J. Biochem, 2003, Vol. 133, No. 3 351-359
© 2003 Japanese Biochemical Society
BIOTECHNOLOGY |
Structure/Function Analysis of an RNA Aptamer for Hepatitis C Virus NS3 Protease
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566
RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (
NS3) were identified in previous studies. These aptamers, G9-I, -II, and –III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of NS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (NEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(–36) base pair implied an A-minor motif involving the intramolecular base triple A(+8)·C(+24)-G(–36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.
+ To whom correspondence should be addressed. Tel: +81-298-61-6085, Fax: +81-298-61-6159, E-mail: satoshi-nishikawa{at}aist.go.jp
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