PROJECT SUMMARY/ABSTRACT My group develops and uses chemical and biochemical tools to investigate the structure and function of noncoding RNAs (ncRNA) and their complexes with proteins (RNPs) and small molecules (smRNAs). In the context of this project, we will specifically focus on two general areas: 1) engineering fragment antigen binding (Fabs) as chaperones for RNA/RNP/smRNA crystallography, cryoelectron microscopy (cryoEM) and as enabling tools for RNA biology, and 2) elaborating mechanisms of RNA function, especially catalytic RNAs (ribozymes). The terrestrial transcriptome contains vast numbers of structured RNAs within both coding and noncoding transcripts that act alone and in concert with proteins to carry out critically important roles in nearly every facet of cellular function. Understanding how these RNAs mediate biological function in health and disease requires knowledge of their three-dimensional architectures. We will deploy a variety of structural biology, biophysical, biochemical, computational and chemical approaches, including kinetic isotope effect analysis and the use of nucleotides from an artificially expanded genetic system. The data that emerge from our recombinant Fab work will engender a robust platform to generate Fabs that bind structured RNA with high affinity and specificity, establish Fabs as powerful reagents in structural and mechanistic RNA biology and yield new macromolecular structures, which have profound impact on our understanding of the underlying biology and serve as a framework to develop and test functional hypotheses. In addition, our in-depth work on catalytic RNA will define for the first time the relationship between catalytic structure and transition state structure, a missing link that has plagued efforts to engineer macromoleclules with novel catalytic functions. Beyond the conceptual advances that emerge, this work promises to deliver to the RNA community powerful Fab reagents for RNA/RNP structural and functional studies, nucleotide analogs to support novel approaches to define RNA catalytic mechanisms, and strategies to render large RNAs more tractable by increasing the density of information.