Integrative Ensemble Modeling reveals RNA conformations targetable by small molecules

Abstract

RNA molecules explore heterogeneous conformational ensembles that are essential for their biological function and molecular recognition, yet this intrinsic flexibility poses a major challenge for structure-based drug discovery. In particular, the absence of well-defined binding pockets in static structures limits the identification of ligandable sites. Here, we present an integrative ensemble-based approach that combines enhanced-sampling molecular dynamics simulations with Nuclear Magnetic Resonance data to characterize the conformational landscape of the HIV-1 TAR RNA at atomic resolution. Starting from extensive sampling, we refined the resulting conformational distribution through maximum-entropy reweighting to achieve quantitative agreement with experimental data. Analysis of the reweighted ensemble reveals a diverse set of conformational substates, including compact arrangements that exhibit pocket features compatible with ligand recognition and overlap with known ligand-bound structures. At the same time, highly ligandable conformations, which are only marginally populated, might nonetheless be critical for RNA recognition. Our results demonstrate that integrative ensemble modeling can reveal pharmacologically relevant RNA conformations that are not apparent from experimental static structures, providing a framework for ensemble-based strategies in RNA-targeted drug discovery.