Kinetics and mechanism of conformational changes in a G-quadruplex of thrombin-binding aptamer induced by Pb2+

It has been shown that guanine-rich DNA can fold into a G-quadruplex with certain metal cations. The spectral characteristics, thermostability, and kinetics for the formation of a Pb²⁺-driven G-quadruplex of thrombin-binding aptamer (TBA) were measured in the current work using a combination of ultraviolet (UV) and circular dichroism (CD) spectroscopy along with stopped-flow technique. CD spectra demonstrated that TBA could fold into a unique G-quadruplex with a strong positive peak at 312 nm. Analysis of the titration data reveals that the binding stoichiometry is 1:1 for the titration of TBA with Pb(NO₃)₂, which is in accordance with the localization of the Pb²⁺ ion between the adjacent G-quartets. Thermal denaturation profiles indicate that the Pb²⁺-induced intramolecular G-quadruplex is more stable than those driven by Na⁺ or K⁺ ions. Kinetic studies suggest that the Pb²⁺-induced folding G-quadruplex of TBA probably proceeds through the rapid formation of an intermediate Pb²⁺-TBA complex, which then isomerizes to the fully folded structure. Conformational changes transpire after the addition of Pb(NO₃)₂ to the Na⁺- or K⁺-induced G-quadruplexes, which may be attributed to the replacement of Na⁺ or K⁺ ions by Pb²⁺ ions and the generation of a more compact structure of the Pb²⁺-TBA structure. The relaxation time, τ, of folding the G-quadruplex is reduced from 1.05 s in the presence of Pb ²⁺ ions alone to 0.34 s under the cooperation of initially added Na⁺ ions, while τ is increased to 8.33 s under the competition of initially added K⁺ ions. © 2011 American Chemical Society.