Project Summary RNA plays a central role in nearly every biological process, acting in turn as a messenger, catalyst, signaling molecule and more. Many of these roles require RNA to fold into specific structures, and a given RNA species can often adopt multiple structures that modulate properties such as protein interactions, ligand binding and catalytic activity. Research in the Widom Lab focuses on developing and applying new spectroscopic methods to study RNA structure and dynamics. We combine bulk, single-molecule and ultrafast spectroscopy in order to obtain a comprehensive picture of RNA folding and interactions over length-scales from Angstroms to microns and time-scales as short as picoseconds. We are currently using these methods to study RNA-protein interactions during pre-messenger RNA (pre-mRNA) splicing, the process in which segments of RNA that do not code for protein are excised, and ligand binding by riboswitches, which regulate gene expression in bacteria. Single-molecule measurements offer a unique window into the heterogeneous and dynamic nature of RNA folding, and ultrafast spectroscopy probes very rapid processes that are inaccessible by other means. However, these are notably low-throughput techniques, with measurements typically being performed on only a single RNA sequence at a time. An entirely new class of scientific questions could be addressed if these techniques could be applied in a high-throughput manner. We will bring this goal to realization by developing a process for performing single-molecule fluorescence measurements on hundreds of different sequences simultaneously. A library of RNA sequences will be prepared containing random bases at sites of interest and the mixture will be subjected to single-molecule fluorescence measurements to monitor structural rearrangements and response to stimuli in real time. Each molecule will then be sequenced in situ in order to determine what sequence gave rise to its single-molecule signal. Our goals for the next 5 years are to optimize this method and to use it to answer questions that can only be addressed via high-throughput approaches. We will investigate the mechanistic impacts of key sequences on pre-mRNA splicing, including variable sequences that fine-tune splicing and conserved sequences that lead to disease when disrupted. We will also measure the folding thermodynamics and kinetics of hundreds of RNA sequences and use the results to benchmark and improve RNA structure prediction algorithms. This research program will greatly increase the throughput of single-molecule fluorescence measurements, enabling detailed biophysical insights to be achieved rapidly across a vast sequence space.