Development of Site-Specific Mg<sup>2+</sup>–RNA Force Field Parameters: A Dream or Reality? Guidelines from Combined Molecular Dynamics and Quantum Mechanics Simulations

The vital contribution of Mg<sup>2+</sup> ions to RNA biology is challenging to dissect at the experimental level. This calls for the integrative support of atomistic simulations, which at the classical level are plagued by limited accuracy. Indeed, force fields intrinsically neglect nontrivial electronic effects that Mg<sup>2+</sup> exerts on its surrounding ligands in varying RNA coordination environments. Here, we present a combined computational study based on classical molecular dynamics (MD) and Density Functional Theory (DFT) calculations, aimed at characterizing (i) the performance of five Mg<sup>2+</sup> force field (FF) models in RNA systems and (ii) how charge transfer and polarization affect the binding of Mg<sup>2+</sup> ions in different coordination motifs. As a result, a total of ∼2.5 μs MD simulations (100/200 ns for each run) for two prototypical Mg<sup>2+</sup>-dependent ribozymes showed remarkable differences in terms of populations of <i>inner-sphere</i> coordination site types. Most importantly, complementary DFT calculations unveiled that differences in charge transfer and polarization among recurrent Mg<sup>2+</sup>–RNA coordination motifs are surprisingly small. In particular, the charge of the Mg<sup>2+</sup> ions substantially remains constant through different coordination sites, suggesting that the common philosophy of developing site-specific Mg<sup>2+</sup> ion parameters is not in line with the physical origin of the Mg<sup>2+</sup>–RNA MD simulations inaccuracies. Overall, this study constitutes a guideline for an adept use of current Mg<sup>2+</sup> models and provides novel insights for the rational development of next-generation Mg<sup>2+</sup> FFs to be employed for atomistic simulations of RNA.