An efficient RNA‐cleaving DNA enzyme can specifically target the 5′‐untranslated region of severe acute respiratory syndrome associated coronavirus (SARS‐CoV)
Identifieur interne : 003A66 ( Main/Exploration ); précédent : 003A65; suivant : 003A67An efficient RNA‐cleaving DNA enzyme can specifically target the 5′‐untranslated region of severe acute respiratory syndrome associated coronavirus (SARS‐CoV)
Auteurs : Shuwen Wu [République populaire de Chine] ; Junqiang Xu [République populaire de Chine] ; Jiangxia Liu [République populaire de Chine] ; Xuan Yan [République populaire de Chine] ; Xiaodong Zhu [République populaire de Chine] ; Gengfu Xiao [République populaire de Chine] ; Lunquan Sun [Australie, République populaire de Chine] ; Po Tien [République populaire de Chine]Source :
- The Journal of Gene Medicine [ 1099-498X ] ; 2007-12.
Descripteurs français
- KwdFr :
- MESH :
- génétique : Virus du SRAS.
- métabolisme : ADN, ADN catalytique, ARN.
- Amorces ADN, Animaux, Cellules Vero, Réaction de polymérisation en chaîne, Régions 5' non traduites, Séquence nucléotidique.
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : DNA, DNA, Catalytic, RNA.
- genetics : SARS Virus.
- Teeft :
- 5' Untranslated Regions, Animals, Base Sequence, Catalytic, Catalytic core, Catalytic motif, Cells copyright, Chlorocebus aethiops, Cleavage, Cleavage activity, Cleavage products, Cleavage reaction, Cleavage reactions, Cleavage site, Copyright, Coronavirus, DNA Primers, Dnazyme, Dnazyme activity, Dnazymes, Efficient dnazyme, Egfp, Egfp expression, Egfp gene, Febs lett, Gene, Gene expression, Genome, Independent experiments, John wiley sons, Life sciences, Loop region, Lower panel, Mammalian cells, Model system, Modern virology research center, Mrna, Mrna expression, Mutant dnazyme, Novel coronavirus, Nuclease degradation, Parental vector, Physiological conditions, Polymerase Chain Reaction, Respiratory syndrome, Sars, Sars genome, Transcription, Transfected, Turnover, Untranslated region, Uorescence microscopy, Uorescent protein, Various concentrations, Vero, Vero Cells, Vero cells, Wuhan university.
Abstract
Background: The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS‐CoV. We report the use of DNAzyme (catalytic DNA) to target the 5′‐untranslated region (5′UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS‐CoV replication. A mono‐DNA enzyme (Dz‐104) possessing the 10–23 catalytic motif was synthesized and tested both in vitro and in cell culture. Materials and methods: SARS‐CoV total RNA was isolated, extracted from the SARS‐CoV‐WHU strain and converted into cDNA. We designed a RNA‐cleaving 10–23 DNAzyme targeting at the loop region of the 5′UTR of SARS‐CoV. The designed DNAzyme, Dz‐104, and its mutant version, Dz‐104 (mut), as a control consist of 9 + 9 arm sequences with a 10–23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5′UTR RNA substrate. A vector containing a fused 5′UTR and enhanced green fluorescent protein (eGFP) was co‐transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence‐activated cell sorting (FACS). Results and conclusions: Our results demonstrated that this DNAzyme could efficiently cleave the SARS‐CoV RNA substrate in vitro and inhibit the expression of the SARS‐CoV 5′UTR‐eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection. Copyright © 2007 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.1111
Affiliations:
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Chine</country><wicri:regionArea>Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072</wicri:regionArea><placeName><settlement type="city">Wuhan</settlement><region type="région">Hubei</region></placeName><orgName type="university">Université de Wuhan</orgName><placeName><settlement type="city">Wuhan</settlement><region type="province">Hubei</region></placeName></affiliation></author><author><name sortKey="Liu, Jiangxia" sort="Liu, Jiangxia" uniqKey="Liu J" first="Jiangxia" last="Liu">Jiangxia Liu</name><affiliation wicri:level="4"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072</wicri:regionArea><placeName><settlement type="city">Wuhan</settlement><region type="région">Hubei</region></placeName><orgName type="university">Université de Wuhan</orgName><placeName><settlement type="city">Wuhan</settlement><region type="province">Hubei</region></placeName></affiliation></author><author><name sortKey="Yan, Xuan" sort="Yan, Xuan" uniqKey="Yan X" first="Xuan" last="Yan">Xuan Yan</name><affiliation wicri:level="4"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072</wicri:regionArea><placeName><settlement type="city">Wuhan</settlement><region type="région">Hubei</region></placeName><orgName type="university">Université de Wuhan</orgName><placeName><settlement type="city">Wuhan</settlement><region type="province">Hubei</region></placeName></affiliation></author><author><name sortKey="Zhu, Xiaodong" sort="Zhu, Xiaodong" uniqKey="Zhu X" first="Xiaodong" last="Zhu">Xiaodong Zhu</name><affiliation wicri:level="1"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Xiao, Gengfu" sort="Xiao, Gengfu" uniqKey="Xiao G" first="Gengfu" last="Xiao">Gengfu Xiao</name><affiliation wicri:level="4"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072</wicri:regionArea><placeName><settlement type="city">Wuhan</settlement><region type="région">Hubei</region></placeName><orgName type="university">Université de Wuhan</orgName><placeName><settlement type="city">Wuhan</settlement><region type="province">Hubei</region></placeName></affiliation></author><author><name sortKey="Sun, Lunquan" sort="Sun, Lunquan" uniqKey="Sun L" first="Lunquan" last="Sun">Lunquan Sun</name><affiliation wicri:level="1"><country xml:lang="fr">Australie</country><wicri:regionArea>Faculty of Medicine, University of New South Wales, Sydney, 2052</wicri:regionArea><wicri:noRegion>2052</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Australie</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Lunquan Sun, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.Po Tien, Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University; Wuhan 430072</wicri:regionArea><wicri:noRegion>Wuhan University; Wuhan 430072</wicri:noRegion></affiliation></author><author><name sortKey="Tien, Po" sort="Tien, Po" uniqKey="Tien P" first="Po" last="Tien">Po Tien</name><affiliation wicri:level="4"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072</wicri:regionArea><placeName><settlement type="city">Wuhan</settlement><region type="région">Hubei</region></placeName><orgName type="university">Université de Wuhan</orgName><placeName><settlement type="city">Wuhan</settlement><region type="province">Hubei</region></placeName></affiliation><affiliation wicri:level="1"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation><affiliation wicri:level="1"><country wicri:rule="url">République populaire de Chine</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Lunquan Sun, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.Po Tien, Modern Virology Research Center, State Key Laboratory of Virology, College of Life Sciences, Wuhan University; Wuhan 430072</wicri:regionArea><wicri:noRegion>Wuhan University; Wuhan 430072</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="ISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><imprint><biblScope unit="vol">9</biblScope><biblScope unit="issue">12</biblScope><biblScope unit="page" from="1080">1080</biblScope><biblScope unit="page" to="1086">1086</biblScope><biblScope unit="page-count">7</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2007-12">2007-12</date></imprint><idno type="ISSN">1099-498X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1099-498X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>5' Untranslated Regions</term><term>Animals</term><term>Base Sequence</term><term>Chlorocebus aethiops</term><term>DNA (metabolism)</term><term>DNA Primers</term><term>DNA, Catalytic (metabolism)</term><term>Polymerase Chain Reaction</term><term>RNA (metabolism)</term><term>SARS Virus (genetics)</term><term>Vero Cells</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN (métabolisme)</term><term>ADN catalytique (métabolisme)</term><term>ARN (métabolisme)</term><term>Amorces ADN</term><term>Animaux</term><term>Cellules Vero</term><term>Réaction de polymérisation en chaîne</term><term>Régions 5' non traduites</term><term>Séquence nucléotidique</term><term>Virus du SRAS (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA</term><term>DNA, Catalytic</term><term>RNA</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN</term><term>ADN catalytique</term><term>ARN</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>5' Untranslated Regions</term><term>Animals</term><term>Base Sequence</term><term>Catalytic</term><term>Catalytic core</term><term>Catalytic motif</term><term>Cells copyright</term><term>Chlorocebus aethiops</term><term>Cleavage</term><term>Cleavage activity</term><term>Cleavage products</term><term>Cleavage reaction</term><term>Cleavage reactions</term><term>Cleavage site</term><term>Copyright</term><term>Coronavirus</term><term>DNA Primers</term><term>Dnazyme</term><term>Dnazyme activity</term><term>Dnazymes</term><term>Efficient dnazyme</term><term>Egfp</term><term>Egfp expression</term><term>Egfp gene</term><term>Febs lett</term><term>Gene</term><term>Gene expression</term><term>Genome</term><term>Independent experiments</term><term>John wiley sons</term><term>Life sciences</term><term>Loop region</term><term>Lower panel</term><term>Mammalian cells</term><term>Model system</term><term>Modern virology research center</term><term>Mrna</term><term>Mrna expression</term><term>Mutant dnazyme</term><term>Novel coronavirus</term><term>Nuclease degradation</term><term>Parental vector</term><term>Physiological conditions</term><term>Polymerase Chain Reaction</term><term>Respiratory syndrome</term><term>Sars</term><term>Sars genome</term><term>Transcription</term><term>Transfected</term><term>Turnover</term><term>Untranslated region</term><term>Uorescence microscopy</term><term>Uorescent protein</term><term>Various concentrations</term><term>Vero</term><term>Vero Cells</term><term>Vero cells</term><term>Wuhan university</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Amorces ADN</term><term>Animaux</term><term>Cellules Vero</term><term>Réaction de polymérisation en chaîne</term><term>Régions 5' non traduites</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Background: The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS‐CoV. We report the use of DNAzyme (catalytic DNA) to target the 5′‐untranslated region (5′UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS‐CoV replication. A mono‐DNA enzyme (Dz‐104) possessing the 10–23 catalytic motif was synthesized and tested both in vitro and in cell culture. Materials and methods: SARS‐CoV total RNA was isolated, extracted from the SARS‐CoV‐WHU strain and converted into cDNA. We designed a RNA‐cleaving 10–23 DNAzyme targeting at the loop region of the 5′UTR of SARS‐CoV. The designed DNAzyme, Dz‐104, and its mutant version, Dz‐104 (mut), as a control consist of 9 + 9 arm sequences with a 10–23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5′UTR RNA substrate. A vector containing a fused 5′UTR and enhanced green fluorescent protein (eGFP) was co‐transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence‐activated cell sorting (FACS). Results and conclusions: Our results demonstrated that this DNAzyme could efficiently cleave the SARS‐CoV RNA substrate in vitro and inhibit the expression of the SARS‐CoV 5′UTR‐eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection. Copyright © 2007 John Wiley & Sons, Ltd.</div></front></TEI><affiliations><list><country><li>Australie</li><li>République populaire de Chine</li></country><region><li>Hubei</li></region><settlement><li>Pékin</li><li>Wuhan</li></settlement><orgName><li>Université de Wuhan</li></orgName></list><tree><country name="République populaire de Chine"><region name="Hubei"><name sortKey="Wu, Shuwen" sort="Wu, Shuwen" uniqKey="Wu S" first="Shuwen" last="Wu">Shuwen Wu</name></region><name sortKey="Liu, Jiangxia" sort="Liu, Jiangxia" uniqKey="Liu J" first="Jiangxia" last="Liu">Jiangxia Liu</name><name sortKey="Sun, Lunquan" sort="Sun, Lunquan" uniqKey="Sun L" first="Lunquan" last="Sun">Lunquan Sun</name><name sortKey="Tien, Po" sort="Tien, Po" uniqKey="Tien P" first="Po" last="Tien">Po Tien</name><name sortKey="Tien, Po" sort="Tien, Po" uniqKey="Tien P" first="Po" last="Tien">Po Tien</name><name sortKey="Tien, Po" sort="Tien, Po" uniqKey="Tien P" first="Po" last="Tien">Po Tien</name><name sortKey="Tien, Po" sort="Tien, Po" uniqKey="Tien P" first="Po" last="Tien">Po Tien</name><name sortKey="Xiao, Gengfu" sort="Xiao, Gengfu" uniqKey="Xiao G" first="Gengfu" last="Xiao">Gengfu Xiao</name><name sortKey="Xu, Junqiang" sort="Xu, Junqiang" uniqKey="Xu J" first="Junqiang" last="Xu">Junqiang Xu</name><name sortKey="Yan, Xuan" sort="Yan, Xuan" uniqKey="Yan X" first="Xuan" last="Yan">Xuan Yan</name><name sortKey="Zhu, Xiaodong" sort="Zhu, Xiaodong" uniqKey="Zhu X" first="Xiaodong" last="Zhu">Xiaodong Zhu</name></country><country name="Australie"><noRegion><name sortKey="Sun, Lunquan" sort="Sun, Lunquan" uniqKey="Sun L" first="Lunquan" last="Sun">Lunquan Sun</name></noRegion><name sortKey="Sun, Lunquan" sort="Sun, Lunquan" uniqKey="Sun L" first="Lunquan" last="Sun">Lunquan Sun</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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