Ligand-binding pockets in RNA, and where to find them [II]

RNAs are critical regulators of gene expression, and their functions are often mediated by complex secondary and tertiary structures. Structured regions in RNA can selectively interact with small molecules – via well-defined ligand binding pockets – to modulate the regulatory repertoire of an RNA. The broad potential to modulate biological function via RNA-ligand interactions remains unrealized, however, due to challenges in identifying RNA motifs with the ability to bind ligands with good physiochemical properties (often termed drug-like). Here, we devise fpocketR, a computational strategy that accurately detects pockets capable of binding drug-like ligands in RNA structures. Remarkably few, less than 60, of such pockets hadbeen previously visualized. We experimentally confirmed the ligandability of novel pockets detected with fpocketR using a fragment-based approach introduced here, Frag-MaP, that detects ligand-binding sites in cells. Analysis of pockets detected by fpocketR and validated by Frag-MaP reveals dozens of newly identified sites able to bind drug-like ligands, supports a model for RNA secondary structural motifs able to bind quality ligands, and creates a broad framework for understanding the RNA ligand-ome. We used photo-crosslinking fully functionalize fragment (FFF) probes and mutational profiling reverse transcription (MaP-RT) to determine the nucleotide position of RNA-ligand interactions. The FFF probes tested include a non-selective control (methyl), a positive control (linezolid), and fragments (6-aminoquinoxaline and Enamine Z4225957206).