Parallel G-quadruplex folds via multiple paths involving G-tract stacking and structuring from coil ensemble

Abstract

G-quadruplexes (G4s) are non-canonical nucleic acid structures that fold through complex processes. Characterization of G4 folding landscape contributes to comprehending G4 roles in gene regulation but is challenging for experiments and computations. Here we investigate the folding of a three-quartet parallel DNA G4 with (GGGA)3GGG sequence using all-atom explicit-solvent enhanced-sampling molecular dynamics (MD) simulations. We suggest an early formation of guanine stacks in the G-tracts, which behave as semi-rigid blocks in the folding process. The parallel G4 folding is initiated by the formation of a collapsed compact coil-like ensemble. Structuring of the G4 from the coil then proceeds via various cross-like, hairpin, slip-stranded, and two-quartet ensembles and can bypass the G-triplex structure. While parallel G-hairpins are extremely unstable when isolated, they are more stable inside the coil structure. Folding of parallel G4 does not appear to involve any salient intermediates and, instead, it is an extremely multiple-pathway process. On the methodology side, we show that the AMBER DNA force field predicts the folded G4 to be less stable than the unfolded ensemble, uncovering substantial force-field issues. Overall, we provide unique atomistic insights into the folding landscape of parallel-stranded G4 but also reveal limitations of the state-of-the-art MD techniques.