Machine learning-augmented molecular dynamics simulations (MD) reveal insights into the disconnect between affinity...

The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.