Rational Design of Dual-Functional Aptamers That Inhibit the Protease and Helicase Activities of HCV NS3
Identifieur interne : 001536 ( Istex/Corpus ); précédent : 001535; suivant : 001537Rational Design of Dual-Functional Aptamers That Inhibit the Protease and Helicase Activities of HCV NS3
Auteurs : Takuya Umehara ; Kotaro Fukuda ; Fumiko Nishikawa ; Michinori Kohara ; Tsunemi Hasegawa ; Satoshi NishikawaSource :
- Journal of Biochemistry [ 0021-924X ] ; 2005.
Abstract
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Δ, was conjugated via an oligo(U) tract to the 3′-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition.
Url:
DOI: 10.1093/jb/mvi042
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Tel: +81-298-61-6085, Fax: +81-298-61-6159, E-mail: satoshi-nishikawa@aist.go.jp</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Journal of Biochemistry</title><title level="j" type="abbrev">J Biochem</title><idno type="ISSN">0021-924X</idno><imprint><publisher>Oxford University Press</publisher><date type="published">2005</date><biblScope unit="vol">137</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="339">339</biblScope><biblScope unit="page" to="347">347</biblScope></imprint><idno type="ISSN">0021-924X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0021-924X</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. 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<contrib contrib-type="author"><name><surname>Umehara</surname><given-names>Takuya</given-names></name>
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<xref rid="CHDFACBH">1</xref>
<xref rid="COR1">*</xref>
</contrib><aff><target target-type="aff" id="CHDFACBH"></target><label>1</label>Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566; <target target-type="aff" id="CHDFFHBA"></target><label>2</label>Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560; and <target target-type="aff" id="CHDJBGBG"></target><label>3</label>Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613</aff></contrib-group><author-notes><corresp id="COR1"><label>*</label>To whom correspondence should be addressed. Tel: +81-298-61-6085, Fax: +81-298-61-6159, E-mail: <ext-link xlink:href="satoshi-nishikawa@aist.go.jp" ext-link-type="email">satoshi-nishikawa@aist.go.jp</ext-link></corresp></author-notes><pub-date pub-type="ppub"><month>3</month><year>2005</year></pub-date><volume>137</volume><issue>3</issue><fpage>339</fpage><lpage>347</lpage><history><date date-type="accepted"><day>26</day><month>12</month><year>2004</year></date><date date-type="received"><day>24</day><month>11</month><year>2004</year></date></history><permissions><copyright-statement>© 2005 The Japanese Biochemical Society.</copyright-statement><copyright-year>2005</copyright-year></permissions><abstract xml:lang="en">
<p>The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Δ, was conjugated <italic>via</italic> an oligo(U) tract to the 3′-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity <italic>in vitro</italic>, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication <italic>in vitro</italic>. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition.</p>
</abstract><kwd-group kwd-group-type="KWD" xml:lang="en">
<kwd>hepatitis C virus</kwd>
<kwd>NS3 helicase</kwd>
<kwd>NS3 protease</kwd>
<kwd>RNA aptamer</kwd>
<kwd>RNA design</kwd>
</kwd-group><custom-meta-wrap><custom-meta><meta-name>hwp-legacy-fpage</meta-name><meta-value>339</meta-value></custom-meta><custom-meta><meta-name>hwp-legacy-dochead</meta-name><meta-value>Regular Paper</meta-value></custom-meta></custom-meta-wrap></article-meta></front>
</article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Rational Design of Dual-Functional Aptamers That Inhibit the Protease and Helicase Activities of HCV NS3</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Rational Design of Dual-Functional Aptamers That Inhibit the Protease and Helicase Activities of HCV NS3</title></titleInfo><name type="personal"><namePart type="given">Takuya</namePart><namePart type="family">Umehara</namePart><affiliation>Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566;</affiliation><affiliation>Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560; and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Kotaro</namePart><namePart type="family">Fukuda</namePart><affiliation>Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566;</affiliation><affiliation>Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560; and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Fumiko</namePart><namePart type="family">Nishikawa</namePart><affiliation>Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566;</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Michinori</namePart><namePart type="family">Kohara</namePart><affiliation>Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Tsunemi</namePart><namePart type="family">Hasegawa</namePart><affiliation>Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560; and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Satoshi</namePart><namePart type="family">Nishikawa</namePart><affiliation>Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566;</affiliation><affiliation>To whom correspondence should be addressed. Tel: +81-298-61-6085, Fax: +81-298-61-6159, E-mail: satoshi-nishikawa@aist.go.jp</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="other" displayLabel="other" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-7474895G-0">other</genre><originInfo><publisher>Oxford University Press</publisher><dateIssued encoding="w3cdtf">2005-03</dateIssued><dateCreated encoding="w3cdtf">2004-12-26</dateCreated><copyrightDate encoding="w3cdtf">2005</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Δ, was conjugated via an oligo(U) tract to the 3′-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition.</abstract><note type="author-notes">*To whom correspondence should be addressed. Tel: +81-298-61-6085, Fax: +81-298-61-6159, E-mail: satoshi-nishikawa@aist.go.jp</note><subject lang="en"><genre>KWD</genre><topic>hepatitis C virus</topic><topic>NS3 helicase</topic><topic>NS3 protease</topic><topic>RNA aptamer</topic><topic>RNA design</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>J Biochem</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0021-924X</identifier><identifier type="PublisherID">jbchem</identifier><identifier type="PublisherID-hwp">jbiochem</identifier><identifier type="PublisherID-nlm-ta">J Biochem</identifier><part><date>2005</date><detail type="volume"><caption>vol.</caption><number>137</number></detail><detail type="issue"><caption>no.</caption><number>3</number></detail><extent unit="pages"><start>339</start><end>347</end></extent></part></relatedItem><relatedItem type="references" displayLabel="MVI042C1"><titleInfo><title>J. 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