Free Energy Landscape of Magnesium Chelation Reveals Dynamic Prechelate Complexes Stabilized by Hybrid Inner–Outer-Sphere RNA–Ion Coordination

Magnesium ions (Mg 2+ ) play a critical role in RNA structure stabilization by forming various coordinated complexes, preferentially interacting with the backbone phosphate groups. Using extensive atomistic and free energy simulations across simple models and RNA structures of varying complexity, we characterized critical components of the RNA–ion atmosphere. Radial distribution function analysis reveals distinct peak positions for direct (inner) and solvent-separated (outer-sphere) Mg 2+ –phosphate coordination layers, aligning with solution X-ray diffraction data. Addressing force field limitations, the free energy calculations quantify the kinetic barriers for Mg 2+ –phosphate binding and benchmark the results against 25 Mg NMR measurement. Importantly, the free energy differences between inner-sphere and outer-sphere Mg 2+ coordination states, as well as between monocoordinated and bicoordinated (chelated) states, are found to be marginal. These findings suggest a dynamic equilibrium among multiple, barrier-separated Mg 2+ coordination states, consistent with prior simulations, NMR spectroscopy, and other spectroscopic studies. Free energy calculations further explore Mg 2+ chelation in a biphosphate-coordinated system, identifying a dynamic ensemble of prechelate complexes, in addition to a chelated and outer-sphere hexa-hydrated state of Mg 2+ . In the prechelated states, Mg 2+ maintains one inner-sphere interaction while simultaneously coordinating with multiple other phosphates in a solvent-separated manner─a hybrid inner–outer-sphere ion-coordination mode. The prechelated complexes spanning different solvent-separated layers undergo frequent transitions, mediated by a unique oxygen exchange mechanism between phosphate groups and water ligands. Insights into the free energy landscape of SAM-I riboswitch aptamer RNA further emphasize the significance of prechelate complexes for complex RNA structure stabilization, where multiple such solvent-separated dynamic phosphate groups are found to influence Mg 2+ –RNA coordination. The comprehensive thermodynamic analysis of Mg 2+ chelation and quantitative characterizations of various RNA–ion coordination modes provide critical insights for advancing RNA modeling and experimental exploration of complex phosphate networks in the RNA structures.