Site‐Selective Ligand Selection by Mutational Profiling for Covalent RNA Targeting

Abstract To facilitate the design of RNA‐targeted covalent probes we report a bottom‐up, structure‐informed approach using mutational profiling. We designed a compact library of covalent probes based on the scaffold of Ribocil, a selective inhibitor targeting the bacterial FMN riboswitch and screened them against the FMN riboswitch aptamer from Fusobacterium nucleatum , Bacillus subtilis , Escherichia coli , and Staphylococcus aureus . This yielded Covacil, a highly base‐selective probe that covalently modifies the FMN riboswitch aptamer at low micromolar concentrations, within 10 minutes. We validate the site‐selectivity and covalency of the probe by competitive photo‐affinity labelling and mass‐spectrometry. When compared to non‐targeted covalent probes, Covacil displayed >1,000‐fold increased selectivity toward a specific base within the FMN riboswitch aptamer. Finally, we apply Covacil to total RNA and demonstrate that it maintains its base‐selective reactivity for the FMN riboswitch within the entire transcriptome.