Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism

G-quadruplexes play important roles in cellular regulatory functions, but despite significant experimental and theoretical efforts, their folding mechanisms remain poorly understood. In this context, we developed a T-jump experiment to access the thermal denaturation and renaturation dynamics of short intramolecular G-quadruplexes in vitro , on the time scale of a few hundred milliseconds. With this new setup, we compared the thermal denaturation and renaturation kinetics of three antiparallel topologies made of the human telomeric sequences d[(5'-GGG(TTAGGG) 3 -3']/Na + and d[5'-AGGG(TTAGGG) 3 -3']/Na + and the thrombin-binding aptamer sequence d[5'-GGTTGGTGTGGTTGG-3']/K + , with those of the parallel topology made of the human CEB25 minisatellite d[5'-AAGGGTGGGTGTAAGTGTGGGTGGGT-3']/Na + . In all cases, exponential kinetics of the order of several hundred milliseconds were observed. Measurements performed for different initial temperatures revealed distinct denaturation and renaturation dynamics, ruling out a simple two-state mechanism. The parallel topology, in which all guanines adopt an anti conformation, displays much slower dynamics than antiparallel topologies associated with very low activation barriers. This behavior can be explained by the constrained conformational space due to the presence of the single-base propeller loops that likely hinders the movement of the coiled DNA strand and reduces the contribution of the entropy during the renaturation process at high temperatures.