Defining the ligandability of bacterial RNAs

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PROJECT SUMMARY Most bioactive small molecules function by targeting proteins. Complex RNA structures are, in principle, also targetable with small molecules. However, apart from bacterial riboswitches, natural products that bind a small number of sites in the ribosome, and a handful of human-designed ligands, we know very little of the capacity of small molecules to bind RNA or to modulate RNA function. During the first year of my postdoctoral training, I demonstrated the feasibility of a new technology, called Frag-JuMP, that simultaneously identifies RNA-binding ligands and maps RNA-ligand binding sites at nucleotide resolution. The current version of the technology requires gene-specific primers. Therefore, one goal of this project is to develop a generic approach for examining the ligand-binding ability of large RNAs. A second goal is to use Frag-JuMP to interrogate ligand-binding by the bacterial ribosomal RNA in live cells to understand fundamental principles that allow ligands to bind selectively to complex RNAs and to identify RNA-ligand binding sites in functional regions of the ribosome. These studies are expected to characterize RNA structures capable of binding small molecules and to identify the subset of these that constitute ligand- binding sites likely to modulate ribosome function. The long-term, sustained impacts of this research and training project include the creation of a comprehensive RNA-ligand discovery strategy that can immediately be used to discover RNA-binding molecules and to provide an unprecedented picture of ligand-binding sites in any cellular RNA. Molecules identified by Frag-JuMP are also readily linkable, facilitating generation of potent bivalent ligands by linking co-binding fragments. The field of small- molecule, RNA-targeted therapeutics is in its infancy and represents an enormous opportunity to manipulate the functions of diverse cellular processes in bacterial and eukaryotic cells. Training will emphasize development of new expertise in RNA chemical probing methods, technologies based on high- throughput sequencing, bioinformatics analyses, cell culture methods, and small-molecule fragment chemistry. This training, combined with my previously developed expertise in chemical synthesis, will equip me with unique, career transforming, skills in chemical biology and structural genomics. Upon completion of this cross-disciplinary training program, I will be equipped for a leadership position at the interface of chemistry and biology, focused on understanding how RNA structure governs biological mechanism and on developing RNA-targeted therapeutics.