Discovery of RNA-Reactive Small Molecules Guides the Design of Electrophilic Modules for RNA-Specific Covalent Binders

RNA is a key drug target that can be modulated by small molecules; however, covalent binders of RNA remain largely unexplored. Using a high-throughput mass spectrometry screen of 2,000 electrophilic compounds, we identified ligands that react with RNA in a binding-dependent manner. RNA reactivity was influenced by both the reactive group and the noncovalent RNA-binding scaffold. In addition to known RNA-reactive electrophiles such as N-acylimidazoles and bis(2-chloroethyl)amines, covalent screening enabled the surprising discovery that common thiol-reactive electrophiles (chloroacetamide) and rarely characterized electrophiles (3-chloropivalamide) cross-linked to RNA. These results suggest that electrophiles commonly used for protein targeting can also covalently modify RNA, potentially contributing to both on- and off-target effects. This insight enabled the design of an RNA-specific covalent compound by modifying a bis-benzimidazole scaffold, originally identified to bind DNA, to react selectively with the expanded triplet repeat RNA, r(CUG)exp, that causes myotonic dystrophy type 1 (DM1). Selectivity appears to arise from differences in the RNA and DNA binding modes, revealing that proper positioning of the electrophile toward the nucleophilic guanine residue is important for efficient covalent bond formation. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules.