The efficiency of duplex loading is not affected by different 5′-end nucleobases. (A) Four siRNA duplexes used in this set of experiments. Each of them bore different nucleotides (A, U, 6-mCEPh-purine or G) at the 5′ end of the guide strand and 5-nitroindole at position 19 of the passenger strand. The guide strand was radiolabeled at the 5′ monophosphate, whereas the passenger strand held a 5′ amino linker that fixes the loading orientation and a 2′-O-methyl modification that blocks passenger ejection. (B) Scheme of RISC assembly. A nonradiolabeled, uncleavable target oligonucleotide was added. (C) A representative result of the native agarose gel assay. (D) Quantification of pre-Ago2-RISC formation. The quantified signals were normalized to the pre-Ago2-RISC value of 6-mCEPh-purine at 120 min. The graph shows the average ± SD from three independent experiments using the same set of reagents. The ANOVA P-values are given in an inset under the graphs. The results of pairwise comparison on the effect of 6-mCEPh-purine compared to natural nucleotides are indicated by asterisks (*) P < 0.05, (**) P < 0.005, (***) P < 0.0005. The detailed results of statistical analyses are summarized in Supplemental Table 1.
