Structure-Based Design of Novel Chemical Modification of the 3′-Overhang for Optimization of Short Interfering RNA Performance

Short interfering RNAs (siRNAs) are broadly used to manipulate gene expression in mammalian cells. Although chemical modification is useful for increasing the potency of siRNAs <i>in vivo</i>, rational optimization of siRNA performance through chemical modification is still a challenge. In this work, we designed and synthesized a set of siRNAs containing modified two-nucleotide 3′-overhangs with the aim of strengthening the interaction between the 3′-end of the siRNA strand and the PAZ domain of Ago2. Their efficiency of binding to the PAZ domain was calculated using a computer modeling program, followed by measurement of RNA–Ago2 interaction in a surface plasmon resonance biochemical assay. The results suggest that increasing the level of binding of the 3′-end of the guiding strand with the PAZ domain, and/or reducing the level of binding of the sense strand through modifying the two-nucleotide 3′-overhangs, affects preferential strand selection and improves siRNA activity, while we cannot exclude the possibility that the modifications at the 3′-end of the sense strand may also affect the recognition of the 5′-end of the guiding strand by the MID domain. Taken together, our work presents a strategy for optimizing siRNA performance through asymmetric chemical modification of 3′-overhangs and also helps to develop the computer modeling method for rational siRNA design