Critical Role of Mg²⁺ Ions in RNA Folding Transitions: Anchoring the A-Minor Twist in the SAM-II Riboswitch

Magnesium ions (Mg²⁺) play a crucial role in stabilizing various RNA tertiary motifs, such as pseudoknots, G-quadruplexes, kissing loops, and A-minor motifs, to name a few. Despite their importance, the precise location and role of Mg²⁺ ions in RNA folding are challenging to characterize both experimentally and computationally. In this study, we employ an all-atom structure-based model integrated with the dynamic counterion condensation (DCC) model to investigate the folding and unfolding transitions of apo SAM-II riboswitch RNA at physiological concentrations of Mg²⁺. Using the Energy Landscape Visualization Method (ELViM), we trace the transitions between conformational phases, focusing on magnesium interactions. ELViM reveals key structural ensembles during the transition from the unfolded to the folded state, facilitated by a partially folded intermediate, which is conformationally similar to that found in early ¹³C-CEST NMR. Interestingly, this study finds the rate-limiting transition from the unfolded state to this intermediate initiated by the formation of an A-minor twist interaction, a stable scaffold in the aptamer domain, stabilized by specific Mg²⁺ coordination. The contact probability map shows that this specific Mg²⁺ bridges a helical region and an internal loop, mitigating electrostatic repulsion at the phosphate level. As a result, a set of hydrogen-bond-mediated interactions between the loop and the minor groove of the helix is stabilized, supporting the formation of the A-minor twist. This study underscores the critical role of Mg²⁺ in driving the rate-limiting event of RNA folding and highlights its strategic location in stabilizing the A-minor twist motif, essential for the global packing and regulatory function of the SAM-II riboswitch aptamer.