Interrogating RNA–Small Molecule Interactions with Structure Probing and Artificial Intelligence-Augmented Molecular Simulations

[Image: see text] While there is increasing interest in the study of RNA as a therapeutic target, efforts to understand RNA-ligand recognition at the molecular level lag far behind our understanding of protein–ligand recognition. This problem is complicated due to the more than 10 orders of magnitude in time scales involved in RNA dynamics and ligand binding events, making it not straightforward to design experiments or simulations. Here, we make use of artificial intelligence (AI)-augmented molecular dynamics simulations to directly observe ligand dissociation for cognate and synthetic ligands from a riboswitch system. The site-specific flexibility profiles from our simulations are compared with in vitro measurements of flexibility using selective 2′ hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). Our simulations reproduce known relative binding affinity profiles for the cognate and synthetic ligands, and pinpoint how both ligands make use of different aspects of riboswitch flexibility. On the basis of our dissociation trajectories, we also make and validate predictions of pairs of mutations for both the ligand systems that would show differing binding affinities. These mutations are distal to the binding site and could not have been predicted solely on the basis of structure. The methodology demonstrated here shows how molecular dynamics simulations with all-atom force-fields have now come of age in making predictions that complement existing experimental techniques and illuminate aspects of systems otherwise not trivial to understand.