"Short interfering RNAs (siRNAs) are a promising nucleic acid-based therapeutic strategy that offer an alternative to traditional small-molecule based drugs for the treatment of a variety of otherwise-untreatable diseases. These small, chemically synthesized RNAs can transiently inhibit the expression of target genes through the use of a native eukaryotic pathway called RNA interference (RNAi). The design of highly active siRNAs is not straightforward. Our strategy to improve siRNA design criteria focuses on the idea that siRNAs enter the RNAi pathway as duplexes but only become functional once one of the siRNA strands has been selected. Ensuring high selectivity of the intended strand is essential for proper siRNA functionality. To address this challenge, this research aimed to (1) understand how features of the siRNA control the relative activities of the two strands of the siRNA duplex, termed functional asymmetry and (2) better understand the mechanism of siRNA asymmetric strand selection. To improve our understanding of the characteristics that drive siRNA strand selection and strand activity, we began our investigation with two characteristics, relative terminal hybridization stability (22062206G) and 50 terminal nucleotide (TN) Rank. These characteristics have been previously shown to cooperatively predict siRNA activity. Our analysis indicates that these characteristics are also predictive of siRNA functional asymmetry. A comparative analysis between individual strand loading and activity shows that the 22062206G reflects a combination of duplex hybridization stabilities (2206Gs) that are important in the formation of RISC and its kinetics. The TN was found to influence siRNA strand activity post-siRNA loading, suggesting a role in maximizing RISC half-life. Taken together, these results indicate that siRNA activity is influenced by siRNA-protein interactions that occur both pre- and post-siRNA strand selection. The mechanism of siRNA loading and the protein-RNA interactions responsible for driving differential strand loading are not fully understood. Based on evidence from other systems, we hypothesized that asymmetric strand selection was driven, at least in part, by preferential binding of different segments of siRNAs by RNAi pathway proteins. Here we demonstrated that one RNAi protein, PACT, preferentially localizes to one siRNA terminus of a duplex known to be functionally asymmetric. This indicates that PACT may thereby influence siRNA asymmetric strand loading during initiation of RNAi. The work presented here identified characteristics of siRNAs duplexes that were found to drive siRNA strand selection and activity. Collectively, these results inform siRNA design and address biological questions about the functional aspects of the RNAi pathway. Future work in this area will continue to investigate how characteristics of siRNAs impact their functionality within the RNAi pathway, ultimately leading towards a highly refined set of rules for siRNA design. A more guided approach to siRNA design will facilitate the development of siRNAs as a therapeutic platform."--Pages ii-iii.
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