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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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CD spectra of the cation-induced G-quadruplexes of PS2.M in the presence of various cations in the buffer (10 mM MES/Tris, pH 6.1) at 25°C. Concentrations of oligonucleotide were 25 µM after mixing.
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gkr1310-F1: CD spectra of the cation-induced G-quadruplexes of PS2.M in the presence of various cations in the buffer (10 mM MES/Tris, pH 6.1) at 25°C. Concentrations of oligonucleotide were 25 µM after mixing.

Mentions: CD is a technique that is quite sensitive to the conformation of anisotropic molecules and chiral super assemblies, and has been used extensively in the study of nucleic acids and G-quadruplex structures. Figure 1 shows the CD spectra of 25 µM PS2.M in the presence of different metal cations. The addition of 0.5 mM NaNO3 shows little effect on the structure of PS2.M. In the presence of K+ ions, the spectrum of PS2.M shows a positive CD peak near 295 nm, characteristic of antiparallel structure (35). Upon the addition of Pb(NO3)2, the wavelength at maximum CD intensity is shifted by almost 20 to 312 nm, and the peak minimum is located at 263 nm, indicating the Pb2+-induced antiparallel G-quadruplex structure (17,23,29,36). The spectrum does not vary with time or oligomer concentration from 1 to 25 µM. This result indicates that there was no transformation to parallel folds at such low concentrations, consistent with the previous study (37). Moreover, after the G-quadruplex was formed in the presence of Pb2+ ions, there were no conformational changes observed upon the addition of NaNO3 or KNO3 at room temperature. On the other hand, spectral changes of PS2.M occur upon the addition of Pb(NO3)2 into the systems containing Na+ or K+ ions. In addition, the CD intensity of the Pb2+-induced G-quadruplex is about three and six times higher than those of induced by K+ and Na+, respectively. These results strongly suggest that there are structural differences between G-quadruplexes induced by Na+, K+ and Pb2+ ions. Previous studies (22,23) have demonstrated that Pb2+-induced G-quadruplexes generally have shorter M–O and O–O bonds than those induced by Na+ or K+ ions. Such compact structures are the result of the unusually high efficiency of Pb2+ ions in stabilizing G-quadruplexes (17,22). The increase in intensity and location shift of the CD spectrum of Pb2+-induced G-quadruplex reveals a change in the dimensions of the G8 cage dimension, which corresponds with the decrease in the cage size as observed in the X-ray crystal structure (22). The overall difference in the CD spectra of the G-quadruplex in the presence of various cations may be attributed to the difference in the coordination number of cations, partial formation of G-quadruplexes with some cations and/or overall tightness of each G-quadruplex.Figure 1.


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)

CD spectra of the cation-induced G-quadruplexes of PS2.M in the presence of various cations in the buffer (10 mM MES/Tris, pH 6.1) at 25°C. Concentrations of oligonucleotide were 25 µM after mixing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1310-F1: CD spectra of the cation-induced G-quadruplexes of PS2.M in the presence of various cations in the buffer (10 mM MES/Tris, pH 6.1) at 25°C. Concentrations of oligonucleotide were 25 µM after mixing.
Mentions: CD is a technique that is quite sensitive to the conformation of anisotropic molecules and chiral super assemblies, and has been used extensively in the study of nucleic acids and G-quadruplex structures. Figure 1 shows the CD spectra of 25 µM PS2.M in the presence of different metal cations. The addition of 0.5 mM NaNO3 shows little effect on the structure of PS2.M. In the presence of K+ ions, the spectrum of PS2.M shows a positive CD peak near 295 nm, characteristic of antiparallel structure (35). Upon the addition of Pb(NO3)2, the wavelength at maximum CD intensity is shifted by almost 20 to 312 nm, and the peak minimum is located at 263 nm, indicating the Pb2+-induced antiparallel G-quadruplex structure (17,23,29,36). The spectrum does not vary with time or oligomer concentration from 1 to 25 µM. This result indicates that there was no transformation to parallel folds at such low concentrations, consistent with the previous study (37). Moreover, after the G-quadruplex was formed in the presence of Pb2+ ions, there were no conformational changes observed upon the addition of NaNO3 or KNO3 at room temperature. On the other hand, spectral changes of PS2.M occur upon the addition of Pb(NO3)2 into the systems containing Na+ or K+ ions. In addition, the CD intensity of the Pb2+-induced G-quadruplex is about three and six times higher than those of induced by K+ and Na+, respectively. These results strongly suggest that there are structural differences between G-quadruplexes induced by Na+, K+ and Pb2+ ions. Previous studies (22,23) have demonstrated that Pb2+-induced G-quadruplexes generally have shorter M–O and O–O bonds than those induced by Na+ or K+ ions. Such compact structures are the result of the unusually high efficiency of Pb2+ ions in stabilizing G-quadruplexes (17,22). The increase in intensity and location shift of the CD spectrum of Pb2+-induced G-quadruplex reveals a change in the dimensions of the G8 cage dimension, which corresponds with the decrease in the cage size as observed in the X-ray crystal structure (22). The overall difference in the CD spectra of the G-quadruplex in the presence of various cations may be attributed to the difference in the coordination number of cations, partial formation of G-quadruplexes with some cations and/or overall tightness of each G-quadruplex.Figure 1.

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.

Show MeSH