Magnesium Regulates\nRNA Ring Dynamics and Folding\nin Subgenomic Flaviviral RNA

Mosquito-borne flaviviruses including\ndengue, Zika, yellow\nfever,\nand regional encephalitis produce a large amount of short subgenomic\nflaviviral RNAs during infection. A segment of these RNAs named as\nxrRNA1 features a multi-pseudoknot (PK)-associated structure, which\nresists the host cell enzyme (XRN1) from degrading the viral RNA.\nWe investigate how this long-range RNA PK folds in the presence of\ncounterions, specifically in a mix of monovalent (K<sup>+</sup>) and\ndivalent (Mg<sup>2+</sup>) salts at physiological concentrations.\nIn this study, we use extensive explicit solvent molecular dynamics\n(MD) simulations to characterize the RNA ion environment of the folded\nRNA conformation, as determined by the crystal structure. This allowed\nus to identify the precise locations of various coordinated RNA–Mg<sup>2+</sup> interactions, including inner-sphere/chelated and outer-sphere\ncoordinated Mg<sup>2+</sup>. Given that RNA folding involves large-scale\nconformational changes, making it challenging to explore through classical\nMD simulations, we investigate the folding mechanism of xrRNA1 using\nan all-atom structure-based RNA model with a hybrid implicit–explicit\ntreatment of the ion environment via the dynamic counterion condensation\nmodel, both with and without physiological Mg<sup>2+</sup> concentration.\nThe study reveals potential folding pathways for this xrRNA1, which\nis consistent with the results obtained from optical tweezer experiments.\nThe equilibrium and free energy simulations both capture a dynamic\nequilibrium between the ring-open and ring-close states of the RNA,\ndriven by a long-range PK interaction. Free energy calculations reveal\nthat with the addition of Mg<sup>2+</sup> ions, the equilibrium shifts\nmore toward the ring-close state. A detailed analysis of the free\nenergy pathways and ion-mediated contact probability map highlights\nthe critical role of Mg<sup>2+</sup> in bridging G50 and A33. This\nMg<sup>2+</sup>-mediated connection helps form the long-range PK which\nin turn controls the transition between the ring-open and ring-close\nstates. The study underscores the critical role of Mg<sup>2+</sup> in the RNA folding transition, highlighting specific locations of\nMg<sup>2+</sup> contributing to the stabilization of long-range PK\nconnections likely to enhance the robustness of Xrn1 resistance of\nflaviviral xrRNAs.