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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 absorbance (303 nm)/time trace of the folding of PS2.M (2.5 µM) induced by Pb(NO3)2 (0.2 mM). All measurements were performed in a buffer containing 10 mM MES/Tris (pH 6.1) at 25°C. The red line shows the non-linear least-squares fit of the data set to three exponentials. The optimized relaxation times are τ1 = 0.029 ± 0.0005 s, τ2 = 0.19 ± 0.003 s and τ3 = 1.25 ± 0.03 s. The residual plot indicates the deviation of the experimental and fitted absorbance changes.
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gkr1310-F5: Representative absorbance (303 nm)/time trace of the folding of PS2.M (2.5 µM) induced by Pb(NO3)2 (0.2 mM). All measurements were performed in a buffer containing 10 mM MES/Tris (pH 6.1) at 25°C. The red line shows the non-linear least-squares fit of the data set to three exponentials. The optimized relaxation times are τ1 = 0.029 ± 0.0005 s, τ2 = 0.19 ± 0.003 s and τ3 = 1.25 ± 0.03 s. The residual plot indicates the deviation of the experimental and fitted absorbance changes.

Mentions: The time-tracing curves of the folding process were obtained by monitoring absorbance changes at 303 nm, the maximum signal of the spectrum of the Pb2+-induced G-quadruplex. In the presence of at least 10-fold excess of Pb(NO3)2, curve fitting analysis shows that three sequential first-order processes (modeling the U→I1→I2→F reaction pathway) are required to adequately reproduce the data sets for the folding of PS2.M (Figure 5). Fitting to single and double exponential rate expressions result in large systematic deviations between the fitted and experimental progress curves. Kinetic and static difference spectra are in reasonable agreement, indicating that the kinetics account for the expected absorbance change. The residual plot shows that the three exponential function fits the experimental curve very well. A set of five curves were analyzed, and the optimal relaxation times are 0.029 ± 0.0005, 0.19 ± 0.003, 1.25 ± 0.03 s. Previous studies showed that the binding of a monovalent cation to G-quadruplex occurs on a millisecond time scale (42,43), hence in this case the first step can be interpreted as a cation binding step, which may be accompanied by partial folding of the oligonucleotide. The second step is proposed to be the folding of the PS2.M chain to give a metastable G-quadruplex, which then rearranges in the third step to give the final folded structure of the G-quadruplex. 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 Pb2+ ions. As expected, the folding rates are independent of the concentration of Pb(NO3)2, suggesting rate-saturation kinetics, which is in accordance with the high stability of the Pb2+-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. In the present case, the proposed mechanism requires at least two intermediates for the Pb2+-induced folding of a G-quadruplex of PS2.M, as shown in Equation 2 below:(2)where (PS2.M)U and (PS2.M–Pb2+)F indicate the unfolded and folded structures of PS2.M, respectively. I1 and I2 refer to the two intermediates.Figure 5.


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 absorbance (303 nm)/time trace of the folding of PS2.M (2.5 µM) induced by Pb(NO3)2 (0.2 mM). All measurements were performed in a buffer containing 10 mM MES/Tris (pH 6.1) at 25°C. The red line shows the non-linear least-squares fit of the data set to three exponentials. The optimized relaxation times are τ1 = 0.029 ± 0.0005 s, τ2 = 0.19 ± 0.003 s and τ3 = 1.25 ± 0.03 s. The residual plot indicates the deviation of the experimental and fitted absorbance changes.
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gkr1310-F5: Representative absorbance (303 nm)/time trace of the folding of PS2.M (2.5 µM) induced by Pb(NO3)2 (0.2 mM). All measurements were performed in a buffer containing 10 mM MES/Tris (pH 6.1) at 25°C. The red line shows the non-linear least-squares fit of the data set to three exponentials. The optimized relaxation times are τ1 = 0.029 ± 0.0005 s, τ2 = 0.19 ± 0.003 s and τ3 = 1.25 ± 0.03 s. The residual plot indicates the deviation of the experimental and fitted absorbance changes.
Mentions: The time-tracing curves of the folding process were obtained by monitoring absorbance changes at 303 nm, the maximum signal of the spectrum of the Pb2+-induced G-quadruplex. In the presence of at least 10-fold excess of Pb(NO3)2, curve fitting analysis shows that three sequential first-order processes (modeling the U→I1→I2→F reaction pathway) are required to adequately reproduce the data sets for the folding of PS2.M (Figure 5). Fitting to single and double exponential rate expressions result in large systematic deviations between the fitted and experimental progress curves. Kinetic and static difference spectra are in reasonable agreement, indicating that the kinetics account for the expected absorbance change. The residual plot shows that the three exponential function fits the experimental curve very well. A set of five curves were analyzed, and the optimal relaxation times are 0.029 ± 0.0005, 0.19 ± 0.003, 1.25 ± 0.03 s. Previous studies showed that the binding of a monovalent cation to G-quadruplex occurs on a millisecond time scale (42,43), hence in this case the first step can be interpreted as a cation binding step, which may be accompanied by partial folding of the oligonucleotide. The second step is proposed to be the folding of the PS2.M chain to give a metastable G-quadruplex, which then rearranges in the third step to give the final folded structure of the G-quadruplex. 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 Pb2+ ions. As expected, the folding rates are independent of the concentration of Pb(NO3)2, suggesting rate-saturation kinetics, which is in accordance with the high stability of the Pb2+-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. In the present case, the proposed mechanism requires at least two intermediates for the Pb2+-induced folding of a G-quadruplex of PS2.M, as shown in Equation 2 below:(2)where (PS2.M)U and (PS2.M–Pb2+)F indicate the unfolded and folded structures of PS2.M, respectively. I1 and I2 refer to the two intermediates.Figure 5.

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