Integrative in silico and in vitro screening of small molecules targeting RNA

Targeting structured RNA elements with small molecules has emerged as a promising yet technically challenging strategy for antiviral drug discovery. Here, we present a comprehensive and experimentally validated workflow for the integrative in silico and in vitro screening of RNA-binding small molecules. The approach is exemplified using conserved RNA elements from the SARS-CoV-2 genome, including the 5'-terminal stem-loop 1 and the programmed -1 ribosomal frameshift pseudoknot, but is broadly applicable to other structured RNAs. The workflow integrates high-resolution RNA structural ensemble generation with virtual screening (VS) and nuclear magnetic resonance (NMR)-based experimental validation. Conformational ensembles generated by fragment-assembly approaches serve as targets for docking chemically diverse fragments and lead-like libraries. Top-ranked compounds are prioritized through consensus scoring and evaluated using ligand- and RNA-observed NMR experiments to confirm binding, characterize interaction modes, and assess specificity. Such ranking allows for NMR-guided fragment optimization, which enables systematic improvement of solubility, affinity, and selectivity through iterative medicinal chemistry in the pharmaceutical pipeline. Detailed procedures are provided for library preparation, ensemble-based VS, hit validation, data interpretation, and progression toward functional assays, together with practical considerations, quality-control parameters, and troubleshooting guidance to ensure reproducibility. By combining computational and experimental strategies that select for high-specificity ligands within a unified framework, this set of protocols accelerates the discovery and optimization of RNA-targeting small molecules and provides a scalable platform for RNA-focused drug discovery. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Target and library preparation for virtual screening Basic Protocol 2: Ensemble-based virtual screening of low-molecular-weight compounds against RNA targets Basic Protocol 3: Comparative hit prioritization and selectivity filtering for RNA-binding small molecules Basic Protocol 4: Preparation of RNA samples for in vitro validation of small-molecule binding Basic Protocol 5: NMR-based in vitro screening of RNA-small molecule interactions Support Protocol: Preparation of ligand stocks and NMR-based quality control Basic Protocol 6: Characterization and prioritization of validated RNA-binding hits.