Characterizing the unfolding activation barrier of c-MYC G-quadruplex by temperature-dependent single-molecule force...

Parallel-stranded G-quadruplexes (G4s) exhibit remarkable kinetic stability. However, it remains elusive whether this kinetic stability arises from an intrinsically high activation energy E_a or a constrained transition pathway. Here, we integrated an objective-based heating system with single-molecule magnetic tweezers to quantify the unfolding dynamics of c-MYC promoter G4s (MYC2345) from 16°C to 51°C. As the temperature increased, the peak unfolding force decreased from 58 ± 4 pN to 40 ± 4 pN, while the unfolding rate k_u increased from (4.6 ± 0.1) × 10^-8 s^-1 to (1.4 ± 0.1) × 10^-5 s^-1. Arrhenius analysis yielded a moderate unfolding activation energy of 137 ± 19 kJ·mol^-1 (∼55 k_BT). Notably, this Arrhenius slope is markedly shallower than that of duplex DNA, demonstrating that G4 unfolding is less sensitive to thermal fluctuation than duplex melting. The kinetic stability of the c-MYC G4 is mainly due to the small transition distance rather than a high enthalpic barrier. Furthermore, the folding rate k_f of G4 exhibits low temperature sensitivity, increasing 1.5-fold between 23°C and 37°C, suggesting that the folding kinetics are dominated by the entropic barrier. Our results provide a direct quantitative measurement of G4 energy landscapes.