Effect of siRNA with an asymmetric RNA/dTdT overhang on RNA interference activity

Small interfering RNAs (siRNAs) guide RNA-induced silencing complexes (RISC) to target mRNAs for sequence-specific silencing. A fundamental aspect of this highly coordinated process is a guide strand-specific loading of the siRNA duplex into the RISC for the accurate target recognition, which is currently dictated by certain duplex parameters such as thermodynamics. Here, we show that minor changes in the overhang structure have profound effects on the extent to which the individual strands of the siRNA duplex participate in RNAi activity. We demonstrate that the two strands of the siRNA are similarly eligible for assembly into RISC for the siRNAs with symmetric overhangs, whereas those with asymmetric RNA/deoxythymidine dinucleotide (dTdT) overhangs exhibit a distinct preference in favor of a strand with an RNA overhang that drives a mature RISC affinity to the desired target. We believe that this additional determinant provides a plausible and simple approach for improving the strand selection, thereby considerably increasing a specificity of target silencing.