The Role of Mg+2 Ion Interactions in Folding of the Twister Ribozyme and PreQ1 Riboswitch Revealed through Umbrella Sampling Combined with Oscillating Chemical Potential Grand Canonical Monte Carlo/Molecular Dynamics Simulations

Ribozymes, RNA molecules that work as enzymes, exhibit self-cleaving properties or catalytic mechanisms for processing of biomolecules. However it is challenging to resolve atomic-details of the folding landscape of ribozymes as they assume the tertiary structures required for catalytic activity. Metal ions, especially Mg +2 ions, are known to neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures. Earlier studies have determined specific binding sites of Mg +2 ions for RNAs, but are limited to folded conformations, such that their role in RNA folding processes is still unclear. Here, we use a computational approach that combines umbrella sampling with oscillating chemical potential Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) simulations to capture the atomic-level details of the intermediate states of RNA folding and identify the ion-RNA interactions that drive the folding pathway. The oscillating excess chemical potential allows for the sampling of Mg +2 ion distributions of the partially folded states of RNA without any bias from initial Mg +2-RNA configurations. We investigate the unfolding of two RNA molecules, the twister ribozyme and preQ 1 riboswitch, along reaction coordinates used in experimental studies. We also simulate the ribozymes at different ion concentrations and capture the effect of ions on the folding potentials of mean force. Results reveal the stability of intermediate states and track the ion-mediated conformational changes. Overall, the present study establishes a better understanding of how Mg +2 ion-interactions contribute to RNA folding