Project Summary Messenger RNAs (mRNAs) encode complex regulatory instructions in their 3' untranslated regions (3'UTRs) that prescribe context-dependent control of translation and decay. Disruption of mRNA post-transcriptional regulation by mutational lesions or shortening of 3'UTRs has been linked to diverse human diseases. RNA molecules fold back on themselves into base-paired secondary structures and occasionally higher-order tertiary structures, and sequence-encoded structures are hypothesized to play key roles in directing 3'UTR regulation. For example, RNA structures can provide specific binding sites for RNA binding proteins and may tune the accessibility of microRNA target sites. RNA structures may also function as molecular switches, with protein or microRNA binding triggering specific 3'UTR structural changes that alter the activity of distal regulatory sites. However, little remains known about 3'UTR structures and their role in post-transcriptional regulation. Existing data indicate that 3'UTRs have heterogenous and dynamic structures, which makes them challenging to study by traditional chemical probing or biophysical methods. My research group has recently developed a new single-molecule chemical probing technology (DANCE-MaP) that makes it possible to accurately define RNA structural ensembles consisting of 2-3 coexisting states in living cells. We will pursue two areas of research that exploit and build on the DANCE-MaP technology: (1) We will apply DANCE- MaP to define 3'UTR folding landscapes and mechanisms of combinatorial regulation by RNA binding proteins and microRNAs. (2) We will develop next-generation technologies that can resolve structurally heterogenous RNAs with greater resolution and provide model-free, physical interpretation of chemical probing data. This work will provide an improved understanding of mRNA regulatory mechanisms, helping advance efforts to therapeutically target 3'UTRs to modulate gene expression, and contribute new structure determination technologies that will be of broad utility to the RNA biology field.