J. Biochem, 2003, Vol. 133, No. 3 263-270
© 2003 Japanese Biochemical Society
BIOTECHNOLOGY |
RNA Aptamers Targeted to Domain II of Hepatitis C Virus IRES That Bind to Its Apical Loop Region
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566; 2 Faculty of Science, Yamagata University, Yamagata 990-8560
The internal ribosome entry site (IRES) is important for translation of hepatitis C virus (HCV) mRNA and has a unique RNA structure containing conserved domains I to IV. To investigate the function of domain II, we selected RNA aptamers that bind to domain II of HCV IRES by applying a simple and convenient selection method using a hybridized tag for fixing domain II RNA on magnetic beads instead of synthesizing long RNA. In addition, we employed surface plasmon resonance (SPR) technology to measure the binding affinity of each generation and to obtain detailed kinetic constants. The selected aptamers have a consensus sequence, 5'-UAUGGCU-3', which is complementary to the apical loop of domain II. The loop–loop interaction between the consensus sequence and domain II was confirmed by mutagenesis and nuclease mapping analyses. Binding affinities were dependent on the local structure containing the conserved sequence. The aptamers could inhibit IRES-dependent translation.
+ 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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