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

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

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Denaturation profiles for the G-quadruplex of PS2.M (2.5 µM) induced by various cations in buffer solution (10 mM MES/Tris, pH 6.1). The UV-melting curves were measured at 303 nm for Pb(NO3)2 (0.5 mM) and at 295 nm for NaNO3 or KNO3 (50 mM).
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gkr1310-F3: Denaturation profiles for the G-quadruplex of PS2.M (2.5 µM) induced by various cations in buffer solution (10 mM MES/Tris, pH 6.1). The UV-melting curves were measured at 303 nm for Pb(NO3)2 (0.5 mM) and at 295 nm for NaNO3 or KNO3 (50 mM).

Mentions: UV-melting experiments were performed to examine the stability of the cation-induced G-quadruplexes (38). As shown in Figure 3, all curves demonstrate monophasic transition with different cooperativities, some broad while others somewhat sharper, depending on the ions used. The UV-melting analysis of PS2.M in the presence of 0.5 mM Pb(NO3)2 reveals a cooperative monophasic melting transition at 72 ± 2°C. Such a high-melting temperature suggests that the Pb2+-induced G-quadruplex of PS2.M is stable under physiological conditions. On the other hand, in the presence of 50 mM NaNO3 or KNO3, the Tm values of the G-quadruplexes are determined to be 42 and 51°C, respectively. The low-melting temperature for the Na+-induced G-quadruplex indicates that the complex should be largely unfolded at the physiological temperature. The higher Tm for the G-quadruplex induced by Pb(NO3)2 than those of NaNO3 or KNO3 is consistent with the higher binding affinity of the Pb2+ ions. In addition, the Tm remains constant over a 10-fold increase in strand concentration, indicating that the G-quadruplex is formed intramolecularly. UV-melting experiments have been previously reported (29,39,40), but the Tm values were different because different metal concentrations were usedFigure 3.


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

Liu W, Zhu H, Zheng B, Cheng S, Fu Y, Li W, Lau TC, Liang H - Nucleic Acids Res. (2012)

Denaturation profiles for the G-quadruplex of PS2.M (2.5 µM) induced by various cations in buffer solution (10 mM MES/Tris, pH 6.1). The UV-melting curves were measured at 303 nm for Pb(NO3)2 (0.5 mM) and at 295 nm for NaNO3 or KNO3 (50 mM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351173&req=5

gkr1310-F3: Denaturation profiles for the G-quadruplex of PS2.M (2.5 µM) induced by various cations in buffer solution (10 mM MES/Tris, pH 6.1). The UV-melting curves were measured at 303 nm for Pb(NO3)2 (0.5 mM) and at 295 nm for NaNO3 or KNO3 (50 mM).
Mentions: UV-melting experiments were performed to examine the stability of the cation-induced G-quadruplexes (38). As shown in Figure 3, all curves demonstrate monophasic transition with different cooperativities, some broad while others somewhat sharper, depending on the ions used. The UV-melting analysis of PS2.M in the presence of 0.5 mM Pb(NO3)2 reveals a cooperative monophasic melting transition at 72 ± 2°C. Such a high-melting temperature suggests that the Pb2+-induced G-quadruplex of PS2.M is stable under physiological conditions. On the other hand, in the presence of 50 mM NaNO3 or KNO3, the Tm values of the G-quadruplexes are determined to be 42 and 51°C, respectively. The low-melting temperature for the Na+-induced G-quadruplex indicates that the complex should be largely unfolded at the physiological temperature. The higher Tm for the G-quadruplex induced by Pb(NO3)2 than those of NaNO3 or KNO3 is consistent with the higher binding affinity of the Pb2+ ions. In addition, the Tm remains constant over a 10-fold increase in strand concentration, indicating that the G-quadruplex is formed intramolecularly. UV-melting experiments have been previously reported (29,39,40), but the Tm values were different because different metal concentrations were usedFigure 3.

Bottom Line: UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C.Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates.Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P R China.

ABSTRACT
DNA sequences with guanine repeats can form G-quartets that adopt G-quadruplex structures in the presence of specific metal ions. Using circular dichroism (CD) and ultraviolet-visible (UV-Vis) spectroscopy, we determined the spectral characteristics and the overall conformation of a G-quadruplex of PS2.M with an oligonucleotide sequence, d(GTG(3)TAG(3)CG(3)TTG(2)). UV-melting curves demonstrate that the Pb(2+)-induced G-quadruplex formed unimolecularly and the highest melting temperature (T(m)) is 72°C. The analysis of the UV titration results reveals that the binding stoichiometry of Pb(2+) ions to PS2.M is two, suggesting that the Pb(2+) ions coordinate between adjacent G-quartets. Binding of ions to G-rich DNA is a complex multiple-pathway process, which is strongly affected by the type of the cations. Kinetic studies suggest that the Pb(2+)-induced folding of PS2.M to G-quadruplex probably proceeds through a three-step pathway involving two intermediates. Structural transition occurs after adding Pb(NO(3))(2) to the Na(+)- or K(+)-induced G-quadruplexes, which may be attributed to the replacement of Na(+) or K(+) by Pb(2+) ions and the generation of a more compact Pb(2+)-PS2.M structure. Comparison of the relaxation times shows that the Na(+)→Pb(2+) exchange is more facile than the K(+)→Pb(2+) exchange process, and the mechanisms for these processes are proposed.

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