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

Abstract

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