Fig 2. Target RNA cleavage activity and serum stability of the selected IRES-targeting siRNAs.

(A) In vitro cleavage assays were performed with 5′ ³²P-radiolabeled 31-nts long HCV IRES (spanning nts 313–343) using Ago2 complexes immunoprecipitated from HeLa cells transfected with each indicated siRNA (20 nM). Sc, scrambled siRNA. (B) RNA target cleavage assay was performed with 5′ ³²P-radiolabeled HCV IRES substrate (spanning nts 1–487) using a recombinant human Ago2 protein and guide strand of indicated siRNAs. As a negative control, siIE22 passenger-strand was used. RNA samples were analyzed by electrophoresis on a denaturing polyacrylamide denaturing gel followed by autoradiography. (C) Serum stability of siRNAs was assessed by incubating siRNAs in 45% plasma for the indicated time periods and subsequent quantification of remained intact siRNA guide-strand by Phosphorimager analysis of northern blots. (D) The cleavage assay was performed as described in (B). Radioactivity of cleaved products was quantified by Phosphorimager analysis and shown below the autoradiogram (mean ± SD of three independent experiments).