(34e) Effect of siRNA Terminal Mismatches On TRBP and Dicer Binding and Silencing Efficacy

RNA interference (RNAi) is initiated in mammalian cells through the delivery of short, interfering RNAs (siRNAs). siRNAs are 21-nt double-stranded RNAs containing 19 complementary internal nucleotides and 2 nt overhangs on each 3'-end. One of the strands, the guide strand, is complementary to the target mRNA and leads to enzymatic destruction of the mRNA. Ideally, the other strand, the passenger strand, is not incorporated into the RNAi pathway. To increase the ratio of guide strands to passenger strands that enter the RNAi pathway, siRNAs are often designed with an intentional thermodynamic bias to make the end of the siRNA containing the guide strand 5'-end less stably hybridized relative to the end containing the passenger strand 5'-end. The proteins of the RNAi pathway are known to sense this asymmetry and preferentially incorporate the guide strand. One manner by which this thermodynamic bias is engineered is to alter the passenger strand sequence to introduce a mismatch with the guide strand 5'-end, making that end of markedly lower stability than the other, fully-hybridized end. However there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, a systematic study was performed by testing siRNAs possessing a terminal mismatch generated either by altering the guide strand (at the 5'-end) or passenger strand (nucleotide 19). The impact of these mismatches on siRNA silencing efficiency and on their binding with the RNA induced silencing complex (RISC) loading complex (RLC) proteins TRBP and Dicer in H1299 and HepG2 cytoplasmic extracts was examined. It was found that siRNA-TRBP binding is largely indicative of eventual silencing efficacy of the siRNAs and that this binding can be significantly reduced by terminal mismatches. Terminal mismatches led to a small increase in Dicer binding, as expected, however, this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always serve to improve target silencing and that care should be taken when designing siRNAs in this way.