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

G-Quadruplexes (G4s) are ubiquitous nucleic acid folding motifs that exhibit structural diversity that is dependent on cationic conditions. In this work, we exploit temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) to elucidate the kinetic and thermodynamic mechanisms by which monovalent cations (K⁺ and Na⁺) impact folding topologies for a simple G-quadruplex sequence (5'-GGG-(TAAGGG)₃-3') with a three-state folding equilibrium. Kinetic measurements indicate that Na⁺ and K⁺ influence G4 formation in two distinctly different ways: the presence of Na⁺ modestly enhances an antiparallel G4 topology through an induced fit (IF) mechanism with a low affinity (K_d = 228 ± 26 mM), while K⁺ drives G4 into a parallel/hybrid topology via a conformational selection (CS) mechanism with much higher affinity (K_d = 1.9 ± 0.2 mM). Additionally, temperature-dependent studies of folding rate constants and equilibrium ratios reveal distinctly different thermodynamic driving forces behind G4 binding to K⁺ (ΔH°_bind > 0, ΔS°_bind > 0) versus Na⁺ (ΔH°_bind < 0, ΔS°_bind < 0), which further illuminates the diversity of the possible pathways for monovalent facilitation of G-quadruplex folding.