Abstract
RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg(2+), neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg(2+) in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg(2+)-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg(2+), as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg(2+) with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg(2+)-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg(2+)-ion interactions contribute to RNA structural properties and stability.