(15b) Mechanism-Based Design of Highly Active Sirnas

Short interfering RNAs (siRNAs) provide a method of post-transcriptional, targeted gene knockdown that is of interest as a therapeutic strategy for the treatment of a variety of diseases including metabolic disorders, cancers, viral infections, and degenerative diseases. Gene knockdown or silencing occurs through interactions of the siRNA with the native eukaryotic RNA interference (RNAi) pathway. siRNA specificity derives from the Watson-Crick base pairing interactions between the bases of the siRNA and its complementary target. The active silencing complex, known as RISC, operates as a multiple turnover enzymatic complex capable of decreasing target transcript levels sufficiently to initiate the therapeutic effect. However, siRNA activity can vary greatly depending upon its sequence and the reasons for this variability are not completely understood, hindering the progression of siRNAs to use as therapeutics. Our goal is to establish the mechanism-based guidelines for selecting siRNA sequences that are most likely to be highly active and specific for a given target.

Structurally, siRNAs are double-stranded RNAs with 19 central base pairs, two-nucleotide 3’ overhangs, and 5’ phosphates. Central to the function of siRNAs is the ability of the RNAi pathway to discriminate which strand of the structurally symmetric siRNA is to be active (guide strand) and which strand is to be degraded (passenger strand). Failure to incorporate the correct strand can cause both off-target effects as well as competition for the protein Argonaute 2, the core protein of RISC. This loading step represents a significant event in the success of an siRNA and must occur properly for maximal siRNA function. In this presentation, we will discuss our findings on the contributing factors that lead to siRNA strand selection and its influence on siRNA activity.