SAR by SHAPE: Towards Small Molecules that Target RNA

Abstract

RNA plays critical roles in diverse biological processes but has proven to be a difficult target for small molecule drug development. I addressed this challenge by developing automated SAR by SHAPE (structure-activity-relationships by selective 2’-hydroxyl acylation analyzed by primer extension), a powerful approach for rapid and generic RNA screening against small molecule libraries. I incorporated three innovations in automated SAR by SHAPE that, together, permit effective small molecule screening campaigns against diverse RNA targets. First, I developed and programmed a magnetic bead-based SHAPE protocol on a liquid handling robot. Second, I created an automated SHAPE analysis pipeline by combining reference-based analysis and novel error-correcting algorithms. Third, I applied single-nucleotide resolution statistical hit detection to identify novel RNA ligands.
In a successful proof-of-concept for automated SAR by SHAPE, I screened nearly 500 small molecules against the Hepatitis C Virus Internal Ribosome Entry Site (HCV IRES), a large, highly structured RNA crucial for viral translation and replication. This screen identified more than 80 previously unknown IRES ligands. Significantly, several of these ligands also inhibited IRES-mediated translation in a dual luciferase assay.