Small RNAs constitute a major class of posttranscriptional regulators in human biology. Synthetic siRNAs have been used extensively for reverse-genetic experiments in mammalian cells and are a promising therapeutic avenue for the treatment of diverse human diseases, including liver cancer, ovarian cancer, and lung cancer. Similarly, inhibition of specific microRNAs (miRNAs), the most abundant form of endogenous small RNAs in humans, can have profound effects on cellular physiology and a growing list of anti-miR compounds for the treatment of diverse forms of cancer have entered clinical and pre-clinical trials. Despite the clear therapeutic opportunities surrounding small RNA biology, gaps in basic knowledge have prevented the field from reaching its full potential. One significant gap is insight into how to control the stability of the RNA-Induced Silencing Complex (RISC), the RNA/protein complex in which small RNAs function. The objective of this application is to apply understanding of the enzymes mediating small RNA metabolism towards development of methods for controlling small RNA stability in human cells. This objective will be achieved by pursuing two specific aims: 1) Develop approaches for optimizing siRNA potency and persistence; and, 2) Develop approaches for destabilizing endogenous miRNAs. Under Aim 1 we will apply our expertise in small RNA biochemistry to generate diverse libraries of siRNAs, from which the most stable sequences can be selected in mammalian cells. This goal is significant because there is currently no way of predicting, much less controlling, siRNA stability Our rational is that the ability to identify siRNAs with extended cellular half-lives would significantly benefit efforts to develop effective therapeutic siRNAs. Under Aim 2 we will determine how anti-miRs influence miRNA stability using assays we developed for monitoring the association of miRNAs with their partner protein, Argonaute2 (Ago2), which forms the core protein subunit of RISC. This is an important goal because, although chemical modifications have been developed to increase the nuclease resistance and improve the PK/PD profile of anit-miRs, exactly how these modifications impact the stability of RISC remains unclear. Our rational is that by focusing on modifications that destabilize the Ago2-miRNA complex and promote miRNA degradation it may be possible to develop anti-miRs that can perform multiple rounds of miRNA destruction. In principle, such catalytic anti-miRs would be effective at far lower doses than conventional anti-miRs. Because the challenge of delivering into target tissues is the major limiting factor in development of therapeutic anti- miRs, insights for improving potency would be a significant advance.