Kinetic and Thermodynamic Control of G-Quadruplex Polymorphism by Na⁺ and K⁺ Cations

G-Quadruplexes (G4s) are ubiquitous nucleic acid foldingmotifsthat exhibit structural diversity that is dependent on cationic conditions.In this work, we exploit temperature-controlled single-molecule fluorescenceresonance energy transfer (smFRET) to elucidate the kinetic and thermodynamicmechanisms by which monovalent cations (K+ and Na+) impact folding topologies for a simple G-quadruplex sequence (5 & PRIME;-GGG-(TAAGGG)(3)-3 & PRIME;) with a three-state folding equilibrium. Kineticmeasurements indicate that Na+ and K+ influenceG4 formation in two distinctly different ways: the presence of Na+ modestly enhances an antiparallel G4 topology through aninduced fit (IF) mechanism with a low affinity (K (d) = 228 & PLUSMN; 26 mM), while K+ drives G4 intoa parallel/hybrid topology via a conformational selection (CS) mechanismwith much higher affinity (K (d) = 1.9 & PLUSMN;0.2 mM). Additionally, temperature-dependent studies of folding rateconstants and equilibrium ratios reveal distinctly different thermodynamicdriving forces behind G4 binding to K+ (& UDelta;H & DEG;(bind) > 0, & UDelta;S & DEG;(bind) > 0) versus Na+ (& UDelta;H & DEG;(bind) < 0, & UDelta;S & DEG;(bind) < 0), which further illuminates the diversityof the possiblepathways for monovalent facilitation of G-quadruplex folding.