Kinetics and Mechanism of Conformational Changes in a G-Quadruplex of Thrombin-Binding Aptamer Induced by Pb²⁺

Abstract

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 Pb(2+)-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(NO(3))(2), which is in accordance with the localization of the Pb(2+) ion between the adjacent G-quartets. Thermal denaturation profiles indicate that the Pb(2+)-induced intramolecular G-quadruplex is more stable than those driven by Na(+) or K(+) ions. Kinetic studies suggest that the Pb(2+)-induced folding G-quadruplex of TBA probably proceeds through the rapid formation of an intermediate Pb(2+)-TBA complex, which then isomerizes to the fully folded structure. Conformational changes transpire after the addition of Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) ions by Pb(2+) ions and the generation of a more compact structure of the Pb(2+)-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.

Kinetics and mechanism of G-quadruplex formation and conformational switch in a G-quadruplex of PS2.M induced by Pb2+

“…In short, the initially added Na + ions have a synergetic effect with Pb 2+ ions on the structural transition of the PS2.M chain, whereas the K + ions have a competitive effect on this transition. This result is quite consistent with those of the previous studies (45).

Figure 7.Proposed mechanism of the structural transition in a G-quadruplex of PS2.M induced by Pb 2+ ions.

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“…These relaxation times are much shorter than those of the G-quadruplexes induced by monovalent cations (34,44), which may be partly due to the higher charge of the Pb 2+ ions. As expected, the folding rates are independent of the concentration of Pb(NO 3 ) 2 , suggesting rate-saturation kinetics, which is in accordance with the high stability of the Pb 2+ -induced G-quadruplex (45). Recently, a simulation study (46) on the binding of cations to G-rich DNA showed that it is a complex multiple pathway process, which is strongly affected by the type of the cations.…”

RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms

“…In contrast, the biologically relevant divalent cations Mg 2+ and Ca 2+ had no effect on RNA G4 stability. However, the data also may suggest that other divalent cations, such as harmful heavy metals (Pb 2+ , Cd 2+ or Hg 2+ ), may bind and disturb G4 structures contributing to their toxicity; such binding is reported for guanine-rich oligos 30; 31 .…”

Spinach RNA aptamer detects lead(ii) with high selectivity

“…Micromolar concentrations of Pb 2+ induce the formation of DNA G-quadruplexes even in the absence of K + or Na + , the two most common cations associated with G-quadruplexes. Pb 2+ -stabilized G-quadruplexes adopt a more compact structure than those stabilized by monovalent cations due to the smaller size and high charge density of Pb 2+ 6a,10,11,12a,b . We wondered whether these observations extend to the RNA G-quadruplex in Spinach.…”