Discovery of RNA-binding fragments using biolayer interferometry

Structured RNAs are increasingly explored as novel pharmacological targets for a range of diseases. Therefore, evaluating methods for RNA-focused hit discovery is crucial. Biolayer Interferometry (BLI), a label-free technique that detects biomolecular interactions by measuring changes in white light interference near the sensor surface, offers high throughput and multiplexing capabilities. While BLI has been widely adopted for protein-targeted screening, its application in RNA-targeted drug discovery remains largely unexplored. In this study, we demonstrate the effective use of BLI to investigate RNA-small molecule interactions using three different riboswitches, which are potential targets for novel antibiotics. Furthermore, we describe the successful use of BLI to identify fragment binders of these RNA targets. We combined the BLI experiments with ligand-based NMR as an orthogonal validation method and were able to identify seven competitive fragment binders of the flavin mononucleotide (FMN) riboswitch, each featuring scaffolds distinct from the previously known ligands.