Unfolding Kinetics of Human Telomeric G‑Quadruplexes Studied by NMR Spectroscopy

Characterization of the unfolding kinetics of G-quadruplexes (G4s) is the key to a better understanding of the biological function of G4s and is important for biomedical research and material design. Of interest is that slight variations of human telomeric sequences can form different types of G4 structures. In general, there is a correlation between unfolding kinetics and thermal stability. Here we examined this correlation by first systematic analysis of the unfolding kinetics of a variety of telomeric G4 structures using the real-time imino proton NMR spectra of DNA hybridization and hydrogen–deuterium exchange (HDX). We then measured the melting temperature (T m) and determined the Gibbs free energy (ΔG) of these G4 structures using differential scanning calorimetry (DSC). Our results showed that both T m and ΔG are slightly structure-dependent, except the T m of the parallel G4 structure is ∼10 °C higher than that of nonparallel G4 structures. The hybridization results showed that the decay times of different imino proton signals for each telomeric G4 structure are quite similar, which are also consistent with the time constant of the central G-tetrad obtained from HDX measurements. It is suggested that global unfolding is the rate-determining step for HDX, and each real-time imino proton NMR measurement can provide the intrinsic unfolding rate constant. The key finding is that the unfolding times of these various G4 structures are quite different and show no correlation between thermal stability and unfolding kinetics. Our results raised an issue that the folding and unfolding kinetics is more relevant for better understanding of biological function of G4 structures.