Unraveling Mg²⁺-RNA binding with atomistic molecular dynamics

Interaction with divalent cations is of paramount importance for RNA structural stability and function. We report here a detailed molecular dynamics study of all the possible binding sites for Mg²⁺ on an RNA duplex, including both direct (inner sphere) and indirect (outer sphere) binding. In order to tackle sampling issues, we develop a modified version of bias-exchange metadynamics, which allows us to simultaneously compute affinities with previously unreported statistical accuracy. Results correctly reproduce trends observed in crystallographic databases. Based on this, we simulate a carefully chosen set of models that allows us to quantify the effects of competition with monovalent cations, RNA flexibility, and RNA hybridization. Our simulations reproduce the decrease and increase of Mg²⁺ affinity due to ion competition and hybridization, respectively, and predict that RNA flexibility has a site-dependent effect. This suggests a nontrivial interplay between RNA conformational entropy and divalent cation binding.