Small-Molecule-Directed RNA Modification via Proximity-Driven Catalysis

Abstract Selective chemical modification of RNA is essential for RNA functionalization, probing RNA structure–function relationships and developing RNA-targeted therapeutics. Existing chemical strategies often rely on guanine accessibility or multiple helper DNA strands, restricting their generality and biological applicability. Inspired by DNA-guided DMAP catalysis and small-molecule binding-induced cross-linking, we report a small-molecule-directed, DMAP-catalyzed, proximity-driven strategy for site-selective RNA functionalization. By appending a catalytic DMAP moiety to RNA-binding ligands, 2′-OH groups are selectively acylated in the presence of azide-bearing acyl donors, enabling subsequent installation of bioorthogonal handles. This approach was validated across diverse RNAs, including Pepper and Clivia RNA aptamers, G-quadruplex Broccoli RNA, and endogenous FMN riboswitch RNA. For a 400 nt Pepper-7SK fusion, selective modification of the Pepper motif was achieved with minimal perturbation to the nucleus localization function of 7SK RNA. Optimized PEG-pentafluorophenyl (PFP) acyl donors provided enhanced reactivity and low background. The method operates catalytically, decouples ligand recognition from the labeling moiety, and enables selective enrichment of target RNAs, offering a versatile platform for RNA functionalization, ligand profiling, and potentially live-cell applications.