SUMMARY OF THE PARENT PROPOSAL RNA functions as the central conduit of information exchange in all cells. RNA molecules encode this information in both their primary sequences and in complex structures that form when RNA folds back on itself. A consistent theme from our MIRA-supported work to date is that biological function mediated by RNA structure is ubiquitous in both eubacterial and eukaryotic organisms. The broad and fundamental importance of RNA structure is clear, but there remains a profound knowledge gap: We generally have only rudimentary understanding of RNA structure across vast regions of most messenger and non-coding RNAs. We need to move beyond modeling RNA structure to directly detecting through-space structural communication and interactions in cells. Our lab takes a near-unique approach, simultaneously focusing on experimental simplicity and directness and on extensive validation in model systems. In essentially every instance where we have applied rigorous and quantitative technologies to study RNA structure-function interrelationships, new insights regarding biological regulatory mechanisms have emerged. We have also shown that RNA elements with higher- order structures are more likely to contain high-information-content clefts and pockets that bind small molecules, broadly informing a vigorous field of RNA-targeted drug discovery. This work is designed to have sustained long-term impact by pursuing three overarching opportunities. First, the project focuses on development of decisive, concise, and broadly implementable technologies for discovering and assessing higher-order structure in RNA. Second, this project applies these direct and experimentally concise methods to challenges of broad importance, from identification of structures essential for replication of pathogenic viruses to understanding how RNA conformational ensembles govern transcriptional gene regulation and pre-mRNA splicing. Third, understanding the propensity of RNA to form higher-order structures – and often clefts and pockets – opens up the possibility of targeting these motifs with small molecules, an opportunity we will exploit with the long-term goal of inventing facile and straightforward strategies for creating RNA-targeted therapeutics.