(530e) Impact Of Target Mrna Structure On Sirna Silencing Efficiency And Immune Stimulation In RNA Interference

RNA interference (RNAi) is a natural phenomenon resulting in potent and primarily specific gene silencing that occurs in most eukaryotes. It is initiated in cells by the presence of short interfering RNAs (siRNAs), which are double-stranded, non-coding RNA molecules ~21 nucleotides long. siRNAs guide the active enzyme complex of RNAi, the RNA induced silencing complex (RISC), to mRNAs possessing regions of sequence complementarity, leading to degradation of the target message. The efficiency of RNAi has been shown to depend not only on the characteristics of the siRNAs but also on the structure of the target mRNA. Features of the siRNA duplex have also been implicated in stimulating an innate immune response, which can complicate their application. Our goal is to define design rules for siRNAs that take into account the target mRNA structure as well as silencing efficiency and the potential for immune stimulation. By using in vitro binding assays, cell culture studies, mRNA secondary structure predictions, and molecular modeling, we examined the mRNA and siRNA sequence and structural features that correlated with silencing efficiency and/or immune stimulation. We found that siRNAs targeting certain predicted structures of the enhanced green fluorescent protein (EGFP) transcript differed in their silencing efficiencies and that these findings agreed with our computational analyses of results from the literature. Furthermore, the correlation between the accessibility of the mRNA structure and the likelihood of good silencing efficiency agreed well with the current mechanistic understanding of biased siRNA strand loading into RISC. Additionally, we have studied the relationships among silencing efficiency, mRNA structure, and activation of the innate immune response, specifically through Protein Kinase R (PKR) and Toll-like Receptor 3 (TLR-3). We have shown that binding of TLR-3 to siRNAs requires an acidic pH and have identified some of the important siRNA features contributing to this interaction. These results have implications regarding the mechanism of RNAi and the requirements for avoiding activation of the innate immune system.