Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom...

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism. The shRNA precursor is processed by Dicer into small interfering RNAs (siRNAs) and subsequently programs the RNAi-induced silencing complex (RISC) to find a complementary target mRNA (mRNA) for post-transcriptional gene silencing. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer to be processed directly by the Ago2 nuclease of the RISC complex. We named this design AgoshRNA as these molecules depend on Ago2 both for processing and subsequent silencing activity. This alternative AgoshRNA processing route yields only a single active RNA strand, an important feature to restrict off-target effects induced by the passenger strand of regular shRNAs. It is therefore important to understand this novel AgoshRNA processing route in mechanistic detail such that one can design the most effective and selective RNA reagents. We performed a systematic analysis of the optimal base pair (bp) composition at the top and bottom of AgoshRNA molecules. In this study, we document the importance of the 5' end nucleotide (nt) and a bottom mismatch. The optimized AgoshRNA design exhibits improved RNAi activity across cell types. These results have important implications for the future design of more specific RNAi therapeutics.