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Intramolecular DNA quadruplexes with different arrangements of short and long loops.

Rachwal PA, Findlow IS, Werner JM, Brown T, Fox KR - Nucleic Acids Res. (2007)

Bottom Line: The stability increases with the number of single T loops, though the arrangement of different length loops has little effect.In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure.Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have examined the folding, stability and kinetics of intramolecular quadruplexes formed by DNA sequences containing four G3 tracts separated by either single T or T4 loops. All these sequences fold to form intramolecular quadruplexes and 1D-NMR spectra suggest that they each adopt unique structures (with the exception of the sequence with all three loops containing T4, which is polymorphic). The stability increases with the number of single T loops, though the arrangement of different length loops has little effect. In the presence of potassium ions, the oligonucleotides that contain at least one single T loop exhibit similar CD spectra, which are indicative of a parallel topology. In contrast, when all three loops are substituted with T4 the CD spectrum is typical of an antiparallel arrangement. In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure. Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

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Arrhenius plots showing the temperature dependence of the kinetic parameters for G3T4-T-T and G3T4-T-T4 (a) and G3T-T4-T and G3T4-T4-T (b). Open symbols were derived from the hysteresis between the melting and annealing profiles; k−1, open circles; k1, open triangles. Filled circles show the time constants obtained from the temperature-jump experiments (k1 + k−1).
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Figure 6: Arrhenius plots showing the temperature dependence of the kinetic parameters for G3T4-T-T and G3T4-T-T4 (a) and G3T-T4-T and G3T4-T4-T (b). Open symbols were derived from the hysteresis between the melting and annealing profiles; k−1, open circles; k1, open triangles. Filled circles show the time constants obtained from the temperature-jump experiments (k1 + k−1).

Mentions: The fluorescence melting experiments shown in Figure 4 were performed at a rate of temperature change of 0.2°C.min−1 and only G3T4 showed hysteresis between the melting and annealing profiles. On increasing the rate to 2°C.min−1 there was a 7–10°C hysteresis for the sequences with two T4 loops in the presence of potassium, though the melting and annealing profiles were identical for the sequences with single T4 loops. The sequences with single T4 loops only displayed hysteresis when the rate of heating was increased to 12°C.min−1, while the melting and annealing curves for G3T were always superimposable. Representative heating and annealing curves at different rates of heating and cooling in the presence of 20 mM potassium are shown in Supplementary material Figure 2 and the different Tm values are summarized in Supplementary Table 1. No hysteresis was observed for any of these sequences in the presence of sodium at even the fastest rate of heating and cooling. Differences between the melting and annealing curves arise because the reaction is not at thermodynamic equilibrium and indicate that either the folding or the unfolding process is slow. The folding (k1) and unfolding (k−1) rate constants for the unimolecular folding reaction can be obtained by analysis of these data as previously described (47,48). Figure 5a shows the melting and annealing profiles for G3T4-T-T and G3T4-T-T4, determined at 12°C.min−1 and 2°C.min−1, respectively, while similar plots for G3T-T4-T and G3T4-T4-T are included in Supplementary material, Figure 3. Figure 6 shows Arrhenius plots for the folding and unfolding rates constructed from these data for G3T-T4-T and G3T4-T4-T (Figure 6a) and G3T4-T-T and G3T4-T-T4 (Figure 6b). The kinetic parameters derived from these Arrhenius plots are presented in Table 3. Several factors are apparent from these kinetic data. Firstly, the association reaction shows unusual temperature dependence, with an apparent negative activation energy, i.e. the reaction is faster a lower temperatures. This has been noted by others and is consistent with the reaction occurring by a nucleation-zipper mechanism (47,48). Secondly, the data for G3T4-T4-T are very similar to G3T4-T-T4 and G3T4-T-T is similar to G3T-T4-T, suggesting that the distribution of the different loops is less important than their length. Thirdly, the unfolding parameters are very similar for all four oligonucleotides, while the folding parameters vary according to the loop lengths. For the association reaction both ln(A) and Ea are less negative for the complexes with longer loops. This kinetic analysis was not performed for G3T as it showed no hysteresis and for G3T4 as the melting and annealing curves were biphasic.Figure 5.


Intramolecular DNA quadruplexes with different arrangements of short and long loops.

Rachwal PA, Findlow IS, Werner JM, Brown T, Fox KR - Nucleic Acids Res. (2007)

Arrhenius plots showing the temperature dependence of the kinetic parameters for G3T4-T-T and G3T4-T-T4 (a) and G3T-T4-T and G3T4-T4-T (b). Open symbols were derived from the hysteresis between the melting and annealing profiles; k−1, open circles; k1, open triangles. Filled circles show the time constants obtained from the temperature-jump experiments (k1 + k−1).
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Related In: Results  -  Collection

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Figure 6: Arrhenius plots showing the temperature dependence of the kinetic parameters for G3T4-T-T and G3T4-T-T4 (a) and G3T-T4-T and G3T4-T4-T (b). Open symbols were derived from the hysteresis between the melting and annealing profiles; k−1, open circles; k1, open triangles. Filled circles show the time constants obtained from the temperature-jump experiments (k1 + k−1).
Mentions: The fluorescence melting experiments shown in Figure 4 were performed at a rate of temperature change of 0.2°C.min−1 and only G3T4 showed hysteresis between the melting and annealing profiles. On increasing the rate to 2°C.min−1 there was a 7–10°C hysteresis for the sequences with two T4 loops in the presence of potassium, though the melting and annealing profiles were identical for the sequences with single T4 loops. The sequences with single T4 loops only displayed hysteresis when the rate of heating was increased to 12°C.min−1, while the melting and annealing curves for G3T were always superimposable. Representative heating and annealing curves at different rates of heating and cooling in the presence of 20 mM potassium are shown in Supplementary material Figure 2 and the different Tm values are summarized in Supplementary Table 1. No hysteresis was observed for any of these sequences in the presence of sodium at even the fastest rate of heating and cooling. Differences between the melting and annealing curves arise because the reaction is not at thermodynamic equilibrium and indicate that either the folding or the unfolding process is slow. The folding (k1) and unfolding (k−1) rate constants for the unimolecular folding reaction can be obtained by analysis of these data as previously described (47,48). Figure 5a shows the melting and annealing profiles for G3T4-T-T and G3T4-T-T4, determined at 12°C.min−1 and 2°C.min−1, respectively, while similar plots for G3T-T4-T and G3T4-T4-T are included in Supplementary material, Figure 3. Figure 6 shows Arrhenius plots for the folding and unfolding rates constructed from these data for G3T-T4-T and G3T4-T4-T (Figure 6a) and G3T4-T-T and G3T4-T-T4 (Figure 6b). The kinetic parameters derived from these Arrhenius plots are presented in Table 3. Several factors are apparent from these kinetic data. Firstly, the association reaction shows unusual temperature dependence, with an apparent negative activation energy, i.e. the reaction is faster a lower temperatures. This has been noted by others and is consistent with the reaction occurring by a nucleation-zipper mechanism (47,48). Secondly, the data for G3T4-T4-T are very similar to G3T4-T-T4 and G3T4-T-T is similar to G3T-T4-T, suggesting that the distribution of the different loops is less important than their length. Thirdly, the unfolding parameters are very similar for all four oligonucleotides, while the folding parameters vary according to the loop lengths. For the association reaction both ln(A) and Ea are less negative for the complexes with longer loops. This kinetic analysis was not performed for G3T as it showed no hysteresis and for G3T4 as the melting and annealing curves were biphasic.Figure 5.

Bottom Line: The stability increases with the number of single T loops, though the arrangement of different length loops has little effect.In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure.Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.

ABSTRACT
We have examined the folding, stability and kinetics of intramolecular quadruplexes formed by DNA sequences containing four G3 tracts separated by either single T or T4 loops. All these sequences fold to form intramolecular quadruplexes and 1D-NMR spectra suggest that they each adopt unique structures (with the exception of the sequence with all three loops containing T4, which is polymorphic). The stability increases with the number of single T loops, though the arrangement of different length loops has little effect. In the presence of potassium ions, the oligonucleotides that contain at least one single T loop exhibit similar CD spectra, which are indicative of a parallel topology. In contrast, when all three loops are substituted with T4 the CD spectrum is typical of an antiparallel arrangement. In the presence of sodium ions, the sequences with two and three single T loops also adopt a parallel folded structure. Kinetic studies on the complexes with one or two T4 loops in the presence of potassium ions reveal that sequences with longer loops display slower folding rates.

Show MeSH
Related in: MedlinePlus