Molecular Simulations to Investigate the Impact of N6-Methylation in RNA Recognition: Improving Accuracy and Precision of Binding Free Energy Prediction.

N6-Methyladenosine (m ⁶ A) is a prevalent RNA post-transcriptional modification that plays crucial roles in RNA stability, structural dynamics, and interactions with proteins. The YT521-B (YTH) family of proteins, which are notable m ⁶ A readers, functions through its highly conserved YTH domain. Recent structural investigations and molecular dynamics (MD) simulations have shed light on the mechanism of recognition of m ⁶ A by the YTHDC1 protein. Despite advancements, using MD to predict the stabilization induced by m ⁶ A on the free energy of binding between RNA and YTH proteins remains challenging due to inaccuracy of the employed force field and limited sampling. For instance, simulations often fail to sufficiently capture the hydration dynamics of the binding pocket. This study addresses these challenges through an innovative methodology that integrates metadynamics, alchemical simulations, and force-field refinement. Importantly, our research identifies hydration of the binding pocket as giving only a minor contribution to the binding free energy and emphasizes the critical importance of precisely tuning force-field parameters to experimental data. By employing a fitting strategy built on alchemical calculations, we refine the m ⁶ A partial charge parameters, thereby enabling the simultaneous reproduction of N6 methylation on both the protein binding free energy and the thermodynamic stability of nine RNA duplexes. Our findings underscore the sensitivity of binding free energies to partial charges, highlighting the necessity for thorough parametrization and validation against experimental observations across a range of structural contexts.