Specificity in RNA Molecular Recognition
A broad goal of our research is to understand how ribonucleases and RNA binding proteins recognize their cognate binding sites, yet accommodate variation in sequence and structure among their numerous RNA targets. With Dr. Eckhard Jankowsky (CWRU) we developed high throughput methods, incorporating next-generation sequencing, that allow the processing rates and binding affinities of thousands of RNAs to be measure simultaneously. The resulting rate and equilibrium constant distributions allow us to globally determine the effects of RNA sequence and structure variation on the free energy landscape of RNA processing and binding reactions. By combining new high-throughput experimental tools with transcriptomic and bioinformatic approaches, we aim to achieve complete and quantitative descriptions protein and small molecule binding to RNA that inform our understanding of their biological function and potential therapeutic applications.
Mechanistic Enzymology of RNA Strand Cleavage
A fundamental goal of enzymology is to learn how active sites stabilize reaction transition states in order to accomplish catalysis. The active site architectures of ribonculeases and ribozymes have been the subject of intense study. However, fundamental questions remain regarding the mechanism (stepwise vs. concerted), catalytic modes (acid/base catalysis, metal ion catalysis, electrostatic stabilization), and trajectory of proton transfers (which atoms donate/accept). In collaboration with Drs. Joseph Piccirilli (University of Chicago)and Darrin York (Rutgers) we created a framework for RNA 2’-O-transphosphorylation reactions that relates biophysical data (kinetic isotope effects and Brønsted coefficients) and transition state structures structure. We are using this framework to learn how interactions with acid/base and metal ion catalysts alter transition states for solution reactions, and we are applying these tools to test catalytic mechanisms and active site interactions proposed for ribozymes and phosphoryl transferases including current and potential drug targets.
Allosteric Regulation and Inhibition of Ribonucleotide Reductase
Ribonucleotide reductase is an important target for cancer chemotherapy due to its essential role in deoxynuclotide metabolism. The allosteric regulation of ribonucleotide reductase is complex, and offers multiple attractive targets for small molecule inhibitors. Together with Dr. Chris Dealwis (CWRU) we using nucleotide analogs and small molecules to pinpoint the key non-covalent interactions that underlie substrate specificity and drive allosteric regulation. Together with the Dealwis lab we have applied a pipeline for identification of small molecule inhibitors and allosteric modulators of human ribonucleotide reductase. The compounds we discovered thus far provide important new insights into ribonucleotide reductase specificity and regulation as well as providing promising anti-cancer agents. Now, we are interested in discovering how binding interactions and protein dynamics are linked in order to design novel small molecules that act as artificial allosteric ligands.