G-Quadruplex Stability from DSC Measurements
Guanine-rich DNA oligonucleotides can adopt G-quadruplex (G4) structures in the presence of specific cations. Folding and unfolding of G4 can be characterized thermodynamically, providing the information on the stability of various G4 conformations. We sh
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Introduction Guanine-rich DNA oligonucleotides can adopt G-quadruplex (G4) structures in the presence of specific cations in vitro and in vivo. Controlling the folding/unfolding of G4 structures is an important strategy for developing novel therapeutics for treatment of cancer and other severe diseases [1–3]. Knowledge of the thermodynamic stability of G4 structures is crucial for understanding their formation propensity and to understand their interactions in vitro and in vivo. Determination of the parameters characterizing the G4 stability allows one to predict the conformational behavior of the G4 forming sequence at different conditions such as temperature and various cation concentrations. Moreover, the Gibbs free energy of folding/unfolding, a common measure of the stability of G4, along with its enthalpy and entropy contributions and the corresponding heat capacity change may be correlated to the DNA structural features and used to separate various contributions due to intra- and intermolecular interactions that drive folding/ unfolding of G4 [4–7].
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_7, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Differential scanning calorimetry (DSC) is the most appropriate technique for investigating G4 thermodynamic stability since it enables determination of thermodynamic parameters of G4 (un) folding both in a model-dependent and model-independent manner. The basic principle of DSC can be illustrated in the following way. During the experiment the temperature in the calorimeter is increased (heating) or decreased (cooling) with constant rate. Calorimeter contains two cells (sample, reference). The temperature regulation system (heaters, Peltier elements) maintains the same temperature in both cells. For example, upon heating the G4 solution (sample cell) the G4 undergoes unfolding (endothermic process ) temperature decrease), so heaters provide more heat to the sample cell than to the reference cell (buffer solution without G4) to keep both cells at the same temperature. By observing the difference in heat flow between the sample and reference cell, differential scanning calorimeters are able to measure the amount of heat absorbed during G4 unfolding. The result of G4 unfolding DSC experiment is a thermogram (curve of heat capacity versus temperature). This curve can be used to calculate enthalpy of G4 unfolding in the model-independent manner, by integrating the corresponding peak. On the other hand, DSC thermograms may be analyzed in a model-dependent manner which is shown schematically in Fig. 1. Measurements performed at different scanning rates suggest whether the observed transitions may be considered reversible (no rate dependence) or kinetically limited (rate dependence). When the thermograms are independent of the heating/cooling rate, they may be described in terms of equilibrium proces
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