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A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

Kumar N, Maiti S - Nucleic Acids Res. (2008)

Bottom Line: We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol.Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition.Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5-18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

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Representative DSC curves for different quadruplexes in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl.
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Figure 3: Representative DSC curves for different quadruplexes in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl.

Mentions: Next we performed differential scanning calorimetry (DSC) to obtain model-independent values which were compared with the thermodynamic data obtained from UV study. The DSC experiments were performed for few sequences which were selected randomly to obtain the van't Hoff and calorimetric enthalpy associated with the unstructured–structured transition. The unstructured–structured transition may be associated with nonzero heat capacity changes. These heat capacity changes arise from the change in solvent exposure for nonpolar and polar groups. These heat capacity changes are determined by the difference between pre- and post-transition baseline of the DSC curves. The net effect of solvent exposure during unstructured–structured transition can sometimes be relatively small due to simultaneous exposure or burial of polar and non-olar groups. Precise nonzero heat capacity changes (ΔCp) associated with quadruplex melting can be obtained at very high oligonucleotide concentration (≈200 µM). But the quadruplex melting experiments performed at high concentrations lead to DNA precipitation at high temperatures. At low concentrations (50 µM) such aggregation problem can be avoided but determination of ΔCp was not possible due to the low signal. Due to this limitation, we performed the analysis with the assumption of zero heat capacity changes. The assumption of negligible heat capacity change associated with unstructured–structured transition is very common in this field, as the process yields heat capacity changes within experimental errors (54,55). The shape of the curve provides the van't Hoff enthalpy and the area under the curve provides calorimetric enthalpy (Figure 3). We observed that ratio of van't Hoff and calorimetric enthalpy is nearly equal to 1, therefore, indicating that structured–unstructured transition follows a two-state process. The thermodynamic parameters obtained from DSC experiments correlated well with the parameters obtained from UV study, further indicating that the model used for the analysis in the UV study is correct (Table 4). Recently using isothermal calorimetry (ITC), heat capacity changes associated with quadruplex formation in a solution containing excess of salt were measured (56). ITC measurements differ from thermal denaturation such as DSC and UV melting. The results obtained from the former technique are valid at temperature selected for the experiment, which is usually lower than Tm, while, the results obtained from the latter technique are reliable at or near the Tm of the unstructured–structured transition. In the ITC experiments, the quadruplex formation is measured under a high salt concentration where the oligonucleotide is subjected to changes in salt conditions; while the thermal melting experiments do not involve such changes in salt conditions. Therefore disagreement in the heat capacity changes obtained through ITC and DSC experiments is quite likely.Figure 3.


A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

Kumar N, Maiti S - Nucleic Acids Res. (2008)

Representative DSC curves for different quadruplexes in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553590&req=5

Figure 3: Representative DSC curves for different quadruplexes in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl.
Mentions: Next we performed differential scanning calorimetry (DSC) to obtain model-independent values which were compared with the thermodynamic data obtained from UV study. The DSC experiments were performed for few sequences which were selected randomly to obtain the van't Hoff and calorimetric enthalpy associated with the unstructured–structured transition. The unstructured–structured transition may be associated with nonzero heat capacity changes. These heat capacity changes arise from the change in solvent exposure for nonpolar and polar groups. These heat capacity changes are determined by the difference between pre- and post-transition baseline of the DSC curves. The net effect of solvent exposure during unstructured–structured transition can sometimes be relatively small due to simultaneous exposure or burial of polar and non-olar groups. Precise nonzero heat capacity changes (ΔCp) associated with quadruplex melting can be obtained at very high oligonucleotide concentration (≈200 µM). But the quadruplex melting experiments performed at high concentrations lead to DNA precipitation at high temperatures. At low concentrations (50 µM) such aggregation problem can be avoided but determination of ΔCp was not possible due to the low signal. Due to this limitation, we performed the analysis with the assumption of zero heat capacity changes. The assumption of negligible heat capacity change associated with unstructured–structured transition is very common in this field, as the process yields heat capacity changes within experimental errors (54,55). The shape of the curve provides the van't Hoff enthalpy and the area under the curve provides calorimetric enthalpy (Figure 3). We observed that ratio of van't Hoff and calorimetric enthalpy is nearly equal to 1, therefore, indicating that structured–unstructured transition follows a two-state process. The thermodynamic parameters obtained from DSC experiments correlated well with the parameters obtained from UV study, further indicating that the model used for the analysis in the UV study is correct (Table 4). Recently using isothermal calorimetry (ITC), heat capacity changes associated with quadruplex formation in a solution containing excess of salt were measured (56). ITC measurements differ from thermal denaturation such as DSC and UV melting. The results obtained from the former technique are valid at temperature selected for the experiment, which is usually lower than Tm, while, the results obtained from the latter technique are reliable at or near the Tm of the unstructured–structured transition. In the ITC experiments, the quadruplex formation is measured under a high salt concentration where the oligonucleotide is subjected to changes in salt conditions; while the thermal melting experiments do not involve such changes in salt conditions. Therefore disagreement in the heat capacity changes obtained through ITC and DSC experiments is quite likely.Figure 3.

Bottom Line: We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol.Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition.Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5-18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

Show MeSH