PROJECT SUMMARY Despite the recent explosion of interest in RNA targeting, therapeutic potential is presently limited by a lack of fundamental understanding of how to achieve selective and functional small molecule targeting. The overarching focus of our research program is to elucidate the key drivers of selectivity in small molecule:RNA recognition and to apply these principles to facilitate development of RNA-targeted chemical probes and therapeutics that modulate RNA function. To begin, we identified physicochemical, structural, and spatial properties of biologically active RNA ligands that are distinct from those of protein-targeted ligands. Synthetic elaboration of RNA binding scaffolds into a library enriched with these properties has led to improved recognition of disease relevant RNA, including viral and long noncoding RNA structures. We used pattern recognition protocols to identify RNA topologies that can be differentially recognized by small molecules and have elaborated this technique to visualize conformational changes. This combined work has led to remarkable successes such as the targeting of enterovirus 71 RNA, where our ligand induced a dramatic conformation change that increased binding of a repressive human protein, decreased viral translation, and inhibited viral replication. Our approach is also showing preliminary success against SARS-CoV2 regulatory RNA. Building off these accomplishments, we propose to develop new libraries and screening methods to understand functional selectivity against a range of more complex tertiary and quaternary structures. Insights into the most critical driving factors will be revealed through pattern recognition / machine learning analysis as well as through an ensemble-based QSAR method that will allow rational targeting of any RNA. Key determinants of biological selectivity will be revealed in high throughput cell-based assays. These discoveries will be further enhanced by elucidation of the structure-dynamics-function relationships of oncogenic long noncoding RNAs. Our tools have lowered barriers to the discovery of selective RNA ligands. The proposed work will finally open a new horizon in RNA-targeting, in which chemical and biological scientists will readily and productively screen for small molecule probes against a wide range of RNA molecules, including those relevant to human disease. Such capabilities will allow the therapeutic potential of RNA to be fully exploited and inherently transform our understanding of molecular biology.