Selective labeling of mature RISC using a siRNA carrying fluorophore–quencher pair

Abstract

RNA interference (RNAi) is an endogenous gene silencing system that has been harnessed to inhibit expression of specific genes through the introduction of short double-stranded RNAs, called small interfering RNAs (siRNAs) into cells. After entry into a cell, an siRNA is assembled into the RNA-induced silencing complex (RISC) and suppresses the target gene translation through interaction between the antisense (guide) strand of the siRNA and the target mRNA. To evaluate the intracellular fate of siRNAs, we performed imaging analyses using siRNAs labeled with newly designed fluorophore–quencher clusters introduced through the base surrogate with D-threoninol as a scaffold. Fluorescence microscopy analyses indicated that mature RISC containing fluorescently labeled antisense strand was localized within P-bodies. Thus, the antisense strand uptake into the RISC machinery was successfully visualized.