Project Summary RNA is now recognized as a compelling therapeutic target because it lies upstream of all protein function and has cellular roles that go beyond protein translation. Similar to proteins, RNA molecules can fold to create complex tertiary structures that form binding pockets capable of specific small-molecule engagement. However, compared to proteins, few small molecules have been rationally designed to target RNA. The central challenges to developing RNA-targeting ligands are, first, the difficulty of identifying ligandable sites on RNA and, second, the lack of direct strategies for converting ligand binding into functional perturbation of mRNA stability, translation and regulation. Both of these challenges can be addressed by a new ligand-engagement technology developed in our lab. This technology enables nucleotide-resolution capture of ligand-RNA interactions via a high-throughput, cell-based screen of a small-molecule library versus an RNA library – the cellular transcriptome. In preliminary work, the ligand-engagement technology has been validated with photo- reactive probes in bacterial cells, and shown to be modular and applicable to diverse chemistries and biological systems. The first goal of this training and research proposal is to extend the technology to human cells and to develop chemistries that yield covalent adducts with RNA specifically at sites of RNA tertiary structures. The second goal of this proposal is to characterize the effects of covalent modification on gene expression for a subset of ligandable RNAs. Completion of these aims will create a concise strategy for linking small molecule engagement with RNA to direct functional modulation of gene expression. This multi-disciplinary project emphasizes training in the fields of technology development, RNA biology, and bioinformatics. Expertise in these fields, combined with my previous experiences as a chemical biologist and proteomicist, will position me at the forefront of RNA-targeted therapeutics development. I envision using this training as a launching point for an independent career as a scientific investigator and visionary leader with a research agenda focused on developing new technologies that modulate RNA function and impact human health.