Discovery of RNA-Reactive Small Molecules Guides 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 RNA-binding scaffold. Electrophilic modules such as 3-chloropivalamide, bis(2-chloroethyl)amine, chloroacetamide, and N-acylimidazole that react with proteins also cross-linked to RNA, especially when paired with aromatic heterocycles, particularly those with a thieno[3,2-c]pyridinium core. 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 Hoechst 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 binding to the RNA major groove near the reactive site. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules.