Structure-Optimized Asymmetric siRNA Increases Predicted AGO2 Binding Affinity to Augment Targeted MMP7 Gene Silencing Efficacy.

RNAi holds significant therapeutic potential, yet the clinical efficacy of siRNA drugs has been constrained by challenges in stability, targeted accumulation, and efficient loading into the RISC. To address this, we hypothesized that rationally extending or shortening the terminal base of the siRNA antisense strand could optimize RISC assembly by enhancing its affinity for AGO2, the key catalytic protein, while minimizing sense strand incorporation. With a focus on the MMP7 gene target, a computational docking model was employed to evaluate a series of antisense-terminally modified, asymmetric siRNAs for their predicted binding affinity to human AGO2. A significant positive correlation was identified between the computed AGO2-binding affinity of these asymmetric siRNAs and their subsequent gene knockdown activity, thereby supporting further evaluation. This work demonstrates that extending or shortening the terminal base of the siRNA antisense strand is a viable approach to engineer next-generation siRNAs with enhanced potency and specificity.