The overarching goal of this project is to develop a detailed biophysical understanding of the mechanism of action of catalytic RNA molecules, or ribozymes. Ribozyme derivatives are currently under development as therapeutics for a number of devastating diseases. A fundamental understanding of ribozyme mechanism is critical in the design of derivatives, with necessary improvements in pharmacological properties that simultaneously maintain the desired catalytic activity. Our thesis, is that, the static structure of a ribozyme is insufficient to understand its mechanism of action, and that structural work must be coupled with studies of the molecule's dynamics, its interactions with multivalent metal ions, and the effects of those metal ions on molecular structure and dynamics. We will apply contemporary magnetic resonance techniques, including heteronuclear NMR spin relaxation and advanced EPR experiments, to provide unique information that bears directly on these issues and, thus, to link structural studies with biochemical energetics. The dynamic properties of ribozyme conformations, as studied by NMR spin relaxation, will be emphasized. In the well characterized hairpin ribozyme, we will investigate the dynamic properties of the molecule using NMR spin relaxation in active vs. inactive sequence variants, and as a function of tertiary structure formation (docking). We will proceed to use an integrated EPR and NMR approach, to delineate the effects of multivalent metal ions, which are crucial for catalysis, but do not participate directly in chemistry in this system, on the ribozyme's structure and dynamics. We are also interested in the U6 snRNA, a catalytically critical component of the eukaryotic mRNA splicing apparatus. In this system, we will investigate the structure of a biochemically-identified metal ion, which appears to participate in reaction chemistry, both in the internal stemloop that forms its immediate binding site, and in larger complexes, including pre-mRNA sequences. Studies of these two systems will provide complementary and synergistic perspectives on the interrelationships among conformational dynamics, metal ion cofactor ligation, and catalytic function in RNA. In short, we propose an integrated biophysical program of technological progress and novel applications, that will significantly advance the ribozyme field toward a molecular-level understanding of these fascinating catalysts.
