Emergence of Dynamic G-Tetraplex Scaffold: Uncovering Low Salt-Induced Conformational Heterogeneity and Folding Mechanism

The topological diversity of human telomeric G-quadruplex structures is intrinsically related to their folding mechanisms, and is significantly modulated by ion-atmospheric conditions. Unlike previous studies that focused on higher Na+ or K+ concentrations, this study explores G-quadruplex folding and dynamics under low NaCl conditions (≤100 mM) using single-molecule FRET microscopy and advanced structure-based DNA simulation techniques. The smFRET data reveal three distinct populations: unfolded, intermediate dynamic triplex, and dynamic tetraplex structural ensemble. The broad distribution of the folded population highlights the dynamic nature of the quadruplex structure under low salt conditions. In agreement with smFRET results, free energy simulations show that with the increase of NaCl concentration, the population shifts towards the folded state, and differentiates all intermediate structural ensembles. The dynamic equilibrium between the triplex and tetraplex scaffolds explains the microscopic basis of conformational heterogeneity within the folded basin. Simulations also reveal that the flexibility of dynamic tetraplex bases depends on the equilibrium distribution of ions underpinning a few ion-mediated dynamic non-native interactions in the G-quadruplex structure. Contrary to the previously held belief that Na+ induces minimal structural heterogeneity, our combined experimental and simulation approaches demonstrate and rationalize the structural variability in G-quadruplexes under low NaCl concentrations.