Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells
Identifieur interne : 001554 ( Main/Exploration ); précédent : 001553; suivant : 001555Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells
Auteurs : Mariola Dutkiewicz [Pologne] ; Agata Ojdowska [Pologne] ; Jakub Kuczynski [Pologne] ; Vanessa Lindig [Allemagne] ; Heinz Zeichhardt [Allemagne] ; Jens Kurreck [Allemagne] ; Jerzy Ciesiołka [Pologne]Source :
- PLoS ONE [ 1932-6203 ] ; 2015.
Descripteurs français
- KwdFr :
- ARN viral (génétique), Antiviraux (pharmacologie), Cellules HeLa, Conformation d'acide nucléique, Données de séquences moléculaires, Entérovirus humain B (), Entérovirus humain B (génétique), Extinction de l'expression des gènes, Humains, Infections à entérovirus (génétique), Infections à entérovirus (virologie), Oligonucléotides antisens (pharmacologie), Petit ARN interférent (pharmacologie), Ribonuclease H (génétique), Régions 5' non traduites (génétique), Séquence nucléotidique.
- MESH :
- génétique : ARN viral, Entérovirus humain B, Infections à entérovirus, Ribonuclease H, Régions 5' non traduites.
- pharmacologie : Antiviraux, Oligonucléotides antisens, Petit ARN interférent.
- virologie : Infections à entérovirus.
- Cellules HeLa, Conformation d'acide nucléique, Données de séquences moléculaires, Entérovirus humain B, Extinction de l'expression des gènes, Humains, Séquence nucléotidique.
English descriptors
- KwdEn :
- 5' Untranslated Regions (genetics), Antiviral Agents (pharmacology), Base Sequence, Enterovirus B, Human (drug effects), Enterovirus B, Human (genetics), Enterovirus Infections (genetics), Enterovirus Infections (virology), Gene Silencing, HeLa Cells, Humans, Molecular Sequence Data, Nucleic Acid Conformation, Oligonucleotides, Antisense (pharmacology), RNA, Small Interfering (pharmacology), RNA, Viral (genetics), Ribonuclease H (genetics).
- MESH :
- chemical , genetics : 5' Untranslated Regions, RNA, Viral, Ribonuclease H.
- chemical , pharmacology : Antiviral Agents, Oligonucleotides, Antisense, RNA, Small Interfering.
- drug effects : Enterovirus B, Human.
- genetics : Enterovirus B, Human, Enterovirus Infections.
- virology : Enterovirus Infections.
- Base Sequence, Gene Silencing, HeLa Cells, Humans, Molecular Sequence Data, Nucleic Acid Conformation.
Abstract
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage
Url:
DOI: 10.1371/journal.pone.0136395
PubMed: 26308932
PubMed Central: 4556297
Affiliations:
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Jerzy" sort="Ciesiolka, Jerzy" uniqKey="Ciesiolka J" first="Jerzy" last="Ciesiołka">Jerzy Ciesiołka</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland</addr-line></nlm:aff><country xml:lang="fr">Pologne</country><wicri:regionArea>Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan</wicri:regionArea><wicri:noRegion>Poznan</wicri:noRegion></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>5' Untranslated Regions (genetics)</term><term>Antiviral Agents (pharmacology)</term><term>Base Sequence</term><term>Enterovirus B, Human (drug effects)</term><term>Enterovirus B, Human (genetics)</term><term>Enterovirus Infections (genetics)</term><term>Enterovirus Infections (virology)</term><term>Gene Silencing</term><term>HeLa Cells</term><term>Humans</term><term>Molecular Sequence Data</term><term>Nucleic Acid Conformation</term><term>Oligonucleotides, Antisense (pharmacology)</term><term>RNA, Small Interfering (pharmacology)</term><term>RNA, Viral (genetics)</term><term>Ribonuclease H (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (génétique)</term><term>Antiviraux (pharmacologie)</term><term>Cellules HeLa</term><term>Conformation d'acide nucléique</term><term>Données de séquences moléculaires</term><term>Entérovirus humain B ()</term><term>Entérovirus humain B (génétique)</term><term>Extinction de l'expression des gènes</term><term>Humains</term><term>Infections à entérovirus (génétique)</term><term>Infections à entérovirus (virologie)</term><term>Oligonucléotides antisens (pharmacologie)</term><term>Petit ARN interférent 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xml:lang="en"><p>RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage <italic>in vitro</italic>. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Lares, Mr" uniqKey="Lares M">MR Lares</name></author><author><name sortKey="Rossi, Jj" uniqKey="Rossi J">JJ Rossi</name></author><author><name sortKey="Ouellet, Dl" uniqKey="Ouellet D">DL Ouellet</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Watts, Jk" uniqKey="Watts J">JK Watts</name></author><author><name sortKey="Corey, Dr" uniqKey="Corey D">DR Corey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, Mm" uniqKey="Fan M">MM Fan</name></author><author><name sortKey="Zhang, Yj" uniqKey="Zhang Y">YJ Zhang</name></author><author><name sortKey="Huang, Zq" uniqKey="Huang Z">ZQ Huang</name></author><author><name sortKey="Liu, J" uniqKey="Liu J">J Liu</name></author><author><name sortKey="Guo, Xm" uniqKey="Guo X">XM Guo</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Jw" uniqKey="Chen J">JW Chen</name></author><author><name sortKey="Zhang, Wb" uniqKey="Zhang W">WB Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Obernosterer, G" uniqKey="Obernosterer G">G Obernosterer</name></author><author><name sortKey="Tafer, H" uniqKey="Tafer H">H Tafer</name></author><author><name sortKey="Martinez, J" uniqKey="Martinez J">J Martinez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tafer, H" uniqKey="Tafer H">H Tafer</name></author><author><name sortKey="Ameres, Sl" uniqKey="Ameres S">SL Ameres</name></author><author><name sortKey="Obernosterer, G" uniqKey="Obernosterer G">G Obernosterer</name></author><author><name sortKey="Gebeshuber, Ca" uniqKey="Gebeshuber C">CA Gebeshuber</name></author><author><name sortKey="Schroeder, R" uniqKey="Schroeder R">R Schroeder</name></author><author><name sortKey="Martinez, J" uniqKey="Martinez J">J Martinez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Westerhout, Em" uniqKey="Westerhout E">EM Westerhout</name></author><author><name sortKey="Berkhout, B" uniqKey="Berkhout B">B Berkhout</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schubert, S" uniqKey="Schubert S">S Schubert</name></author><author><name sortKey="Grunweller, A" uniqKey="Grunweller A">A Grunweller</name></author><author><name sortKey="Erdmann, Va" uniqKey="Erdmann V">VA Erdmann</name></author><author><name sortKey="Kurreck, J" uniqKey="Kurreck J">J Kurreck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Krueger, U" uniqKey="Krueger U">U Krueger</name></author><author><name sortKey="Bergauer, T" uniqKey="Bergauer T">T Bergauer</name></author><author><name sortKey="Kaufmann, B" uniqKey="Kaufmann B">B Kaufmann</name></author><author><name sortKey="Wolter, I" uniqKey="Wolter I">I Wolter</name></author><author><name sortKey="Pilk, S" uniqKey="Pilk S">S Pilk</name></author><author><name sortKey="Heider Fabian, M" uniqKey="Heider Fabian M">M Heider-Fabian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dutkiewicz, M" uniqKey="Dutkiewicz M">M Dutkiewicz</name></author><author><name sortKey="Grunert, Hp" uniqKey="Grunert H">HP Grunert</name></author><author><name sortKey="Zeichhardt, H" uniqKey="Zeichhardt H">H Zeichhardt</name></author><author><name sortKey="Lena, Sw" uniqKey="Lena S">SW Lena</name></author><author><name sortKey="Wengel, J" uniqKey="Wengel J">J Wengel</name></author><author><name sortKey="Kurreck, J" uniqKey="Kurreck J">J Kurreck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brown, Km" uniqKey="Brown K">KM Brown</name></author><author><name sortKey="Chu, Cy" uniqKey="Chu C">CY Chu</name></author><author><name sortKey="Rana, Tm" uniqKey="Rana T">TM Rana</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Racaniello, Vr" uniqKey="Racaniello V">VR Racaniello</name></author><author><name sortKey="Knipe, H P" uniqKey="Knipe H">H.P. 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sort="Ojdowska, Agata" uniqKey="Ojdowska A" first="Agata" last="Ojdowska">Agata Ojdowska</name></country><country name="Allemagne"><region name="Berlin"><name sortKey="Lindig, Vanessa" sort="Lindig, Vanessa" uniqKey="Lindig V" first="Vanessa" last="Lindig">Vanessa Lindig</name></region><name sortKey="Kurreck, Jens" sort="Kurreck, Jens" uniqKey="Kurreck J" first="Jens" last="Kurreck">Jens Kurreck</name><name sortKey="Zeichhardt, Heinz" sort="Zeichhardt, Heinz" uniqKey="Zeichhardt H" first="Heinz" last="Zeichhardt">Heinz Zeichhardt</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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