Kinetic and thermodynamic characterization of telomeric G-quadruplex by nonequilibrium capillary electrophoresis: application to G-quadruplex/duplex competition

In this paper, nonequilibrium capillary electrophoresis (NECE) was attempted for the first time to investigate a dual equilibrium system, where the intramolecular G-quadruplex folding was in competition with the intermolecular duplex formation. Samples of an equilibrium mixture of human telomeric DNA and its complementary strands were separated in capillaries under nonequilibrium conditions without [K.sup.+]. Polyethylene oxide was added to the running buffer facilitating the separation of single-stranded DNA, duplex, and G-quadruplex. Thus, the folding/unfolding rate constants of the G-quadruplex and the association/dissociation constants of the duplex could be simultaneously derived from the same experiment. Results indicated that the duplex formation induced minimal influence on the G-quadruplex folding. On the basis of the kinetic characterization of the G-quadruplex at varying temperatures, the thermodynamic parameters of the G-quadruplex could also be determined. Thus, the NECE method provided a new avenue for studying the kinetics and thermodynamics of nucleic acids within dual equilibrium systems with significant advantages of extreme-low sample cost ([~10.sup.-18] mol) and high repeatability.