Covalent Probes Reveal Small-Molecule Binding Pockets in Structured RNA and Enable Bioactive Compound Design

The SARS-CoV-2 frameshift stimulation element (FSE) is a critical RNA structure that is essential for viral replication and represents a promising target for antiviral intervention. Here, Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) covalent target validation and binding site mapping was applied, to identify small-molecule binding pockets within the FSE and ultimately develop a ligandability map. These studies employed ∼ 190 Chem-CLIP fragments, including the fluoroquinolone merafloxacin, previously shown to interact with this element. Covalent mapping defined merafloxacin’s binding pocket at a nucleotide-level resolution and revealed interactions that, along with structure-based design, efficient one-pot on-plate synthesis and competitive displacement assays, enabled the development of bioactive compounds with antiviral activity. Complementary chemical probing with dimethyl sulfate (DMS) in the presence of a bioactive ligand, coupled to Deconvolution of RNA Alternative Conformations (DRACO), revealed that compound binding increased the reactivity of specific nucleotides with DMS, indicative of changes in local RNA folding. These results highlight the importance of combining Chem-CLIP and DMS profiling to differentiate direct ligand binding from ligand-induced changes in RNA structure. In addition, in silico pocket analysis of FSE structures derived from cryogenic-electron microscopy (cryo-EM) studies identified four recurring cavities, including the experimentally determined merafloxacin and Chem-CLIP fragments binding pockets. Altogether, the findings advance our understanding of RNA–ligand interactions and support a strategy to design and discover small molecules that bind RNA structures.