(228bn) Specific siRNA-Target Interactions Influence siRNA Activity

Short-interfering RNAs (siRNAs) direct targeted post-transcriptional gene knockdown through the native eukaryotic RNAi pathway. siRNAs are widely used in research settings and several siRNA-based therapies are progressing through clinical trials. Still, siRNA design is not straightforward; this is due to an incomplete understanding of the RNAi mechanism and how chemically synthesized siRNAs interact with the various proteins of the RNAi pathway. Here we investigated how the 5â?? terminal nucleotide, siRNA hybridization stability, and siRNA-mRNA interactions impact siRNA strand loading and activity in cultured HeLa cells. We found that low hybridization stability at the 5â?? terminus of the intended guide strand (Nucleotides: 1-2) correlates with greater siRNA loading. Post-loading, low stability siRNA-mRNA interactions within the seed region of the guide strand (Nucleotides: 3-4) and high stability interactions near the 3â?? terminus (Nucleotides: 17-18) correlate with greater siRNA activity. Further, our results indicate that 5â?? terminal nucleotide sequence impacts the activity of an siRNA post-loading. In this presentation, we will discuss these results and how these parameters influence the design of highly active siRNAs.