The 6-mCEPh-purine modification enhances siRNA activity. (A) Luc sequence. (B) Luc1 sequence. Comparison of RNAi activity of luciferase-targeting siRNAs, each bearing A or 6-mCEPh-purine at the 5′-end of the guide strand. Relative levels of luciferase luminescence in luciferase-expressing HeLa cells after treatment with various concentrations of transfected siRNA. Data are represented as the means ± SD, n = 5, The 6-mCEPh-purine P value was calculated by comparison with 5′-A. P-value (a) Two-way ANOVA, (###) P < 0.001 (b): unpaired t-test. (C) Ago2-associated guide strand copy number of 5′-A and 5′-6-mCEPh-purine siRNAs. The amount of RISC-loaded guide strand was determined by immunoprecipitation of AGO2 from HeLa cells and qRT-PCR. siRNAs were transfected into cells at concentrations of 5 pM and 50 pM. Copies of guide strands were calculated per cell lysate. Data are represented as the means ± SD, n = 3. The 6-mCEPh-purine P value was calculated by comparison with 5′-A. P < 0.001 (a): Two-way ANOVA, (###) P < 0.001 (b), (##) P = 0.009 (b): unpaired t-test. (D) Time-course of Ago2-associated guide strand copy number. Ago2-associated guide strand copy number of 5′-A and 5′-6-mCEPh-purine siRNA was determined at 1, 2, and 3 d after 50 pM siRNA treatment. Data are represented as the means ± SD, n = 6.
