fig3: Inhibition analysis of IRES-mediated translation in vitro by aptamers. Luciferase activity in the absence of an aptamer was used as a control (100%). Values represent results from at least three independent experiments. A, B and C are equivalent to the groups in Figure 2.

Mentions: In order to survey the effect of aptamers on IRES-dependent translation activity, we performed an in vitro translation assay of IRES–luciferase mRNA containing the 3′-UTR of the HCV genome after the luciferase gene. Translation was examined using rabbit reticulocyte lysate and 0.5 μg of mRNA in the presence of aptamers (Figure 3). Aptamer 3-07 strongly decreased the luciferase activity to 10% of the control levels. Aptamers 3-03 and 3-04 reduced activity to 30–40%, and other aptamers had almost no effect. Aptamers 3-15 and 3-08 without the consensus sequence did not have an inhibitory effect. This result indicates that translation inhibition is dependent on the presence of the consensus sequence and inhibition efficiency is not related to the binding efficiency. Accordingly, we focused on aptamer 3-07 and we detected dose-dependent inhibition by aptamer 3-07 (IC50 ∼0.6 μM) using 0.5 μg of mRNA. No effect was observed using luciferase mRNA without the IRES (data not shown). This result shows that translation inhibition is dependent on aptamer 3-07 and is a specific effect on the IRES. As the inhibitory effect varied among aptamers, the aptamer structure seems to be important for translation inhibition. Recently, antisense RNA-targeted domain IIId showed more strong inhibition (IC50 < 10 nM) compared to our aptamer (13), but experimental conditions are different.

Kikuchi K, Umehara T, Fukuda K, Kuno A, Hasegawa T, Nishikawa S - Nucleic Acids Res. (2005)

Bottom Line: This convergence suggests that domain IIId is preferentially selected in an RNA-RNA interaction.One of the aptamers inhibited translation both in vitro and in vivo.Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.

Affiliation: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

Abstract: The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III-IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA-RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.