biomedical and bioelectronics applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Crystal-Structure-Guided Design of Self-Assembling RNA Nanotriangles.

Science.gov (United States)

Boerneke, Mark A; Dibrov, Sergey M; Hermann, Thomas

2016-03-14

RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Å resolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

Science.gov (United States)

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

RNA interference and Register Machines (extended abstract

Directory of Open Access Journals (Sweden)

Masahiro Hamano

2012-11-01

Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs

Energy Technology Data Exchange (ETDEWEB)

Mutisya, Daniel; Hardcastle, Travis; Cheruiyot, Samwel K.; Pallan, Pradeep S.; Kennedy, Scott D.; Egli, Martin; Kelley, Melissa L.; Smith, Anja van Brabant; Rozners, Eriks

2017-06-27

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA–DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.

Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions

International Nuclear Information System (INIS)

Kim, Insil; Muto, Yutaka; Watanabe, Satoru; Kitamura, Aya; Futamura, Yasuhiro; Yokoyama, Shigeyuki; Hosono, Kazumi; Kawai, Gota; Takaku, Hiroshi; Dohmae, Naoshi; Takio, Koji; Sakamoto, Hiroshi; Shimura, Yoshiro

2000-01-01

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5- 2 H]uridine phosphoramidite, and synthesized a series of 2 H-labeled RNAs, in which all of the uridine residues except one were replaced by [5- 2 H]uridine in the target sequence, GU 8 C. By observing the H5-H6 TOCSY cross peaks of the series of 2 H-labeled RNAs complexed with the Sxl RBD1-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU 2 GU 8 , AU 8 , and UAU 8 , were assigned by comparison with those of GU 8 C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex

Interactions of a didomain fragment of the Drosophila Sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions

Energy Technology Data Exchange (ETDEWEB)

Kim, Insil; Muto, Yutaka; Watanabe, Satoru; Kitamura, Aya; Futamura, Yasuhiro; Yokoyama, Shigeyuki [University of Tokyo, Department of Biophysics and Biochemistry, Graduate School of Science (Japan); Hosono, Kazumi; Kawai, Gota; Takaku, Hiroshi [Chiba Institute of Technology, Department of Industrial Chemistry (Japan); Dohmae, Naoshi; Takio, Koji [Institute of Physical and Chemical Research (RIKEN) (Japan); Sakamoto, Hiroshi [Kobe University, Department of Biology, Faculty of Science (Japan); Shimura, Yoshiro [Biomolecular Engineering Research Institute (Japan)

2000-06-15

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sxl) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-{sup 2}H]uridine phosphoramidite, and synthesized a series of {sup 2}H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-{sup 2}H]uridine in the target sequence, GU{sub 8}C. By observing the H5-H6 TOCSY cross peaks of the series of {sup 2}H-labeled RNAs complexed with the Sxl RBD1-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU{sub 2}GU{sub 8}, AU{sub 8}, and UAU{sub 8}, were assigned by comparison with those of GU{sub 8}C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

Ammonia disinfection of hatchery waste for elimination of single-stranded RNA viruses.

Science.gov (United States)

Emmoth, Eva; Ottoson, Jakob; Albihn, Ann; Belák, Sándor; Vinnerås, Björn

2011-06-01

Hatchery waste, an animal by-product of the poultry industry, needs sanitation treatment before further use as fertilizer or as a substrate in biogas or composting plants, owing to the potential presence of opportunistic pathogens, including zoonotic viruses. Effective sanitation is also important in viral epizootic outbreaks and as a routine, ensuring high hygiene standards on farms. This study examined the use of ammonia at different concentrations and temperatures to disinfect hatchery waste. Inactivation kinetics of high-pathogenic avian influenza virus H7N1 and low-pathogenic avian influenza virus H5N3, as representatives of notifiable avian viral diseases, were determined in spiked hatchery waste. Bovine parainfluenza virus type 3, feline coronavirus, and feline calicivirus were used as models for other important avian pathogens, such as Newcastle disease virus, infectious bronchitis virus, and avian hepatitis E virus. Bacteriophage MS2 was also monitored as a stable indicator. Coronavirus was the most sensitive virus, with decimal reduction (D) values of 1.2 and 0.63 h after addition of 0.5% (wt/wt) ammonia at 14 and 25°C, respectively. Under similar conditions, high-pathogenic avian influenza H7N1 was the most resistant, with D values of 3.0 and 1.4 h. MS2 was more resistant than the viruses to all treatments and proved to be a suitable indicator of viral inactivation. The results indicate that ammonia treatment of hatchery waste is efficient in inactivating enveloped and naked single-stranded RNA viruses. Based on the D values and confidence intervals obtained, guidelines for treatment were proposed, and one was successfully validated at full scale at a hatchery, with MS2 added to hatchery waste.

Programmed self-assembly of DNA/RNA for biomedical applications

Science.gov (United States)

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.

siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract.

Directory of Open Access Journals (Sweden)

John A H Hoerter

Full Text Available RNA interference (RNAi is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence any gene of interest by the introduction of synthetic small-interfering (siRNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET. We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

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Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

2015-03-04

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

Science.gov (United States)

Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

2003-10-01

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

Science.gov (United States)

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Structure of noncoding RNA is a determinant of function of RNA binding proteins in transcriptional regulation

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Oyoshi Takanori

2012-01-01

Full Text Available Abstract The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs, resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT activity in human cells. Transcription regulator EWS (Ewing's sarcoma, which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

Crystal structure of the bacteriophage Qβ coat protein in complex with the RNA operator of the replicase gene.

Science.gov (United States)

Rumnieks, Janis; Tars, Kaspars

2014-03-06

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein-RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA. © 2013.

Recognition of RNA by amide modified backbone nucleic acids: molecular dynamics simulations of DNA-RNA hybrids in aqueous solution.

Science.gov (United States)

Nina, Mafalda; Fonné-Pfister, Raymonde; Beaudegnies, Renaud; Chekatt, Habiba; Jung, Pierre M J; Murphy-Kessabi, Fiona; De Mesmaeker, Alain; Wendeborn, Sebastian

2005-04-27

Thermodynamic and structural properties of a chemically modified DNA-RNA hybrid in which a phosphodiester linkage is replaced by a neutral amide-3 linkage (3'-CH(2)-CONH-5') were investigated using UV melting experiments, molecular dynamics simulations in explicit water, and continuum solvent models. van't Hoff analysis of the experimental UV melting curves suggests that the significant increase of the thermodynamic stability of a 15-mer DNA-RNA with seven alternated amide-3 modifications (+11 degrees C) is mainly due to an increased binding enthalpy. To further evaluate the origin in the observed affinities differences, the electrostatic contribution to the binding free energy was calculated by solving the Poisson-Boltzmann equation numerically. The nonelectrostatic contribution was estimated as the product of a hydrophobic surface tension coefficient and the surface area that is buried upon double strand formation. Structures were taken from 10 ns molecular dynamics simulations computed in a consistent fashion using explicit solvent, counterions, and the particle-mesh Ewald procedure. The present preliminary thermodynamic study suggests that the favorable binding free energy of the amide-3 DNA single strand to the complementary RNA is equally driven by electrostatic and nonpolar contributions to the binding compared to their natural analogues. In addition, molecular dynamics simulations in explicit water were performed on an amide-3 DNA single strand and the corresponding natural DNA. Results from the conformations cluster analysis of the simulated amide-3 DNA single strand ensembles suggest that the 25% of the population sampled within 10 ns has a pre-organized conformation where the sugar C3' endo pucker is favored at the 3'-flanking nucleotides. These structural and thermodynamic features contribute to the understanding of the observed increased affinities of the amide-3 DNA-RNA hybrids at the microscopic level.

Complete Genome Sequence of a Double-Stranded RNA Virus from Avocado

OpenAIRE

Villanueva, Francisco; Sabanadzovic, Sead; Valverde, Rodrigo A.; Navas-Castillo, Jesús

2012-01-01

A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV).

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

International Nuclear Information System (INIS)

Takeda, N.; Kuhn, R.J.; Yang, C.F.; Takegami, T.; Wimmer, E.

1986-01-01

An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T 1 -resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized 32 P-RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex.

Science.gov (United States)

Xu, Ning; Gkountela, Sofia; Saeed, Khalid; Akusjärvi, Göran

2009-11-01

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

Directory of Open Access Journals (Sweden)

Barik Sailen

2001-12-01

Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Natural and artificial binders of polyriboadenylic acid and their effect on RNA structure

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Giovanni N. Roviello

2015-06-01

Full Text Available The employment of molecular tools with nucleic acid binding ability to specifically control crucial cellular functions represents an important scientific area at the border between biochemistry and pharmaceutical chemistry. In this review we describe several molecular systems of natural or artificial origin, which are able to bind polyriboadenylic acid (poly(rA both in its single-stranded or structured forms. Due to the fundamental role played by the poly(rA tail in the maturation and stability of mRNA, as well as in the initiation of the translation process, compounds able to bind this RNA tract, influencing the mRNA fate, are of special interest for developing innovative biomedical strategies mainly in the field of anticancer therapy.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

Science.gov (United States)

Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

2015-10-01

Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Control of DNA strand displacement kinetics using toehold exchange.

Science.gov (United States)

Zhang, David Yu; Winfree, Erik

2009-12-02

DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

Probing electronic coupling between adenine bases in RNA strands from synchrotron radiation circular dichroism experiments

DEFF Research Database (Denmark)

Nielsen, Lisbeth Munksgård; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

2012-01-01

Circular dichroism spectra (176–330 nm) of RNA adenine oligomers, (rA)n (n = 1–10, 12, 15, and 20), reveal electronic coupling between two bases in short strands. The number of interacting bases in long strands is more and larger than that reported previously for the corresponding DNA strands....

Complete Genome Sequence of a Double-Stranded RNA Virus from Avocado

Science.gov (United States)

Villanueva, Francisco; Sabanadzovic, Sead; Valverde, Rodrigo A.

2012-01-01

A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV). PMID:22205720

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Science.gov (United States)

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci

DEFF Research Database (Denmark)

Alkhnbashi, Omer S.; Costa, Fabrizio; Shah, Shiraz Ali

2014-01-01

Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR...... array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci...... by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection...

Induction of virus resistance by exogenous application of double-stranded RNA.

Science.gov (United States)

Mitter, Neena; Worrall, Elizabeth A; Robinson, Karl E; Xu, Zhi Ping; Carroll, Bernard J

2017-10-01

Exogenous application of double-stranded RNA (dsRNA) for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However, the instability of dsRNA sprayed onto plants is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only 5 days. Innovative approaches, such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release, are emerging as key disruptive technologies. Knowledge is still limited about the mechanism of entry, transport and processing of exogenously applied dsRNA in plants. Cost of dsRNA and regulatory framework will be key influencers towards practical adoption of this technology. Copyright © 2017 Elsevier B.V. All rights reserved.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

Science.gov (United States)

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

Science.gov (United States)

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

Science.gov (United States)

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Unifying evolutionary and thermodynamic information for RNA folding of multiple alignments

DEFF Research Database (Denmark)

Seemann, Ernst Stefan; Gorodkin, Jan; Backofen, Rolf

2008-01-01

Computational methods for determining the secondary structure of RNA sequences from given alignments are currently either based on thermodynamic folding, compensatory base pair substitutions or both. However, there is currently no approach that combines both sources of information in a single...... the corresponding probability of being single stranded. Furthermore, we found that structurally conserved RNA motifs are mostly supported by folding energies. Other problems (e.g. RNA-folding kinetics) may also benefit from employing the principles of the model we introduce. Our implementation, PETfold, was tested...

Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

Directory of Open Access Journals (Sweden)

Rodrigo Jácome

Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

Sub-ensemble monitoring of DNA strand displacement using multiparameter single-molecule FRET

OpenAIRE

Baltierra Jasso, Laura; Morten, Michael; Magennis, Steven William

2018-01-01

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constan...

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

Science.gov (United States)

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

International Nuclear Information System (INIS)

Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

2007-01-01

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms.

Science.gov (United States)

Bitko, Vira; Musiyenko, Alla; Bayfield, Mark A; Maraia, Richard J; Barik, Sailen

2008-08-01

The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

KAUST Repository

Fornander, Louise H

2012-02-22

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans.

NARCIS (Netherlands)

Steiner, F.A.; Okihara, K.L.; Hoogstrate, S.W.; Sijen, T.; Ketting, R.F.

2009-01-01

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA

Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

Science.gov (United States)

Belotserkovskii, Boris P.; Neil, Alexander J.; Saleh, Syed Shayon; Shin, Jane Hae Soo; Mirkin, Sergei M.; Hanawalt, Philip C.

2013-01-01

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816–12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation. PMID:23275544

Biophysical characterization of the association of histones with single-stranded DNA.

Science.gov (United States)

Wang, Ying; van Merwyk, Luis; Tönsing, Katja; Walhorn, Volker; Anselmetti, Dario; Fernàndez-Busquets, Xavier

2017-11-01

Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin. Copyright © 2017 Elsevier B.V. All rights reserved.

Electron attachment to DNA single strands: gas phase and aqueous solution.

Science.gov (United States)

Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

2007-01-01

The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

Science.gov (United States)

Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

2018-02-01

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

Science.gov (United States)

Zgaga, Z

1991-08-01

UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

Correlation of RNA secondary structure statistics with thermodynamic stability and applications to folding.

Science.gov (United States)

Wu, Johnny C; Gardner, David P; Ozer, Stuart; Gutell, Robin R; Ren, Pengyu

2009-08-28

The accurate prediction of the secondary and tertiary structure of an RNA with different folding algorithms is dependent on several factors, including the energy functions. However, an RNA higher-order structure cannot be predicted accurately from its sequence based on a limited set of energy parameters. The inter- and intramolecular forces between this RNA and other small molecules and macromolecules, in addition to other factors in the cell such as pH, ionic strength, and temperature, influence the complex dynamics associated with transition of a single stranded RNA to its secondary and tertiary structure. Since all of the factors that affect the formation of an RNAs 3D structure cannot be determined experimentally, statistically derived potential energy has been used in the prediction of protein structure. In the current work, we evaluate the statistical free energy of various secondary structure motifs, including base-pair stacks, hairpin loops, and internal loops, using their statistical frequency obtained from the comparative analysis of more than 50,000 RNA sequences stored in the RNA Comparative Analysis Database (rCAD) at the Comparative RNA Web (CRW) Site. Statistical energy was computed from the structural statistics for several datasets. While the statistical energy for a base-pair stack correlates with experimentally derived free energy values, suggesting a Boltzmann-like distribution, variation is observed between different molecules and their location on the phylogenetic tree of life. Our statistical energy values calculated for several structural elements were utilized in the Mfold RNA-folding algorithm. The combined statistical energy values for base-pair stacks, hairpins and internal loop flanks result in a significant improvement in the accuracy of secondary structure prediction; the hairpin flanks contribute the most.

Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

Science.gov (United States)

Caldecott, K W

2001-05-01

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

Science.gov (United States)

Pompey, Justine M; Foda, Bardees; Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

Directory of Open Access Journals (Sweden)

Justine M Pompey

Full Text Available Dicer enzymes process double-stranded RNA (dsRNA into small RNAs that target gene silencing through the RNA interference (RNAi pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

Temporary electron localization and scattering in disordered single strands of DNA

International Nuclear Information System (INIS)

Caron, Laurent; Sanche, Leon

2006-01-01

We present a theoretical study of the effect of structural and base sequence disorders on the transport properties of nonthermal electron scattering within and from single strands of DNA. The calculations are based on our recently developed formalism to treat multiple elastic scattering from simplified pseudomolecular DNA subunits. Structural disorder is shown to increase both the elastic scattering cross section and the attachment probability on the bases at low energy. Sequence disorder, however, has no significant effect

Formation of double-strand breaks in DNA of γ-irradiated bacteria depending on the function of fast repair processes of DNA single-strand breaks

International Nuclear Information System (INIS)

Petrov, S.I.; Gaziev, A.I.

1980-01-01

The formation of double-strand breaks in DNA of γ-irradiated ( 60 Co)Ex coli bacteria depending on the function of fast repair processes of DNA single-strand breaks, is investigated. The profiles of sedimentation of DNA Ex coli cells, irradiated at 0-2 deg C in the salt medium and in EDTA-borate buffer, are presented. It is shown that when irradiating cells in EDTA-borate buffer, the output of single- and double strand breaks in DNA is much higher than in the case of their irradiation in the minimum salt medium. The dependence of output of single-strand and double-strand breaks depending on the radiatier doze of E coli cells in the salt medium and EDTA-borate buffer, is studied. The supposition is made on the presence of a regulative interaction between the accumulation of DNA single-breaks and their repair with the formation of double-strand breaks. The functionating of fast and superfast repair processes considerably affects the formation of double-strand breaks in DNA of a bacterium cell. A considerable amount of double-breaks registered immediately after irradiation forms due to a close position of single-strand breaks on the opposite DNA strands

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

International Nuclear Information System (INIS)

Kabilova, Tatyana O; Chernolovskaya, Elena L; Kovtonyuk, Larisa V; Zonov, Evgeniy V; Ryabchikova, Elena I; Popova, Nelly A; Nikolin, Valeriy P; Kaledin, Vasiliy I; Zenkova, Marina A; Vlassov, Valentin V

2014-01-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment. The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA. In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3′-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC. The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

International Nuclear Information System (INIS)

Reigadas, Sandrine; Ventura, Michel; Andreola, Marie-Line; Michel, Justine; Gryaznov, Sergei; Tarrago-Litvak, Laura; Litvak, Simon; Astier-Gin, Therese

2003-01-01

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC 50 values lower than 1 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA

Efficacy of double-stranded RNA against white spot syndrome virus (WSSV non-structural (orf89, wsv191 and structural (vp28, vp26 genes in the Pacific white shrimp Litopenaeus vannamei

Directory of Open Access Journals (Sweden)

César M. Escobedo-Bonilla

2015-04-01

Full Text Available White spot syndrome virus (WSSV is a major pathogen in shrimp aquaculture. RNA interference (RNAi is a promising tool against viral infections. Previous works with RNAi showed different antiviral efficacies depending on the silenced gene. This work evaluated the antiviral efficacy of double-stranded (ds RNA against two non-structural (orf89, wsv191 WSSV genes compared to structural (vp26, vp28 genes to inhibit an experimental WSSV infection. Gene orf89 encodes a putative regulatory protein and gene white spot virus (wsv191 encodes a nonspecific nuclease; whereas genes vp26 and vp28 encode envelope proteins, respectively. Molecules of dsRNA against each of the WSSV genes were intramuscularly injected (4 μg per shrimp into a group of shrimp 48 h before a WSSV challenge. The highest antiviral activity occurred with dsRNA against orf89, vp28 and vp26 (cumulative mortalities 10%, 10% and 21%, respectively. In contrast, the least effective treatment was wsv191 dsRNA (cumulative mortality 83%. All dead animals were WSSV-positive by one-step PCR, whereas reverse-transcription PCR of all surviving shrimp confirmed inhibition of virus replication. This study showed that dsRNA against WSSV genes orf89, vp28 and vp26 were highly effective to inhibit virus replication and suggest an essential role in WSSV infection. Non-structural WSSV genes such as orf89 can be used as novel targets to design therapeutic RNAi molecules against WSSV infection.

Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices.

Science.gov (United States)

Robertson, Neil M; Hizir, Mustafa Salih; Balcioglu, Mustafa; Wang, Rui; Yavuz, Mustafa Selman; Yumak, Hasan; Ozturk, Birol; Sheng, Jia; Yigit, Mehmet V

2015-09-15

In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR-10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

OpenAIRE

Stroud, A. L.

2012-01-01

Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

Directory of Open Access Journals (Sweden)

Oleg M Ganichkin

Full Text Available Selenocysteine tRNAs (tRNA(Sec exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec. tRNA(Sec lacking the single-stranded 3'-acceptor end ((ΔGCCARNA(Sec yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCARNA(Sec resembles the structure of human tRNA(Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Secin vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec.We provide the most highly resolved structure of a tRNA(Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec support its interaction with proteins.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

Science.gov (United States)

Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

2018-03-05

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Self containment, a property of modular RNA structures, distinguishes microRNAs.

Directory of Open Access Journals (Sweden)

Miler T Lee

2008-08-01

Full Text Available RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

International Nuclear Information System (INIS)

Lloyd, Richard E.

2015-01-01

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Lloyd, Richard E., E-mail: rlloyd@bcm.edu

2015-05-15

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

Science.gov (United States)

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

Investigation of RNA Structure by High-Throughput SHAPE-Based Probing Methods

DEFF Research Database (Denmark)

Poulsen, Line Dahl

of highthroughput SHAPE-based approaches to investigate RNA structure based on novel SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection (SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA genome, and the data is used to construct a genome-wide structural...... that they are functional. The SHAPES method is further applied to the hepatitis C virus (HCV), where the data is used to refine known and predicted structures. Over the past years, the interest of studying RNA structure in their native environment has been increased, and to allow studying RNA structure inside living cells...... using the SHAPE Selection approach, I introduce a biotinylated probing reagent. This chemical can cross cell membranes and reacts with RNA inside the cells, allowing the structural conformations to be studied in the context of physiological relevant conditions in living cells. The methods and results...

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

International Nuclear Information System (INIS)

Boye, E.; Krisch, R.E.

1980-01-01

Induction and repair of double-and single-strand DNA breaks have been measured after decays of 125 I and 3 H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-( 125 I)iodo-2'-deoxyuridine or with (methyl- 3 H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125 I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3 H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10 -14 (double-strand breaks) and 2.82 x 10 -12 (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all. (Author)

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

International Nuclear Information System (INIS)

Vende, Patrice; Tortorici, M. Alejandra; Taraporewala, Zenobia F.; Patton, John T.

2003-01-01

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans.

Science.gov (United States)

Steiner, Florian A; Okihara, Kristy L; Hoogstrate, Suzanne W; Sijen, Titia; Ketting, René F

2009-02-01

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Energy Technology Data Exchange (ETDEWEB)

Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

2010-10-11

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Science.gov (United States)

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Terahertz time-domain spectroscopy and imaging of artificial RNA

DEFF Research Database (Denmark)

Fischer, Bernd M.; Hoffmann, Matthias; Helm, Hanspeter

2005-01-01

We use terahertz time-domain spectroscopy (THz-TDS) to measure the far-infrared dielectric function of two artificial RNA single strands, composed of polyadenylic acid (poly-A) and polycytidylic acid (poly-C). We find a significant difference in the absorption between the two types of RNA strands......, and we show that we can use this difference to record images of spot arrays of the RNA strands. Under controlled conditions it is possible to use the THz image to distinguish between the two RNA strands. We discuss the requirements to sample preparation imposed by the lack of sharp spectral features...

A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi.

Directory of Open Access Journals (Sweden)

Timothy T Perkins

2009-07-01

Full Text Available High-density, strand-specific cDNA sequencing (ssRNA-seq was used to analyze the transcriptome of Salmonella enterica serovar Typhi (S. Typhi. By mapping sequence data to the entire S. Typhi genome, we analyzed the transcriptome in a strand-specific manner and further defined transcribed regions encoded within prophages, pseudogenes, previously un-annotated, and 3'- or 5'-untranslated regions (UTR. An additional 40 novel candidate non-coding RNAs were identified beyond those previously annotated. Proteomic analysis was combined with transcriptome data to confirm and refine the annotation of a number of hpothetical genes. ssRNA-seq was also combined with microarray and proteome analysis to further define the S. Typhi OmpR regulon and identify novel OmpR regulated transcripts. Thus, ssRNA-seq provides a novel and powerful approach to the characterization of the bacterial transcriptome.

Hole hopping rates in single strand oligonucleotides

Energy Technology Data Exchange (ETDEWEB)

Borrelli, Raffaele [Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO (Italy); Capobianco, Amedeo [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy); Peluso, Andrea, E-mail: apeluso@unisa.it [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy)

2014-08-31

Highlights: • DNA hole transfer rates have been computed. • Delocalized adenine domains significantly affect hole transfer rates in DNA. • Franck–Condon weighted density of state from DFT normal modes. • DNA application in molecular electronics. - Abstract: The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck–Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck–Condon density of states extends over a wide range of hole site energy difference, 0–1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

International Nuclear Information System (INIS)

Valles, Steven M.; Oi, David H.; Becnel, James J.; Wetterer, James K.; LaPolla, John S.; Firth, Andrew E.

2016-01-01

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species. - Highlights: • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

Energy Technology Data Exchange (ETDEWEB)

Valles, Steven M., E-mail: steven.valles@ars.usda.gov [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Oi, David H.; Becnel, James J. [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Wetterer, James K. [Wilkes Honors College, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458 (United States); LaPolla, John S. [Department of Biological Sciences, Towson University, 8000 York Road, Towson, MD 21252 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom)

2016-09-15

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species. - Highlights: • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

Science.gov (United States)

Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

2015-01-01

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

Directory of Open Access Journals (Sweden)

Brad Thomas Townsley

2015-05-01

Full Text Available Next Generation Sequencing (NGS is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE libraries and can easily extend to full transcript coverage shotgun (SHO type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

The fidelity of reverse transcription differs in reactions primed with RNA versus DNA primers

NARCIS (Netherlands)

Oude Essink, B. B.; Berkhout, B.

1999-01-01

Reverse transcriptase enzymes (RT) convert single-stranded retroviral RNA genomes into double-stranded DNA. The RT enzyme can use both RNA and DNA primers, the former being used exclusively during initiation of minus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by

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

Science.gov (United States)

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.

Small catalytic RNA: Structure, function and application

Energy Technology Data Exchange (ETDEWEB)

Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

1991-04-01

We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

International Nuclear Information System (INIS)

Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

2012-01-01

The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

Science.gov (United States)

Holton, Nathaniel W; Andrews, Joel F; Gassman, Natalie R

2017-09-05

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

NARCIS (Netherlands)

Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

Science.gov (United States)

Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

2016-01-01

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Repair and gamma radiation-induced single- and double-strand breaks in DNA of Escherichia coli

International Nuclear Information System (INIS)

Petrov, S.I.

1981-01-01

Studies in the kinetics of repair of γ-radiation-induced single- and double-strand breaks in DNA of E. coli cells showed that double-strand DNA breaks are rejoined by the following two ways. The first way is conditioned by repair of single-strand breaks and represents the repair of ''oblique'' double-strand breaks in DNA, whereas the second way is conditioned by functioning of the recombination mechanisms and, to all appearance, represents the repair of ''direct'' double-strand breaks in DNA

Positive-Strand RNA Viruses Infecting the Red Imported Fire Ant, Solenopsis invicta

Directory of Open Access Journals (Sweden)

Steven M. Valles

2012-01-01

Full Text Available The imported fire ants, Solenopsis invicta and S. richteri were introduced into the USA between 1918 and 1945. Since that time, they have expanded their USA range to include some 138 million hectares. Their introduction has had significant economic consequences with costs associated with damage and control efforts estimated at 6 billion dollars annually in the USA. The general consensus of entomologists and myrmecologists is that permanent, sustainable control of these ants in the USA will likely depend on self-sustaining biological control agents. A metagenomics approach successfully resulted in discovery of three viruses infecting S. invicta. Solenopsis invicta virus 1 (SINV-1, SINV-2, and SINV-3 are all positive, single-stranded RNA viruses and represent the first viral discoveries in any ant species. Molecular characterization, host relationships, and potential development and use of SINV-1, SINV-2, and SINV-3 as biopesticides are discussed.

Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription

NARCIS (Netherlands)

Berkhout, B.; Vastenhouw, N. L.; Klasens, B. I.; Huthoff, H.

2001-01-01

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There

Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response

OpenAIRE

Marques, Joao T.; Rebouillat, Dominique; Ramana, Chilakamarti V.; Murakami, Junko; Hill, Jason E.; Gudkov, Andrei; Silverman, Robert H.; Stark, George R.; Williams, Bryan R. G.

2005-01-01

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus la...

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

International Nuclear Information System (INIS)

Valles, Steven M.; Hashimoto, Yoshifumi

2009-01-01

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number (FJ528584)), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

Cytoplasmic Z-RNA

International Nuclear Information System (INIS)

Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

1987-01-01

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

Science.gov (United States)

Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

2015-06-12

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

Directory of Open Access Journals (Sweden)

Baodong Wu

2009-03-01

Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

Small catalytic RNA: Structure, function and application

Energy Technology Data Exchange (ETDEWEB)

Monforte, J.A.

1991-04-01

We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

Regulatory RNA design through evolutionary computation and strand displacement.

Science.gov (United States)

Rostain, William; Landrain, Thomas E; Rodrigo, Guillermo; Jaramillo, Alfonso

2015-01-01

The discovery and study of a vast number of regulatory RNAs in all kingdoms of life over the past decades has allowed the design of new synthetic RNAs that can regulate gene expression in vivo. Riboregulators, in particular, have been used to activate or repress gene expression. However, to accelerate and scale up the design process, synthetic biologists require computer-assisted design tools, without which riboregulator engineering will remain a case-by-case design process requiring expert attention. Recently, the design of RNA circuits by evolutionary computation and adapting strand displacement techniques from nanotechnology has proven to be suited to the automated generation of DNA sequences implementing regulatory RNA systems in bacteria. Herein, we present our method to carry out such evolutionary design and how to use it to create various types of riboregulators, allowing the systematic de novo design of genetic control systems in synthetic biology.

Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

Energy Technology Data Exchange (ETDEWEB)

Umemura, Kazuo, E-mail: meicun2006@163.com; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

2016-12-01

Highlights: • Conjugates of protein, DNA, and SWNTs were observed by AFM in liquid. • Non-uniform binding of proteins was visualized in liquid. • Thickness of DNA molecules on SWNT surfaces was well characterized in liquid. - Abstract: Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA–SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA–SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA–SWNT hybrids. The morphology of the SSB–ssDNA–SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB–ssDNA–SWNT hybrids showed much larger variance than the ssDNA–SWNT hybrids.

MEIOB targets single-strand DNA and is necessary for meiotic recombination.

Directory of Open Access Journals (Sweden)

Benoit Souquet

Full Text Available Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB. This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/- spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/- meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

Science.gov (United States)

Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

2011-07-06

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

Reconstruction of ancestral RNA sequences under multiple structural constraints

OpenAIRE

Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldisp?hl, J?r?me

2016-01-01

Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given...

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

Science.gov (United States)

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

International Nuclear Information System (INIS)

Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M.

2016-01-01

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

Energy Technology Data Exchange (ETDEWEB)

Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M., E-mail: sean.burrows@oregonstate.edu

2016-02-25

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

A Novel Single-Strand RNAi Therapeutic Agent Targeting the (Pro)renin Receptor Suppresses Ocular Inflammation.

Science.gov (United States)

Kanda, Atsuhiro; Ishizuka, Erdal Tan; Shibata, Atsushi; Matsumoto, Takahiro; Toyofuku, Hidekazu; Noda, Kousuke; Namba, Kenichi; Ishida, Susumu

2017-06-16

The receptor-associated prorenin system (RAPS) refers to the pathogenic mechanism whereby prorenin binding to the (pro)renin receptor [(P)RR] dually activates the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling. Here we revealed significant upregulation of prorenin and soluble (P)RR levels in the vitreous fluid of patients with uveitis compared to non-inflammatory controls, together with a positive correlation between these RAPS components and monocyte chemotactic protein-1 among several upregulated cytokines. Moreover, we developed a novel single-strand RNAi agent, proline-modified short hairpin RNA directed against human and mouse (P)RR [(P)RR-PshRNA], and we determined its safety and efficacy in vitro and in vivo. Application of (P)RR-PshRNA in mice caused significant amelioration of acute (uveitic) and chronic (diabetic) models of ocular inflammation with no apparent adverse effects. Our findings demonstrate the significant implication of RAPS in the pathogenesis of human uveitis and the potential usefulness of (P)RR-PshRNA as a therapeutic agent to reduce ocular inflammation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

Energy Technology Data Exchange (ETDEWEB)

Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun, E-mail: yunatswu@swu.edu.cn

2016-04-15

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. - Highlights: • Amplified and sensitive detection of microRNA from tumor cells is achieved. • Signal amplification is realized by two cascaded strand displacement reactions. • The developed sensor is selective and label-free without involving any enzymes.

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4

NARCIS (Netherlands)

van der Avoort, H. G.; van der Ende, A.; van Arkel, G. A.; Weisbeek, P. J.

1984-01-01

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

Directory of Open Access Journals (Sweden)

Simon Roux

2016-12-01

Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

Science.gov (United States)

Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

2017-12-05

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer.

Science.gov (United States)

Seamon, Kyle J; Bumpus, Namandjé N; Stivers, James T

2016-11-08

Sterile alpha motif and HD domain protein 1 (SAMHD1) is a unique enzyme that plays important roles in nucleic acid metabolism, viral restriction, and the pathogenesis of autoimmune diseases and cancer. Although much attention has been focused on its dNTP triphosphohydrolase activity in viral restriction and disease, SAMHD1 also binds to single-stranded RNA and DNA. Here we utilize a UV cross-linking method using 5-bromodeoxyuridine-substituted oligonucleotides coupled with high-resolution mass spectrometry to identify the binding site for single-stranded nucleic acids (ssNAs) on SAMHD1. Mapping cross-linked amino acids on the surface of existing crystal structures demonstrated that the ssNA binding site lies largely along the dimer-dimer interface, sterically blocking the formation of the homotetramer required for dNTPase activity. Surprisingly, the disordered C-terminus of SAMHD1 (residues 583-626) was also implicated in ssNA binding. An interaction between this region and ssNA was confirmed in binding studies using the purified SAMHD1 583-626 peptide. Despite a recent report that SAMHD1 possesses polyribonucleotide phosphorylase activity, we did not detect any such activity in the presence of inorganic phosphate, indicating that nucleic acid binding is unrelated to this proposed activity. These data suggest an antagonistic regulatory mechanism in which the mutually exclusive oligomeric state requirements for ssNA binding and dNTP hydrolase activity modulate these two functions of SAMHD1 within the cell.

RNA-Pareto: interactive analysis of Pareto-optimal RNA sequence-structure alignments.

Science.gov (United States)

Schnattinger, Thomas; Schöning, Uwe; Marchfelder, Anita; Kestler, Hans A

2013-12-01

Incorporating secondary structure information into the alignment process improves the quality of RNA sequence alignments. Instead of using fixed weighting parameters, sequence and structure components can be treated as different objectives and optimized simultaneously. The result is not a single, but a Pareto-set of equally optimal solutions, which all represent different possible weighting parameters. We now provide the interactive graphical software tool RNA-Pareto, which allows a direct inspection of all feasible results to the pairwise RNA sequence-structure alignment problem and greatly facilitates the exploration of the optimal solution set.

Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.

Directory of Open Access Journals (Sweden)

Nicholas J Schurch

Full Text Available The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3' untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3' polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1 gene and 3' UTR re-annotation (including extension of one 3' UTR by 5.9 kb; (2 disentangling of gene expression in complex regions; (3 clearer interpretation of small RNA expression and (4 identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

Science.gov (United States)

Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

2017-06-01

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction

Science.gov (United States)

Puton, Tomasz; Kozlowski, Lukasz P.; Rother, Kristian M.; Bujnicki, Janusz M.

2013-01-01

We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks. PMID:23435231

CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction.

Science.gov (United States)

Puton, Tomasz; Kozlowski, Lukasz P; Rother, Kristian M; Bujnicki, Janusz M

2013-04-01

We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks.

Molecular dynamics simulation of a DNA containing a single strand break

Energy Technology Data Exchange (ETDEWEB)

Yamaguchi, H.; Siebers, G.; Furukawa, A.; Otagiri, N.; Osman, R

2002-07-01

Molecular dynamics simulations were performed for a dodecamer DNA containing a single strand break (SSB), which has been represented by a 3'-OH deoxyribose and 5'-OH phosphate in the middle of the strand. Molecular force field parameters of the 5'-OH phosphate region were determined from an ab initio calculation at the HF/6-31G level using the program package GAMESS. The DNA was placed in a periodic boundary box with water molecules and Na+ counter-ions to produce a neutralised system. After minimisation, the system was heated to 300 K, equilibrated and a production run at constant NTP was executed for 1 ns using AMBER 4.1. Snapshots of the SSB-containing DNA and a detailed analysis of the equilibriated average structure revealed surprisingly small conformational changes compared to normal DNA. However, dynamic properties calculated using the essential dynamics method showed some features that may be important for the recognition of this damage by repair enzymes. (author)

Branched RNA: A New Architecture for RNA Interference

Directory of Open Access Journals (Sweden)

Anna Aviñó

2011-01-01

Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

DNA single strand break in fibroblast from Down syndrome patients

International Nuclear Information System (INIS)

Rozga, B.

1992-01-01

The radiosensitivity of tree trisomic (trisomia +21) strains of human fibroblasts to gamma radiation has been investigated in vitro and the causes of induction and repair of single strand DNA breaks in these cells have been estimated. The single strand breaks in DNA of normal and trisomic cells have been found to be ameliorated with an approximately equal efficiency. Repair has been found to be three times slower in trisomic cells compared to their normal relevant, most likely due to their elevated sensitivity to ionizing radiation and the following mortality of trisomic cells, and/or the potential occurrence of a great number of chromosome aberrations in cells irradiated in vitro. (author). 28 refs, 4 figs, 1 tab

Sequence analysis of L RNA of Lassa virus

International Nuclear Information System (INIS)

Vieth, Simon; Torda, Andrew E.; Asper, Marcel; Schmitz, Herbert; Guenther, Stephan

2004-01-01

The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses

Single-strand breaks in supercoiled DNA induced by vacuum-UV radiation in aqueous solution

Energy Technology Data Exchange (ETDEWEB)

Takakura, Kaoru; Ishikawa, Mitsuo; Hieda, Kotaro; Kobayashi, Katsumi; Ito, Atsushi; Ito, Takashi

1986-09-01

The induction of single-strand breaks in the DNA of plasmid pBR 322 by vacuum-UV radiation above 145 nm in aqueous solutions was studied in relation to the production of OH-radicals in water. The similarity and dissimilarity were examined on the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH-radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single-strand breaks.

Single-strand breaks in supercoiled DNA induced by vacuum-UV radiation in aqueous solution

International Nuclear Information System (INIS)

Takakura, Kaoru; Ishikawa, Mitsuo; Hieda, Kotaro; Kobayashi, Katsumi; Ito, Atsushi; Ito, Takashi

1986-01-01

The induction of single-strand breaks in the DNA of plasmid pBR 322 by vacuum-UV radiation above 145 nm in aqueous solutions was studied in relation to the production of OH-radicals in water. The similarity and dissimilarity were examined on the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH-radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single-strand breaks. (author)

3′-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary cis-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation

OpenAIRE

Sharma, Nidhi; O'Donnell, Brian J.; Flanegan, James B.

2005-01-01

The 5′ cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5′ cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was no...

Defective processing of methylated single-stranded DNA by E. coli alkB mutants

Science.gov (United States)

Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

2000-01-01

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

Science.gov (United States)

Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

2017-06-01

Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

Science.gov (United States)

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

Science.gov (United States)

Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

2016-02-25

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

CRISPR-Cas9 Structures and Mechanisms.

Science.gov (United States)

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro

Science.gov (United States)

Sharwood, Robert E.; Hotto, Amber M.; Bollenbach, Thomas J.; Stern, David B.

2011-01-01

Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3′-to-5′ exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNAArg, raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S–AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1. PMID:21148395

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

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Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

2017-02-01

The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

Science.gov (United States)

Rey, Félix A.

2017-01-01

The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

Directory of Open Access Journals (Sweden)

Danilo Dubrau

2017-02-01

Full Text Available The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132, which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

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Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-04-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

A universal and label-free impedimetric biosensing platform for discrimination of single nucleotide substitutions in long nucleic acid strands.

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Mills, Dawn M; Martin, Christopher P; Armas, Stephanie M; Calvo-Marzal, Percy; Kolpashchikov, Dmitry M; Chumbimuni-Torres, Karin Y

2018-06-30

We report a label-free universal biosensing platform for highly selective detection of long nucleic acid strands. The sensor consists of an electrode-immobilized universal stem-loop (USL) probe and two adaptor strands that form a 4J structure in the presence of a specific DNA/RNA analyte. The sensor was characterized by electrochemical impedance spectroscopy (EIS) using K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] redox couple in solution. An increase in charge transfer resistance (R CT ) was observed upon 4J structure formation, the value of which depends on the analyte length. Cyclic voltammetry (CV) was used to further characterize the sensor and monitor the electrochemical reaction in conjunction with thickness measurements of the mixed DNA monolayer obtained using spectroscopic ellipsometry. In addition, the electron transfer was calculated at the electrode/electrolyte interface using a rotating disk electrode. Limits of detection in the femtomolar range were achieved for nucleic acid targets of different lengths (22 nt, 60 nt, 200 nt). The sensor produced only a background signal in the presence of single base mismatched analytes, even in hundred times excess in concentration. This label-free and highly selective biosensing platform is versatile and can be used for universal detection of nucleic acids of varied lengths which could revolutionize point of care diagnostics for applications such as bacterial or cancer screening. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

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Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

The validity of sedimentation data from high molecular weight DNA and the effects of additives on radiation-induced single-strand breakage

International Nuclear Information System (INIS)

Dugle, D.L.

1979-10-01

The optimization of many of the factors governing reproducible sedimentation behaviour of high molecular weight single-strand DNA in a particular alkaline sucrose density gradient system is described. A range of angular momenta is defined for which a constant strand breakage efficiency is required, despite a rotor speed effect which increases the measured molecular weights at decreasing rotor speeds for larger DNA molecules. The possibility is discussed that the bimodal control DNA profiles obtained after sedimentation at 11 500 rev/min (12 400 g) or less represent structural subunits of the chromatid. The random induction of single-strand DNA breaks by ionizing radiation is demonstrated by the computer-derived fits to the experimental profiles. The enhancement of single-strand break (SSB) yields in hypoxic cells by oxygen, para-nitroacetophenone (PNAP), or any of the three nitrofuran derivatives used was well correlated with increased cell killing. Furthermore, reductions in SSB yields for known hydroxyl radical (OH.) scavengers correlates with the reactivities of these compounds toward OH.. This supports the contention that some type of OH.-induced initial lesion, which may ultimately be expressed as an unrepaired or misrepaired double-strand break, constitutes a lethal event. (author)

Single-strand DNA molecule translocation through nanoelectrode gaps

International Nuclear Information System (INIS)

Zhao Xiongce; Payne, Christina M; Cummings, Peter T; Lee, James W

2007-01-01

Molecular dynamics simulations were performed to investigate the translocation of single-strand DNA through nanoscale electrode gaps under the action of a constant driving force. The application behind this theoretical study is a proposal to use nanoelectrodes as a screening gap as part of a rapid genomic sequencing device. Preliminary results from a series of simulations using various gap widths and driving forces suggest that the narrowest electrode gap that a single-strand DNA can pass is ∼1.5 nm. The minimum force required to initiate the translocation within nanoseconds is ∼0.3 nN. Simulations using DNA segments of various lengths indicate that the minimum initiation force is insensitive to the length of DNA. However, the average threading velocity of DNA varies appreciably from short to long DNA segments. We attribute such variation to the different nature of drag force experienced by the short and long DNA segments in the environment. It is found that DNA molecules deform significantly to fit in the shape of the nanogap during the translocation

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

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Xie, Ping

2015-10-09

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

Processing of nuclear viroids in vivo: an interplay between RNA conformations.

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María-Eugenia Gas

2007-11-01

Full Text Available Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+ polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+ RNAs of nuclear viroids (family Pospiviroidae occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+ RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase.

The RNA of turnip yellow mosaic virus exhibits icosahedral order

International Nuclear Information System (INIS)

Larson, Steven B.; Lucas, Robert W.; Greenwood, Aaron; McPherson, Alexander

2005-01-01

Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses

Towards Antiviral shRNAs Based on the AgoshRNA Design.

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Ying Poi Liu

Full Text Available RNA interference (RNAi can be induced by intracellular expression of a short hairpin RNA (shRNA. Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA. Dicer is also involved in microRNA (miRNA processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2 protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these "AgoshRNA" molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.

The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.

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Raman, Pravrutha; Zaghab, Soriayah M; Traver, Edward C; Jose, Antony M

2017-08-21

Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Repair of X-ray-induced single-strand breaks by a cell-free system

International Nuclear Information System (INIS)

Seki, Shuji; Ikeda, Shogo; Tsutui, Ken; Teraoka, Hirobumi

1990-01-01

Repair of X-ray-induced single-strand breaks of DNA was studied in vitro using an exonuclease purified from mouse ascites sarcoma (SR-C3H/He) cells. X-ray-dose-dependent unscheduled DNA synthesis was primed by the exonuclease. Repair of X-ray-induced single-strand breaks in pUC19 plasmid DNA was demonstrated by agarose gel electrophoresis after incubating the damaged DNA with the exonuclease, DNA polymerase (Klenow fragment of DNA polymerase I or DNA polymerase β purified from SR-C3H/He cells), four deoxynucleoside triphosphates, ATP and DNA ligase (T4 DNA ligase or DNA ligase I purified from calf thymus). The present results suggested that the exonuclease is involved in the initiation of repair of X-ray-induced single-strand breaks in removing 3' ends of X-ray-damaged DNA. (author)

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

Energy Technology Data Exchange (ETDEWEB)

Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.; Scuderi, M. José; Bashkin, James K.; Luo, Ming; Dutch, Rebecca Ellis

2018-02-07

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.

IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit

Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

Science.gov (United States)

Vernal, Javier; Serpa, Viviane I; Tavares, Carolina; Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

2007-05-01

An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

OpenAIRE

Khodakov, Dmitriy A.; Khodakova, Anastasia S.; Huang, David M.; Linacre, Adrian; Ellis, Amanda V.

2015-01-01

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within doubl...

DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

OpenAIRE

Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

2012-01-01

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up...

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

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Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Lack of close relationship between three strains of human rhinoviruses as determined by their RNA sequences.

Science.gov (United States)

Yin, F H; Lonberg-Holm, K; Chan, S P

1973-07-01

The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA.

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers

International Nuclear Information System (INIS)

Livneh, Z.

1986-01-01

Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins

Normal formation and repair of γ-radiation-induced single and double strand DNA breaks in Down syndrome fibroblasts

International Nuclear Information System (INIS)

Steiner, M.E.; Woods, W.G.

1982-01-01

Fibroblasts from patients with Down syndrome (Trisomy 21) were examined for repair capability of γ-radiation-induced single strand and double strand DNA breaks. Formation and repair of DNA breaks were determined by DNA alkaline and non-denaturing elution techniques. Down syndrome fibroblasts were found to repair single strand and double strand breaks as well as fibroblasts from normal controls. (orig.)

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

Science.gov (United States)

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

Science.gov (United States)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

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Dever Thomas E

2008-03-01

Full Text Available Abstract Background Double-stranded (ds RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs. Fish and amphibian PKR genes have not been described so far. Results Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2α in yeast. Conclusion Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA

Free energy minimization to predict RNA secondary structures and computational RNA design.

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Churkin, Alexander; Weinbrand, Lina; Barash, Danny

2015-01-01

Determining the RNA secondary structure from sequence data by computational predictions is a long-standing problem. Its solution has been approached in two distinctive ways. If a multiple sequence alignment of a collection of homologous sequences is available, the comparative method uses phylogeny to determine conserved base pairs that are more likely to form as a result of billions of years of evolution than by chance. In the case of single sequences, recursive algorithms that compute free energy structures by using empirically derived energy parameters have been developed. This latter approach of RNA folding prediction by energy minimization is widely used to predict RNA secondary structure from sequence. For a significant number of RNA molecules, the secondary structure of the RNA molecule is indicative of its function and its computational prediction by minimizing its free energy is important for its functional analysis. A general method for free energy minimization to predict RNA secondary structures is dynamic programming, although other optimization methods have been developed as well along with empirically derived energy parameters. In this chapter, we introduce and illustrate by examples the approach of free energy minimization to predict RNA secondary structures.

Electronic coupling between photo-excited stacked bases in DNA and RNA strands with emphasis on the bright states initially populated

DEFF Research Database (Denmark)

Nielsen (Baggesen), Lisbeth Munksgård; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

2013-01-01

. In this review paper, we focus on the bright states initially populated and discuss their nature based on information obtained from systematic absorption and circular dichroism experiments on single strands of different lengths. Our results from the last five years are compared with those from other groups......, and are discussed in the context of successive deexcitation schemes. Pieces to the puzzle have come from different experiments and theory but a complete description has yet to emerge. As such the story about DNA/RNA photophysical decay mechanisms resembles the tale about the blind men and the elephant where all see...

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea.

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Hajime Muraguchi

Full Text Available The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC. To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

Genome-wide annotation of porcine microRNA genes and transcriptome profiling during Actinobacillus infection

DEFF Research Database (Denmark)

Nielsen, Mathilde

MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project was to ex......MicroRNAs are small single stranded non-coding RNA molecules which contributes to the regulation of gene expression by primarily binding to the 3´end of protein coding mRNA, hereby inhibiting the translation process or promting degradation of the mRNA. The main focus of this PhD project...

Genetic and biochemical identification of a novel single-stranded DNA binding complex in Haloferax volcanii

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Amy eStroud

2012-06-01

Full Text Available Single-stranded DNA binding proteins play an essential role in DNA replication and repair. They use oligosaccharide-binding folds, a five-stranded ß-sheet coiled into a closed barrel, to bind to single-stranded DNA thereby protecting and stabilizing the DNA. In eukaryotes the single-stranded DNA binding protein is known as replication protein A (RPA and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed single-stranded DNA-binding protein (SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3 exist in operons with a novel gene specific to Euryarchaeota, this gene encodes a protein that we have termed rpa-associated protein (RPAP. The rpap genes encode proteins belonging to COG3390 group and feature oligosaccharide-binding folds, suggesting that they might cooperate with RPA in binding to single-stranded DNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only ∆rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins. We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA binding complex that is unique to Euryarchaeota.

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination

Science.gov (United States)

Chao, Mei; Wang, Tzu-Chi; Lin, Chia-Chi; Yung-Liang Wang, Robert; Lin, Wen-Bin; Lee, Shang-En; Cheng, Ying-Yu; Yeh, Chau-Ting; Iang, Shan-Bei

2017-01-01

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription. PMID:28977829

Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

Science.gov (United States)

Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs

Science.gov (United States)

Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

2015-01-01

Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and lead to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. PMID:25589350

Small RNA sequence analysis of adenovirus VA RNA-derived miRNAs reveals an unexpected serotype-specific difference in structure and abundance.

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Wael Kamel

Full Text Available Human adenoviruses (HAds encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs. We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3'-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5'-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3'-mivaRNAs with a slight variation of the position of the 5' terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5'-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

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Audry, Julien; Maestroni, Laetitia; Delagoutte, Emmanuelle; Gauthier, Tiphaine; Nakamura, Toru M; Gachet, Yannick; Saintomé, Carole; Géli, Vincent; Coulon, Stéphane

2015-07-14

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres. © 2015 The Authors.

Yield of single-strand breaks in the DNA of E. coli 10 msec after irradiation

Energy Technology Data Exchange (ETDEWEB)

Fox, R A; Fielden, E M; Sapora, O [Institute of Cancer Research, Sutton (UK). Surrey Branch

1976-04-01

The rapid mixing of 0.3M alkali with a suspension of E.coli B/r 6 +- 3 and 144 +- 3 msec after irradiation with electrons (4.3 MeV, 0 to 50 krad) has been used to make a comparison of the yields of single strand breaks in the presence and absence of oxygen. No significant difference was observed between the numbers of single strand breaks appearing at 6 and 144 msec after irradiation. Assuming that mixing with alkali inactivates the cellular repair enzymes within several milliseconds, these results indicate that enzymic repair does not operate within this time scale. It seems probable that radiation chemical processes are responsible for the initial oxygen effect on single strand breaks.

Repair of single-strand breaks in normal and trisomic lymphocytes

International Nuclear Information System (INIS)

Leonard, J.C.; Merz, T.

1982-01-01

Recently, Athanasiou and colleagues (1981) reported a deficiency in the capacity of lymphocytes from persons with Down's syndrome to repair single-strand DNA breaks. They found that 1 h after exposure to 160 Gray, repair processes had restored the sedimentation profile of DNA from normal lymphocytes to control values, whereas the relative average molecular weight of DNA from irradiated lymphocytes from persons with Down's syndrome showed no increase during the repair interval. They have suggested that their data, in conjunction with the earlier data concerning the frequencies of induced chromosomal aberrations in lymphocytes from persons with Down's syndrome, reflect a decreased efficiency in some aspect of DNA repair in trisomic cells. However, for further studies of this hypothesis, it is more appropriate to study the rejoining of DNA single-strand breaks after doses comparable to those used in tests for chromosomal aberrations. (orig.)

An Approach to Detect and Study DNA Double-Strand Break Repair by Transcript RNA Using a Spliced-Antisense RNA Template.

Science.gov (United States)

Keskin, Havva; Storici, Francesca

2018-01-01

A double-strand break (DSB) is one of the most dangerous DNA lesion, and its repair is crucial for genome stability. Homologous recombination is considered the safest way to repair a DNA DSB and requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome for accurate repair. Can transcript RNA serve as donor template for DSB repair? Here, we describe an approach that we developed to detect and study DNA repair by transcript RNA. Key features of the method are: (i) use of antisense (noncoding) RNA as template for DSB repair by RNA, (ii) use of intron splicing to distinguish the sequence of the RNA template from that of the DNA that generates the RNA template, and (iii) use of a trans and cis system to study how RNA repairs a DSB in homologous but distant DNA or in its own DNA, respectively. This chapter provides details on how to use a spliced-antisense RNA template to detect and study DSB repair by RNA in trans or cis in yeast cells. Our approach for detection of DSB repair by RNA in cells can be applied to cell types other than yeast, such as bacteria, mammalian cells, or other eukaryotic cells. © 2018 Elsevier Inc. All rights reserved.

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

Energy Technology Data Exchange (ETDEWEB)

Kim, Seongman; Chul Ahn, Byung; O' Callaghan, Dennis J. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States); Kim, Seong Kee, E-mail: skim1@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States)

2012-10-25

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

International Nuclear Information System (INIS)

Kim, Seongman; Chul Ahn, Byung; O’Callaghan, Dennis J.; Kim, Seong Kee

2012-01-01

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)–UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

Lack of a Close Relationship Between Three Strains of Human Rhinoviruses as Determined by Their RNA Sequences 1

Science.gov (United States)

Yin, Fay H.; Lonberg-Holm, K.; Chan, S. P.

1973-01-01

The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA. PMID:4126194

UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

Energy Technology Data Exchange (ETDEWEB)

Rao, K.N.; Swaminathan, S.; Burley, S. K.

2008-12-11

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

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Lu Tingting

2012-12-01

Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions

Evolution of primary and secondary structures in 5S and 5.8S rRNA

International Nuclear Information System (INIS)

Curtiss, W.C.

1986-01-01

The secondary structure of Bombyx mori 5S rRNA was studied using the sing-strand specific S1 nuclease and the base pair specific cobra venom ribonuclease. The RNA was end-labeled with [ 32 P] at either the 5' or 3' end and sequenced using enzymatic digestion techniques. These enzymatic data coupled with thermodynamic structure prediction were used to generate a secondary structure for 5S rRNA. A computer algorithm has been implemented to aid in the comparison of a large set of homologous RNAs. Eukaryotic 5S rRNA sequences from thirty four diverse species were compared by (1) alignment or the sequences, (2) the positions of substitutions were located with respect to the aligned sequence and secondary structure, and (3) the R-Y model of base stacking was used to study stacking pattern relationships in the structure. Eukaryotic 5S rRNA was found to have significant sequence variation throughout much of the molecule while maintaining a relatively constant secondary structure. A detailed analysis of the sequence and structure variability in each region of the molecule is presented

Yield of single-strand breaks in the DNA of E.coli 10 msec after irradiation

International Nuclear Information System (INIS)

Fox, R.A.; Fielden, E.M.; Sapora, O.

1976-01-01

The rapid mixing of 0.3M alkali with a suspension of E.coli B/r 6 +- 3 and 144 +- 3 msec after irradiation with electrons (4.3 MeV, 0 to 50 krad) has been used to make a comparison of the yields of single strand breaks in the presence and absence of oxygen. No significant difference was observed between the numbers of single strand breaks appearing at 6 and 144 msec after irradiation. Assuming that mixing with alkali inactivates the cellular repair enzymes within several milliseconds, these results indicate that enzymic repair does not operate within this time scale. It seems probable that radiation chemical processes are responsible for the initial oxygen effect on single strand breaks. (U.K.)

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

Energy Technology Data Exchange (ETDEWEB)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

2015-04-22

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

Repair of single-strand breaks induced in the DNA of Proteus mirabilis by excision repair after UV-irradiation

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

Single-strand breaks have been produced in the DNA of P. mirabilis after UV-irradiation in dependence on the incident UV-doses. It has been found that there exists a discrepancy between the single-strand breaks estimated from sedimentation in alkaline sucrose gradients and the expected single-strand breaks approximated from measurements of dimer excision. The low number in incision breaks observed by sedimentation experiments is an indication that the cells are able to repair the excision-induced breaks as fast as they are formed. Toluenized cells have been used for investigation of the incision step independently of subsequent repair processes. In presence of NMN the appearance of more single-strand breaks in the DNA has been observed. Furthermore, the number of incision breaks in toluenized cells increased in presence of exogenous ATP. The completion of the excision repair process has been investigated by observing the rejoining of incision breaks. After irradiation with UV-doses higher than approximately 240 erg/mm 2 the number of single-strand breaks remaining unrepaired in the DNA increased. Studies of the influence of nutrition conditions on the repair process have shown approximately the same capacity for repair of single-strand breaks in growth medium as well as in buffer. Progress in the excision repair was also followed by investigation of the DNA synthesized at the template-DNA containing the pyrimidine dimers. In comparison with E. coli, P. mirabilis showed a somewhat lower efficiency for the repair of single-strand breaks during the excision repair. (author)

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Science.gov (United States)

Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

2014-09-25

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Analysis of bacterial core communities in the central Baltic by comparative RNA-DNA-based fingerprinting provides links to structure-function relationships.

Science.gov (United States)

Brettar, Ingrid; Christen, Richard; Höfle, Manfred G

2012-01-01

Understanding structure-function links of microbial communities is a central theme of microbial ecology since its beginning. To this end, we studied the spatial variability of the bacterioplankton community structure and composition across the central Baltic Sea at four stations, which were up to 450 km apart and at a depth profile representative for the central part (Gotland Deep, 235 m). Bacterial community structure was followed by 16S ribosomal RNA (rRNA)- and 16S rRNA gene-based fingerprints using single-strand conformation polymorphism (SSCP) electrophoresis. Species composition was determined by sequence analysis of SSCP bands. High similarities of the bacterioplankton communities across several hundred kilometers were observed in the surface water using RNA- and DNA-based fingerprints. In these surface communities, the RNA- and DNA-based fingerprints resulted in very different pattern, presumably indicating large difference between the active members of the community as represented by RNA-based fingerprints and the present members represented by the DNA-based fingerprints. This large discrepancy changed gradually over depth, resulting in highly similar RNA- and DNA-based fingerprints in the anoxic part of the water column below 130 m depth. A conceivable mechanism explaining this high similarity could be the reduced oxidative stress in the anoxic zone. The stable communities on the surface and in the anoxic zone indicate the strong influence of the hydrography on the bacterioplankton community structure. Comparative analysis of RNA- and DNA-based community structure provided criteria for the identification of the core community, its key members and their links to biogeochemical functions.

Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

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Francis M. F. Nunes

2013-01-01

Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

Science.gov (United States)

Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

2013-01-04

RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

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Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

2018-04-18

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

Landscape and variation of RNA secondary structure across the human transcriptome.

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Wan, Yue; Qu, Kun; Zhang, Qiangfeng Cliff; Flynn, Ryan A; Manor, Ohad; Ouyang, Zhengqing; Zhang, Jiajing; Spitale, Robert C; Snyder, Michael P; Segal, Eran; Chang, Howard Y

2014-01-30

In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA

International Nuclear Information System (INIS)

Branch, A.D.; Benenfeld, B.J.; Robertson, H.D.

1985-01-01

The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA fingerprinting and secondary analysis. Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (i) both crosslinks occur in an identical eight-base context, with the sequence 5 GGGAA 3 on one side and the sequence 5 UAC 3 on the other; (ii) both crosslinks connect bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as single-stranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

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Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

NARCIS (Netherlands)

Vriend, Lianne E. M.; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M.

2016-01-01

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided

Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy

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Akira Kitamura

2018-07-01

Full Text Available Normal function and abnormal aggregation of transactivation response (TAR DNA/RNA-binding protein 43 kDa (TDP-43 are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS, and frontotemporal lobar degeneration (FTLD. The binding of TDP-43 to single-stranded DNA (ssDNA or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure Kd with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the Kd of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS, which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA. Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

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Pandita, Raj K; Chow, Tracy T; Udayakumar, Durga; Bain, Amanda L; Cubeddu, Liza; Hunt, Clayton R; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E; Khanna, Kum Kum; Shay, Jerry W; Pandita, Tej K

2015-03-01

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR. ©2015 American Association for Cancer Research.

Reconstruction of ancestral RNA sequences under multiple structural constraints.

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Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldispühl, Jérôme

2016-11-11

Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surface.

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Rant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2006-05-15

We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment.

Four RNA families with functional transient structures.

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Zhu, Jing Yun A; Meyer, Irmtraud M

2015-01-01

Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks.

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Pachkowski, Brian; Nakamura, Jun

2013-01-01

Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.

A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

Science.gov (United States)

Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

2012-01-01

We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

Interspecific Transmission of Double-Stranded RNA and Hypovirulence from Sclerotinia sclerotiorum to S. minor.

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Melzer, M S; Ikeda, S S; Boland, G J

2002-07-01

ABSTRACT Interspecific transmission of a hypovirulence-associated double-stranded RNA (dsRNA) and hypovirulent phenotype was attempted from hypovirulent isolate Ss275 of Sclerotinia sclerotiorum to five virulent isolates of S. minor. dsRNA and the hypovirulent phenotype were successfully transmitted to one of the five isolates, Sm10. Three putative converted isolates of Sm10 were slow growing and developed atypical colony morphologies characteristic of the hypovirulent phenotype. These isolates were assayed for virulence and produced significantly smaller lesions than isolate Sm10 on detached leaves of Romaine lettuce. One of these putative converted isolates, designated Sm10T, was tested to confirm interspecific transmission of dsRNA. In northern hybridizations, dsRNA isolated from Sm10T hybridized with a digoxigenin-labeled cDNA probe prepared from dsRNA isolated from Ss275. Random amplified polymorphic DNA analysis confirmed that isolate Sm10T was derived from Sm10 and not from Ss275 or a hybrid of the two species. The dsRNA and hypovirulent phenotype were subsequently transmitted intraspecifically from Sm10T to Sm8. To our knowledge, this is the first report of interspecific transmission of dsRNA and an associated hypovirulent phenotype between fungal plant pathogens by hyphal anastomosis.

RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures

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Wasik Szymon

2010-05-01

Full Text Available Abstract Background Recent discoveries concerning novel functions of RNA, such as RNA interference, have contributed towards the growing importance of the field. In this respect, a deeper knowledge of complex three-dimensional RNA structures is essential to understand their new biological functions. A number of bioinformatic tools have been proposed to explore two major structural databases (PDB, NDB in order to analyze various aspects of RNA tertiary structures. One of these tools is RNA FRABASE 1.0, the first web-accessible database with an engine for automatic search of 3D fragments within PDB-derived RNA structures. This search is based upon the user-defined RNA secondary structure pattern. In this paper, we present and discuss RNA FRABASE 2.0. This second version of the system represents a major extension of this tool in terms of providing new data and a wide spectrum of novel functionalities. An intuitionally operated web server platform enables very fast user-tailored search of three-dimensional RNA fragments, their multi-parameter conformational analysis and visualization. Description RNA FRABASE 2.0 has stored information on 1565 PDB-deposited RNA structures, including all NMR models. The RNA FRABASE 2.0 search engine algorithms operate on the database of the RNA sequences and the new library of RNA secondary structures, coded in the dot-bracket format extended to hold multi-stranded structures and to cover residues whose coordinates are missing in the PDB files. The library of RNA secondary structures (and their graphics is made available. A high level of efficiency of the 3D search has been achieved by introducing novel tools to formulate advanced searching patterns and to screen highly populated tertiary structure elements. RNA FRABASE 2.0 also stores data and conformational parameters in order to provide "on the spot" structural filters to explore the three-dimensional RNA structures. An instant visualization of the 3D RNA

Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

Energy Technology Data Exchange (ETDEWEB)

Cheng, A.; Wong, S; Yuan, Y

2009-01-01

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

NARCIS (Netherlands)

Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

2001-01-01

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

The first phlebo-like virus infecting plants: a case study on the adaptation of negative-stranded RNA viruses to new hosts.

Science.gov (United States)

Navarro, Beatriz; Minutolo, Maria; De Stradis, Angelo; Palmisano, Francesco; Alioto, Daniela; Di Serio, Francesco

2018-05-01

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

International Nuclear Information System (INIS)

Rubach, Jon K.; Wasik, Brian R.; Rupp, Jonathan C.; Kuhn, Richard J.; Hardy, Richard W.; Smith, Janet L.

2009-01-01

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA

Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

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Herington Adrian C

2008-10-01

Full Text Available Abstract Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS, which spans the promoter and untranslated regions of the ghrelin gene (GHRL. Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2. Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis, as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA genes, including 5' capping, polyadenylation, extensive splicing and short open reading

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

Science.gov (United States)

Awate, Sanket; Brosh, Robert M

2017-06-08

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

Crystallographic and Modeling Studies of RNase III Suggest a Mechanism for Double-Stranded RNA Cleavage | Center for Cancer Research

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Background: Ribonuclease III belongs to the family of Mg2+-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

Science.gov (United States)

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Reconstruction of ancestral RNA sequences under multiple structural constraints

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Olivier Tremblay-Savard

2016-11-01

Full Text Available Abstract Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. Results We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Conclusions Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

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Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

2013-03-01

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

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Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

2012-10-19

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

Sparse RNA folding revisited: space-efficient minimum free energy structure prediction.

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Will, Sebastian; Jabbari, Hosna

2016-01-01

RNA secondary structure prediction by energy minimization is the central computational tool for the analysis of structural non-coding RNAs and their interactions. Sparsification has been successfully applied to improve the time efficiency of various structure prediction algorithms while guaranteeing the same result; however, for many such folding problems, space efficiency is of even greater concern, particularly for long RNA sequences. So far, space-efficient sparsified RNA folding with fold reconstruction was solved only for simple base-pair-based pseudo-energy models. Here, we revisit the problem of space-efficient free energy minimization. Whereas the space-efficient minimization of the free energy has been sketched before, the reconstruction of the optimum structure has not even been discussed. We show that this reconstruction is not possible in trivial extension of the method for simple energy models. Then, we present the time- and space-efficient sparsified free energy minimization algorithm SparseMFEFold that guarantees MFE structure prediction. In particular, this novel algorithm provides efficient fold reconstruction based on dynamically garbage-collected trace arrows. The complexity of our algorithm depends on two parameters, the number of candidates Z and the number of trace arrows T; both are bounded by [Formula: see text], but are typically much smaller. The time complexity of RNA folding is reduced from [Formula: see text] to [Formula: see text]; the space complexity, from [Formula: see text] to [Formula: see text]. Our empirical results show more than 80 % space savings over RNAfold [Vienna RNA package] on the long RNAs from the RNA STRAND database (≥2500 bases). The presented technique is intentionally generalizable to complex prediction algorithms; due to their high space demands, algorithms like pseudoknot prediction and RNA-RNA-interaction prediction are expected to profit even stronger than "standard" MFE folding. SparseMFEFold is free

Engineered proteins with PUF scaffold to manipulate RNA metabolism

Science.gov (United States)

Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M.

2013-01-01

Pumilio/fem-3 mRNA binding factor (FBF) proteins are characterized by a sequence-specific RNA-binding domain. This unique single-stranded RNA recognition module, whose sequence specificity can be reprogrammed, has been fused with functional modules to engineer protein factors with various functions. Here we summarize the advancement in developing RNA regulatory tools and opportunities for the future. PMID:23731364

The Ku Heterodimer and the Metabolism of Single-Ended DNA Double-Strand Breaks

NARCIS (Netherlands)

A. Balestrini (Alessia); D. Ristic (Dejan); I. Dionne (Isabelle); X.Z. Liu (Xiao); C. Wyman (Claire); R.J. Wellinger (Raymund); J.H.J. Petrini (John)

2013-01-01

textabstractSingle-ended double-strand breaks (DSBs) are a common form of spontaneous DNA break, generated when the replisome encounters a discontinuity in the DNA template. Given their prevalence, understanding the mechanisms governing the fate(s) of single-ended DSBs is important. We describe the

Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response

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Marques, Joao T.; Rebouillat, Dominique; Ramana, Chilakamarti V.; Murakami, Junko; Hill, Jason E.; Gudkov, Andrei; Silverman, Robert H.; Stark, George R.; Williams, Bryan R. G.

2005-01-01

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication. PMID:16103161

Kinetics of end-to-end collision in short single-stranded nucleic acids.

Science.gov (United States)

Wang, Xiaojuan; Nau, Werner M

2004-01-28

A novel fluorescence-based method, which entails contact quenching of the long-lived fluorescent state of 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO), was employed to measure the kinetics of end-to-end collision in short single-stranded oligodeoxyribonucleotides of the type 5'-DBO-(X)n-dG with X = dA, dC, dT, or dU and n = 2 or 4. The fluorophore was covalently attached to the 5' end and dG was introduced as an efficient intrinsic quencher at the 3' terminus. The end-to-end collision rates, which can be directly related to the efficiency of intramolecular fluorescence quenching, ranged from 0.1 to 9.0 x 10(6) s(-1). They were strongly dependent on the strand length, the base sequence, as well as the temperature. Oligonucleotides containing dA in the backbone displayed much slower collision rates and significantly higher positive activation energies than strands composed of pyrimidine bases, suggesting a higher intrinsic rigidity of oligoadenylate. Comparison of the measured collision rates in short single-stranded oligodeoxyribonucleotides with the previously reported kinetics of hairpin formation indicates that the intramolecular collision is significantly faster than the nucleation step of hairpin closing. This is consistent with the configurational diffusion model suggested by Ansari et al. (Ansari, A.; Kuznetsov, S. V.; Shen, Y. Proc.Natl. Acad. Sci. USA 2001, 98, 7771-7776), in which the formation of misfolded loops is thought to slow hairpin formation.

Ratiometric FRET-based detection of DNA and micro-RNA in solution

International Nuclear Information System (INIS)

Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

2009-01-01

A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

International Nuclear Information System (INIS)

Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

2011-01-01

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi targets based on Gibbs free energy

Directory of Open Access Journals (Sweden)

Tiago Campos Pereira

2007-01-01

Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte.

Directory of Open Access Journals (Sweden)

Renata Bolognesi

Full Text Available RNA interference (RNAi has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte larvae via oral delivery of synthetic double-stranded RNA (dsRNA in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7 as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

Science.gov (United States)

Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

2009-01-01

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

Science.gov (United States)

Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

2011-04-06

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

A plasmonic colorimetric strategy for visual miRNA detection based on hybridization chain reaction

Science.gov (United States)

Miao, Jie; Wang, Jingsheng; Guo, Jinyang; Gao, Huiguang; Han, Kun; Jiang, Chengmin; Miao, Peng

2016-08-01

In this work, a novel colorimetric strategy for miRNA analysis is proposed based on hybridization chain reaction (HCR)-mediated localized surface plasmon resonance (LSPR) variation of silver nanoparticles (AgNPs). miRNA in the sample to be tested is able to release HCR initiator from a solid interface to AgNPs colloid system by toehold exchange-mediated strand displacement, which then triggers the consumption of fuel strands with single-stranded tails for HCR. The final produced long nicked double-stranded DNA loses the ability to protect AgNPs from salt-induced aggregation. The stability variation of the colloid system can then be monitored by recording corresponding UV-vis spectrum and initial miRNA level is thus determined. This sensing system involves only four DNA strands which is quite simple. The practical utility is confirmed to be excellent by employing different biological samples.

The nucleotide sequence of RNA1 of Lettuce big-vein virus, genus Varicosavirus, reveals its relation to nonsegmented negative-strand RNA viruses.

Science.gov (United States)

Sasaya, Takahide; Ishikawa, Koichi; Koganezawa, Hiroki

2002-06-05

The complete nucleotide sequence of RNA1 from Lettuce big-vein virus (LBVV), the type member of the genus Varicosavirus, was determined. LBVV RNA1 consists of 6797 nucleotides and contains one large ORF that encodes a large (L) protein of 2040 amino acids with a predicted M(r) of 232,092. Northern blot hybridization analysis indicated that the LBVV RNA1 is a negative-sense RNA. Database searches showed that the amino acid sequence of L protein is homologous to those of L polymerases of nonsegmented negative-strand RNA viruses. A cluster dendrogram derived from alignments of the LBVV L protein and the L polymerases indicated that the L protein is most closely related to the L polymerases of plant rhabdoviruses. Transcription termination/polyadenylation signal-like poly(U) tracts that resemble those in rhabdovirus and paramyxovirus RNAs were present upstream and downstream of the coding region. Although LBVV is related to rhabdoviruses, a key distinguishing feature is that the genome of LBVV is segmented. The results reemphasize the need to reconsider the taxonomic position of varicosaviruses.

Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop

International Nuclear Information System (INIS)

Kolonko, Nadine; Bannach, Oliver; Aschermann, Katja; Hu, Kang-Hong; Moors, Michaela; Schmitz, Michael; Steger, Gerhard; Riesner, Detlev

2006-01-01

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

International Nuclear Information System (INIS)

Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

2015-01-01

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

Energy Technology Data Exchange (ETDEWEB)

Xiao, Xiao [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Gang, Yi [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province (China); Wang, Honghong [No. 518 Hospital of Chinese People’s Liberation Army, Xi’an 710043, Shaanxi Province (China); Wang, Jiayin [The Genome Institute, Washington University in St. Louis, St. Louis, MO 63108 (United States); Zhao, Lina [Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Xu, Li, E-mail: lxuhelen@163.com [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China); Liu, Zhiguo, E-mail: liuzhiguo@fmmu.edu.cn [State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province (China)

2015-02-06

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

The Crystal Structure of the RNA-Dependent RNA Polymerase from Human Rhinovirus: A Dual Function Target for Common Cold Antiviral Therapy

Energy Technology Data Exchange (ETDEWEB)

Love, Robert A.; Maegley, Karen A.; Yu, Xiu; Ferre, RoseAnn; Lingardo, Laura K.; Diehl, Wade; Parge, Hans E.; Dragovich, Peter S.; Fuhrman, Shella A. (Pfizer)

2010-11-16

Human rhinoviruses (HRV), the predominant members of the Picornaviridae family of positive-strand RNA viruses, are the major causative agents of the common cold. Given the lack of effective treatments for rhinoviral infections, virally encoded proteins have become attractive therapeutic targets. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) denoted 3D{sup pol}, which is responsible for replicating the viral genome and for synthesizing a protein primer used in the replication. Here the crystal structures for three viral serotypes (1B, 14, and 16) of HRV 3D{sup pol} have been determined. The three structures are very similar to one another, and to the closely related poliovirus (PV) 3D{sup pol} enzyme. Because the reported PV crystal structure shows significant disorder, HRV 3D{sup pol} provides the first complete view of a picornaviral RdRp. The folding topology of HRV 3D{sup pol} also resembles that of RdRps from hepatitis C virus (HCV) and rabbit hemorrhagic disease virus (RHDV) despite very low sequence homology.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

Science.gov (United States)

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

International Nuclear Information System (INIS)

Annamalai, Padmanaban; Hsu, Y.-H.; Liu, Y.-P.; Tsai, C.-H.; Lin, N.-S.

2003-01-01

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication

Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

OpenAIRE

Wu, Tiyun; Heilman-Miller, Susan L.; Levin, Judith G.

2007-01-01

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop stru...

Effects of RNA branching on the electrostatic stabilization of viruses

NARCIS (Netherlands)

Erdemci-Tandogan, Gonca; Wagner, Jef; Schoot, Paul van der|info:eu-repo/dai/nl/102140618; Podgornik, Rudolf; Zandi, Roya

2016-01-01

Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although

Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA directed against malaria histone deacetylase

International Nuclear Information System (INIS)

Sriwilaijaroen, N.; Boonma, S.; Attasart, P.; Pothikasikorn, J.; Panyim, S.; Noonpakdee, W.

2009-01-01

Acetylation and deacetylation of histones play important roles in transcription regulation, cell cycle progression and development events. The steady state status of histone acetylation is controlled by a dynamic equilibrium between competing histone acetylase and deacetylase (HDAC). We have used long PfHDAC-1 double-stranded (ds)RNA to interfere with its cognate mRNA expression and determined the effect on malaria parasite growth and development. Chloroquine- and pyrimethamine-resistant Plasmodium falciparum K1 strain was exposed to 1-25 μg of dsRNA/ml of culture for 48 h and growth was determined by [ 3 H]-hypoxanthine incorporation and microscopic examination. Parasite culture treated with 10 μg/ml pfHDAC-1 dsRNA exhibited 47% growth inhibition when compared with either untreated control or culture treated with an unrelated dsRNA. PfHDAC-1 dsRNA specifically blocked maturation of trophozoite to schizont stages and decreased PfHDAC-1 transcript 44% in treated trophozoites. These results indicate the potential of HDAC-1 as a target for development of novel antimalarials.

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis.

Science.gov (United States)

Glavac, Damjan; Potocnik, Uros; Podpecnik, Darja; Zizek, Teofil; Smerkolj, Sava; Ravnik-Glavac, Metka

2002-04-01

We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments. Copyright 2002 Wiley-Liss, Inc.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

Science.gov (United States)

Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

2013-03-15

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

RNA binding specificity of Ebola virus transcription factor VP30.

Science.gov (United States)

Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

2016-09-01

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

Solubilization of Single-walled Carbon Nanotubes with Single- stranded DNA Generated from Asymmetric PCR

Directory of Open Access Journals (Sweden)

Chunhai Fan

2007-07-01

Full Text Available Carbon nanotubes (CNTs can be effectively dispersed and functionalized bywrapping with long single-stranded DNA (ssDNA synthesized by asymmetric PCR. ThessDNA-CNTs attached on surface of glass carbon electrode made it possible forelectrochemical analysis and sensing, which was demonstrated by reduction of H2O2 onhemoglobin/ssDNA-CNTs modified electrodes. This research showed the potentialapplication of DNA-functionalised CNTs in construction of future electrochemicalbiosensors.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

Science.gov (United States)

Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

2017-03-01

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

Science.gov (United States)

Luo, Qingying; Liu, Lin; Yang, Cai; Yuan, Jing; Feng, Hongtao; Chen, Yan; Zhao, Peng; Yu, Zhiqiang; Jin, Zongwen

2018-03-01

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

Dissociation of single-strand DNA: single-walled carbon nanotube hybrids by Watson-Crick base-pairing.

Science.gov (United States)

Jung, Seungwon; Cha, Misun; Park, Jiyong; Jeong, Namjo; Kim, Gunn; Park, Changwon; Ihm, Jisoon; Lee, Junghoon

2010-08-18

It has been known that single-strand DNA wraps around a single-walled carbon nanotube (SWNT) by pi-stacking. In this paper it is demonstrated that such DNA is dissociated from the SWNT by Watson-Crick base-pairing with a complementary sequence. Measurement of field effect transistor characteristics indicates a shift of the electrical properties as a result of this "unwrapping" event. We further confirm the suggested process through Raman spectroscopy and gel electrophoresis. Experimental results are verified in view of atomistic mechanisms with molecular dynamics simulations and binding energy analyses.

Different responses to muon implantation in single- and double-stranded DNA

International Nuclear Information System (INIS)

Hubbard, Penny L.; Tani, Akiko; Oganesyan, Vasily S.; Butt, Julea N.; Cottrell, Stephen P.; Jayasooriya, Upali A.

2006-01-01

A model-free analysis of the longitudinal muon spin relaxation of muons implanted into single- and double-stranded DNA samples is reported. These samples show distinctly different responses to implanted muons with discontinuities of the integrated asymmetries at temperatures where these molecules are likely to have onset of molecular and electron dynamics

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs.

Science.gov (United States)

Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

2014-06-01

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

MatureBayes: a probabilistic algorithm for identifying the mature miRNA within novel precursors.

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Katerina Gkirtzou

Full Text Available BACKGROUND: MicroRNAs (miRNAs are small, single stranded RNAs with a key role in post-transcriptional regulation of thousands of genes across numerous species. While several computational methods are currently available for identifying miRNA genes, accurate prediction of the mature miRNA remains a challenge. Existing approaches fall short in predicting the location of mature miRNAs but also in finding the functional strand(s of miRNA precursors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a computational tool that incorporates a Naive Bayes classifier to identify mature miRNA candidates based on sequence and secondary structure information of their miRNA precursors. We take into account both positive (true mature miRNAs and negative (same-size non-mature miRNA sequences examples to optimize sensitivity as well as specificity. Our method can accurately predict the start position of experimentally verified mature miRNAs for both human and mouse, achieving a significantly larger (often double performance accuracy compared with two existing methods. Moreover, the method exhibits a very high generalization performance on miRNAs from two other organisms. More importantly, our method provides direct evidence about the features of miRNA precursors which may determine the location of the mature miRNA. We find that the triplet of positions 7, 8 and 9 from the mature miRNA end towards the closest hairpin have the largest discriminatory power, are relatively conserved in terms of sequence composition (mostly contain a Uracil and are located within or in very close proximity to the hairpin loop, suggesting the existence of a possible recognition site for Dicer and associated proteins. CONCLUSIONS: This work describes a novel algorithm for identifying the start position of mature miRNA(s produced by miRNA precursors. Our tool has significantly better (often double performance than two existing approaches and provides new insights about the potential use

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

2011-01-24

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Changes in secondary structure of poliovirus ribonucleic acid

International Nuclear Information System (INIS)

Koza, J.

1975-01-01

Infectious single-stranded RNA isolated from mature purified poliovirus was separated into three fractions by means of chromatography on an ''evaporated'' calcium phosphate column. RNA molecules with a higher degree of secondary structure were detected in two of the fractions as a result of the chromatography. These RNA molecules (1) were resistant to hydrolysis by pancreatic ribonuclease A, (2) retained unchanged the original infectivity for actinomycin D-pretreated cells, (3) were resistant to ultraviolet-light inactivation and (4) were partially resistant to formaldehyde inactivation

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

Science.gov (United States)

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stochastic sampling of the RNA structural alignment space.

Science.gov (United States)

Harmanci, Arif Ozgun; Sharma, Gaurav; Mathews, David H

2009-07-01

A novel method is presented for predicting the common secondary structures and alignment of two homologous RNA sequences by sampling the 'structural alignment' space, i.e. the joint space of their alignments and common secondary structures. The structural alignment space is sampled according to a pseudo-Boltzmann distribution based on a pseudo-free energy change that combines base pairing probabilities from a thermodynamic model and alignment probabilities from a hidden Markov model. By virtue of the implicit comparative analysis between the two sequences, the method offers an improvement over single sequence sampling of the Boltzmann ensemble. A cluster analysis shows that the samples obtained from joint sampling of the structural alignment space cluster more closely than samples generated by the single sequence method. On average, the representative (centroid) structure and alignment of the most populated cluster in the sample of structures and alignments generated by joint sampling are more accurate than single sequence sampling and alignment based on sequence alone, respectively. The 'best' centroid structure that is closest to the known structure among all the centroids is, on average, more accurate than structure predictions of other methods. Additionally, cluster analysis identifies, on average, a few clusters, whose centroids can be presented as alternative candidates. The source code for the proposed method can be downloaded at http://rna.urmc.rochester.edu.

RNA Interference and its therapeutic applications

Directory of Open Access Journals (Sweden)

Srinivasa Rao T

2011-10-01

Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

Sequence-based separation of single-stranded DNA using nucleotides in capillary electrophoresis: focus on phosphate.

Science.gov (United States)

Zhang, Xueru; McGown, Linda B

2013-06-01

DNA analysis has widespread applicability in biology, medicine, biotechnology, and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5'-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here, we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whether the mechanism involves specific interactions between the phosphates and the DNA strands or is a result of other properties of the separation medium. Phosphate generally has been avoided in DNA separations by capillary gel electrophoresis because its high ionic strength exacerbates Joule heating. Our results suggest that phosphate compounds should be examined for separation of DNA based on sequence. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single slit interference made easy with a strand of hair and a laser

Science.gov (United States)

Messer, Rebecca

2018-01-01

Students can easily measure the width of a strand of their own hair with a monochromatic light source such as a laser. This inexpensive activity engages students in an application of single slit diffraction using Babinet's principle.

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity

NARCIS (Netherlands)

Buck, H. M.; Koole, L. H.; van Genderen, M. H.; Smit, L.; Geelen, J. L.; Jurriaans, S.; Goudsmit, J.

1990-01-01

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human

A new wine Torulaspora delbrueckii killer strain with broad antifungal activity and its toxin-encoding double-stranded RNA virus

Science.gov (United States)

Ramírez, Manuel; Velázquez, Rocío; Maqueda, Matilde; López-Piñeiro, Antonio; Ribas, Juan C.

2015-01-01

Wine Torulaspora delbrueckii strains producing a new killer toxin (Kbarr-1) were isolated and selected for wine making. They killed all the previously known Saccharomyces cerevisiae killer strains, in addition to other non-Saccharomyces yeasts. The Kbarr-1 phenotype is encoded by a medium-size 1.7 kb dsRNA, TdV-Mbarr-1, which seems to depend on a large-size 4.6 kb dsRNA virus (TdV-LAbarr) for stable maintenance and replication. The TdV-Mbarr-1 dsRNA was sequenced by new generation sequencing techniques. Its genome structure is similar to those of S. cerevisiae killer M dsRNAs, with a 5′-end coding region followed by an internal A-rich sequence and a 3′-end non-coding region. Mbarr-1 RNA positive strand carries cis acting signals at its 5′ and 3′ termini for transcription and replication respectively, similar to those RNAs of yeast killer viruses. The ORF at the 5′ region codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No relevant sequence identity was found either between the full sequence of Mbarr-1 dsRNA and other yeast M dsRNAs, or between their respective toxin-encoded proteins. However, a relevant identity of TdV-Mbarr-1 RNA regions to the putative replication and packaging signals of most of the M-virus RNAs suggests that they are all evolutionarily related. PMID:26441913

Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA.

Directory of Open Access Journals (Sweden)

Audrey Dieudonné

Full Text Available Scavenger receptors and Toll-like receptors (TLRs cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (dsRNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

Science.gov (United States)

Yu, Xiong; Egelman, Edward H.

2010-01-01

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

Radiation-induced DNA single-strand scission and its rejoining in spermatogonia and spermatozoa of mouse

International Nuclear Information System (INIS)

Ono, T.; Okada, S.

1977-01-01

Gamma-ray-induced DNA single-strand scissions and the ability to repair the scissions in spermatogonia from young mice and in spermatozoa from adult mice were studied quantitatively by an alkaline sucrose density-gradient centrifugation method. The average size of DNAs in non-irradiated spermatogonia was 2.6-3.0xx10 8 daltons, similar to those of a spermatid-rich population, and the size of DNA in non-irradiated spermatozoa was 1.2x10 8 daltons. In spermatogonia, the radiosensitivity of DNA was 0.42 single-strand breaks/10 12 daltons of DNA/rad in oxic conditions and only 0.24 under anoxic conditions. In spermatozoa the break efficiency of DNA was 0.22 single-strand breaks/10 12 daltons of DNA/rad under oxic conditions and altered little under anoxic irradiation. The DNA scissions were efficiently repaired in spermatogonia within 10 min, whereas the breaks in spermatozoa were not rejoined at all even after two days of post-irradiation time. The radiosensitivities of DNA, repair capability and non- and/or slowreparable DNA scissions were compared in spermatogonium-rich, spermatid-rich and spermatozoanrich populations

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

International Nuclear Information System (INIS)

Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

1987-01-01

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

Pleolipoviridae, a newly proposed family comprising archaeal pleomorphic viruses with single-stranded or double-stranded DNA genomes.

Science.gov (United States)

Pietilä, Maija K; Roine, Elina; Sencilo, Ana; Bamford, Dennis H; Oksanen, Hanna M

2016-01-01

Viruses infecting archaea show a variety of virion morphotypes, and they are currently classified into more than ten viral families or corresponding groups. A pleomorphic virus morphotype is very common among haloarchaeal viruses, and to date, several such viruses have been isolated. Here, we propose the classification of eight such viruses and formation of a new family, Pleolipoviridae (from the Greek pleo for more or many and lipos for lipid), containing three genera, Alpha-, Beta-, and Gammapleolipovirus. The proposal is currently under review by the International Committee on Taxonomy of Viruses (ICTV). The members of the proposed family Pleolipoviridae infect halophilic archaea and are nonlytic. They share structural and genomic features and differ from any other classified virus. The virion of pleolipoviruses is composed of a pleomorphic membrane vesicle enclosing the genome. All pleolipoviruses have two major structural protein species, internal membrane and spike proteins. Although the genomes of the pleolipoviruses are single- or double-stranded, linear or circular DNA molecules, they share the same genome organization and gene synteny and show significant similarity at the amino acid level. The canonical features common to all members of the proposed family Pleolipoviridae show that they are closely related and thus form a new viral family.

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

DEFF Research Database (Denmark)

Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Rapid NMR screening of RNA secondary structure and binding

International Nuclear Information System (INIS)

Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald

2015-01-01

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA

Rapid NMR screening of RNA secondary structure and binding

Energy Technology Data Exchange (ETDEWEB)

Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de [Johann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)

2015-09-15

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-l-homocysteine

International Nuclear Information System (INIS)

Pioszak, Augen A.; Murayama, Kazutaka; Nakagawa, Noriko; Ebihara, Akio; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

2005-01-01

Three structures of a putative RNA 5-methyluridine methyltransferase from T. thermophilus, including its complex with S-adenosyl-l-homocysteine, are presented. The structures reveal the mode of cofactor binding, architecture of the putative active site, and the presence of a deep cleft adjacent to the active site that may bind RNA. The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-l-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

RNA signal amplifier circuit with integrated fluorescence output.

Science.gov (United States)

Akter, Farhima; Yokobayashi, Yohei

2015-05-15

We designed an in vitro signal amplification circuit that takes a short RNA input that catalytically activates the Spinach RNA aptamer to produce a fluorescent output. The circuit consists of three RNA strands: an internally blocked Spinach aptamer, a fuel strand, and an input strand (catalyst), as well as the Spinach aptamer ligand 3,5-difluoro-4-hydroxylbenzylidene imidazolinone (DFHBI). The input strand initially displaces the internal inhibitory strand to activate the fluorescent aptamer while exposing a toehold to which the fuel strand can bind to further displace and recycle the input strand. Under a favorable condition, one input strand was able to activate up to five molecules of the internally blocked Spinach aptamer in 185 min at 30 °C. The simple RNA circuit reported here serves as a model for catalytic activation of arbitrary RNA effectors by chemical triggers.

Structure of the EMMPRIN N-terminal domain 1: Dimerization via [beta]-strand swapping

Energy Technology Data Exchange (ETDEWEB)

Luo, Jinquan; Teplyakov, Alexey; Obmolova, Galina; Malia, Thomas; Wu, Sheng-Jiun; Beil, Eric; Baker, Audrey; Swencki-Underwood, Bethany; Zhao, Yonghong; Sprenkle, Justin; Dixon, Ken; Sweet, Raymond; Gilliland, Gary L.; (Centocor)

2010-09-27

ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

Science.gov (United States)

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.