Dynamics of the Unfolding of Riboswitch in Free Energy Landscape: A Theoretical Study

Riboswitches are regulatory RNA elements that control gene expression by undergoing conformational changes upon metabolite binding. Understanding their folding and unfolding dynamics is key to elucidating their regulatory roles and therapeutic potential. This study investigates the unfolding dynamics of the adenosine deaminase (add) A-riboswitch aptamer using steered molecular dynamics (SMD) simulations with constant-velocity (CV) pulling. The Jarzynski equality is applied to derive potential of mean force (PMF) profiles and force-extension curves for ligand-bound and unbound states, revealing distinct conformational transitions. CV-SMD coupled with Jarzynski equality enables quantitative reconstruction of free-energy profiles from multiple non-equilibrium realizations and computes the thermodynamic parameters. Unfolding occurs through a sequential multistep pathway, with adenine binding stabilizing the folded state and enhancing energetic stability. Force-extension data show a linear relationship between simulation time and molecular extension, while analyses of radius of gyration (Rg) and root mean square deviation (RMSD) highlight ligand-induced structural stability. Lower pulling velocities capture intermediate states more effectively than higher velocities. These results provide computational insights into RNA folding mechanisms consistent with experimental observations.