(620ae) Predictors of siRNA Functional Asymmetry (Rapid Fire)

RNA interference is a regulatory pathway that uses a small RNA trigger to direct gene knockdown through the destruction of mRNA containing complementary nucleotide sequences. The pathway can be initiated by exogenous short interfering RNAs (siRNAs), double-stranded RNAs with specific structures. For siRNAs to function properly they must be capable of base pairing with their target, meaning that one of the strands must be removed before forming the active complex. Proper strand selection is functionally critical to ensure targeting of only the intended mRNA target. The process of strand selection occurs through the orientation of the siRNA duplex within a multiprotein loading complex, followed by selection of one strand and destruction of the other. Once activated, this RNA-protein complex is known as the RNA induced silencing complex (RISC). However, not all siRNAs form highly active RISCs.

Focusing on known functional interactions of the proteins of the RNA interference pathway, we have characterized siRNA strand activity based on two criteria, the difference in the relative terminal hybridization stability (ΔΔG) and the terminal nucleotide sequence classification (TN). Using a cell culture model, we investigated differences in relative siRNA strand activity, referred to as functional asymmetry, and differences in RISC loading. We found that the TN is a stronger predictor of siRNA functional asymmetry than ΔΔG, but both characteristics do provide complementary information when predicting siRNA functional asymmetry. Specifically, ΔΔG has the strongest impact on functional asymmetry when siRNA strands have identical 5’ terminal nucleotides. Our results also indicate that TN effects siRNA activity at the level of RISC formation and strand selection, similar to the literature, while, ΔΔG is less predictive of siRNA strand selection and therefore may have a larger influence on RISC activation and turnover. We will present our findings on siRNA functional asymmetry and the correlation of siRNA activity with siRNA strand selection.