2004 Fiscal Year Final Research Report Summary
DEVELOPMENT OF NEW RNAAPTAMERS AGAINST HCV
| Project/Area Number |
15590108
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Drug development chemistry
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| Research Institution | NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY |
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Principal Investigator |
NISHIKAWA Satoshi NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY, AGE DIMENSION RESEARCH CENTER, DEPUTY DIRECTOR, 年齢軸生命工学研究センター, 副研究センター長 (60357704)
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| Project Period (FY) |
2003 – 2004
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| Keywords | APTAMER / HCV / NS3 PROTEASE / NS3 HELICASE / IRES / FRET / RIBOZYME |
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| Research Abstract |
Infecting 3% of the world's population, Hepatitis C Virus(HCV) is the major infectious agent leading to non-A and non-B hepatitis. Non-structural protein 3(NS3) of HCV has two distinct activities, protease and helicase, which are essential for HCV proliferation. The translation of HCV starts at the internal ribosome entry site(IRES) and IRES is well conserved in HCV strains. Accordingly these proteins and RNA site are all good targets of anti-HCV drugs. In vitro selection, namely, SELEX(systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating APTMERs that have high affinity for target molecules from a randomized oligonucleotide pool. In order to develop new drug-lead compounds to block HCV, we did selection and obtained RNA aptamers specific to NS3 protease, NS3 helicase and HCV IRES, respectively. In the first step we established an assay system of anti-NS3 protease aptamer in cultured cells. Peptide substrate bearing the sequence of NS3 protease cleavage site sandwiched with fluorescent proteins CFP and YEP. In the next step we constructed efficient aptamer expression vector in cells by connecting cis-acting ribozyme and the aptamer. Efficient protease inhibition was detected by repeating this ribozyme-aptamer unit. We also rationally designed dual-functional aptamers by fusing anti-protease and anti-helicase aptamers. By conjugation of two different aptamers, both inhibition ability increased, especially against helicase four to five-fold inhibition was detected. As a result of selection against IRES, we found domain II of IRES is important target site in HCV RNA genome.
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Research Products
(26 results)
