RNA G-Quadruplex Emerges from a Coil-Like Ensemble via Multiple Pathways

RNA G-quadruplexes (rG4s) are vital structural elements in gene regulation and genome stability. In untranslated regions of mRNAs, rG4s influence translation efficiency and mRNA localization. Additionally, rG4s of long non-coding RNAs and telomeric RNA play roles in RNA processing and cellular aging. Despite their significance, atomic-level folding mechanisms of rG4s remain poorly understood due to their complexity. We used enhanced-sampling all-atom molecular dynamics simulations to model the folding of an r(GGGA) ₃ GGG sequence into a parallel-stranded rG4. The folding pathways suggest that RNA initially adopts a compact coil- like ensemble, marked by dynamic guanine stacking and pairing. The three-quartet rG4 builds up gradually from the coil via diverse routes involving strand rearrangements and guanine incorporations. While the folding mechanism is multi-pathway, various two-quartet rG4s seem to be a common transitory ensemble for most routes. Our simulations also exposed force-field imbalances, with the predicted folding free energy of +12.5 kcal/mol deviating from experiments. Additionally, the enhanced sampling protocol, combining well-tempered metadynamics with solute tempering, faced productivity challenges on the multidimensional free-energy surface. Overall, this study provides atomistic insights into rG4 folding, emphasizing compact coil-like ensembles as key precursors, while revealing limitations in simulating non-canonical RNA structures.