Effects of Chemical Modifications on siRNA Strand Selection in Mammalian Cells

Small interfering RNAs (siRNAs) enable efficient gene silencing through RNA interference (RNAi) mechanisms. The RNAi machinery relies on an RNA-guided nuclease, Argonaute-2 (Ago2), which preferentially selects a single strand from an siRNA duplex. Complementarity between the selected strand and an RNA target strand leads to silencing through cleavage. The U.S. Food and Drug Administration's recent approval of two siRNA drugs has reignited optimism for RNAi therapeutics. Despite this recent success in the field, off-target effects are still a major concern; however, chemical modifications have shown promise in mitigating some off-target gene silencing. To evaluate the impact of novel chemical modifications on strand selection, we developed a quantitative polymerase chain reaction-based assay that is compatible with several pre-existing siRNA libraries and was used to characterize chemically modified siRNAs. siRNAs bearing azobenzene and propargyl modifications at the central region of the passenger strand significantly improved strand selection. On the other hand, folic acid-modified siRNAs improved strand selection best when placed at the 3' terminus. This study highlights the development and utility of a convenient method to evaluate the impact that novel chemical modifications have on strand-specific gene silencing of siRNAs.