PROJECT SUMMARY/ABSTRACT The high stability of local structure for RNA and DNA has profound and widespread impacts on life. The simplicity of base pairing provides a powerful strategy for enzymes and machines to recognize specific sequences, and structured RNAs use the high stability to fold incrementally. However, the ease and stability of base pairing also increases the odds and the consequences of misfolding, requiring RNA chaperones. More broadly, essentially all cellular processes that require structural rearrangements of RNA or DNA also require proteins to accelerate these rearrangements. This framework provides the broad theme of our research. Over the next several years, our focuses will be in three main areas. (1) Specificity of CRISPR-Cas enzymes. The overarching goal is to understand the molecular origins, strategies, and limits of specificity of these enzymes for their target sequences. We will explore the hypothesis that the affinity of the enzymes for their DNA or RNA targets is decreased by mismatches between the guide RNA and the target strand by an amount that can be understood from the intrinsic properties of the R-loop or RNA helix. With these affinity penalties as a starting point, we will explore the strategies that nature has used to generate specificity, and we will probe the origins of enhanced specificity in designed enzyme variants. (2) RNA chaperone activity of DEAD-box RNA helicase proteins. Over the past two decades, we have delineated how DEAD-box helicases can function as general RNA chaperones, using local RNA unwinding to promote folding and structural rearrangements of structured RNAs. Our goals for the next few years are to explore whether a helicase that functions as a general RNA chaperone can be converted to a specific chaperone by modular replacement of its intrinsically disordered C-tail. Preliminary results indicate that this substitution produces a functional chimeric protein, enabling the proposed structural and functional studies. (3) DNA and RNA compaction and folding. We will leverage a disulfide crosslinking approach that we developed in 2022 to build on our understanding of the electrostatics of nucleic acid helices and to probe RNA folding. We will extend our observations of trivalent cation-mediated attraction between DNA helices by testing polyamines and RNA helices, and we will extend the approach by measuring repulsion or attraction between nucleosomes. We will also adapt the approach to probe sharp bending of RNA junctions. In each research area, we strive to answer basic research questions that are likely to give important and generalizable insights. Our work also has implications for understanding and treating diseases, as defects in these proteins are linked to many diseases including cancer, and CRISPR-Cas enzymes have emerged as key tools to combat genetic diseases.