Towards Accurate Prediction of Protonation Equilibrium of Nucleic Acids

The role of protonated nucleotides in modulating the pH-dependent properties of nucleic acids is one of the emerging frontiers in the field of nucleic acid biology. The recent development of a constant pH molecular dynamics simulation (CPHMD^MSλD) framework for simulating nucleic acids has provided a tool for realistic simulations of pH-dependent dynamics. We enhanced the CPHMD^MSλD framework with pH-based replica exchange (pH-REX), which significantly improves the sampling of both titration and spatial coordinates. The results from our pK_a calculations for the GAAA tetraloop, which was predicted with lower accuracy previously due to sampling challenges, demonstrates that pH-REX reduces the average unsigned error (AUE) to 0.7 pK_a units, and the error of the most poorly predicted residue A17 was drastically reduced from 2.9 to 1.2 pK_a unit. Lastly, we show that pH-REX CPHMD^MSλD simulations can be used to identify the dominant conformation of nucleic acid structures in alternate pH environments. This work suggests that pH-REX CPHMD^MSλD simulations provide a practical tool for predicting nucleic acid protonation equilibrium from first-principles, and offering structural and mechanistic insight into the study of pH-dependent properties of nucleic acids.