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

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.