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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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Representative 3D plots of the wavelength and time dependence of spectrophotometric changes accompanying the Pb2+-induced folding of PS2.M. Pb(NO3)2 (20 µM) was mixed with PS2.M (5.0 µM) in buffer solution (10 mM MES/Tris, pH 6.1). The temperature was kept at 25°C.
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gkr1310-F4: Representative 3D plots of the wavelength and time dependence of spectrophotometric changes accompanying the Pb2+-induced folding of PS2.M. Pb(NO3)2 (20 µM) was mixed with PS2.M (5.0 µM) in buffer solution (10 mM MES/Tris, pH 6.1). The temperature was kept at 25°C.

Mentions: To our knowledge, there are few kinetic studies on the formation pathways of G-quadruplexes (41). Considering that the time scale of the folding of G-quadruplex is very short, we used stopped-flow mixing technique to acquire the spectrophotometric changes that accompany the Pb2+-driven G-quadruplex formation. Figure 4 shows representative 3D plots of the wavelength and time dependence of the UV difference spectra for the folding process of ∼2.5 µM of the oligonucleotide induced by 10 µM of Pb(NO3)2. Spectra were obtained by rapid mixing at 0.1 s intervals and the folding process was completed within 5 s. The growth of a negative peak at around 303 nm indicates the formation of the Pb2+-induced G-quadruplex. Analysis of the repetitive scanning spectra shows an initial rapid step with slight absorbance change at 0.03 s after mixing, this is followed by slower steps with larger absorbance changes.Figure 4.


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)

Representative 3D plots of the wavelength and time dependence of spectrophotometric changes accompanying the Pb2+-induced folding of PS2.M. Pb(NO3)2 (20 µM) was mixed with PS2.M (5.0 µM) in buffer solution (10 mM MES/Tris, pH 6.1). The temperature was kept at 25°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1310-F4: Representative 3D plots of the wavelength and time dependence of spectrophotometric changes accompanying the Pb2+-induced folding of PS2.M. Pb(NO3)2 (20 µM) was mixed with PS2.M (5.0 µM) in buffer solution (10 mM MES/Tris, pH 6.1). The temperature was kept at 25°C.
Mentions: To our knowledge, there are few kinetic studies on the formation pathways of G-quadruplexes (41). Considering that the time scale of the folding of G-quadruplex is very short, we used stopped-flow mixing technique to acquire the spectrophotometric changes that accompany the Pb2+-driven G-quadruplex formation. Figure 4 shows representative 3D plots of the wavelength and time dependence of the UV difference spectra for the folding process of ∼2.5 µM of the oligonucleotide induced by 10 µM of Pb(NO3)2. Spectra were obtained by rapid mixing at 0.1 s intervals and the folding process was completed within 5 s. The growth of a negative peak at around 303 nm indicates the formation of the Pb2+-induced G-quadruplex. Analysis of the repetitive scanning spectra shows an initial rapid step with slight absorbance change at 0.03 s after mixing, this is followed by slower steps with larger absorbance changes.Figure 4.

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
Related in: MedlinePlus