Revealing the Energetics of Ligand-Quadruplex Interactions Using Isothermal Titration Calorimetry
The thermodynamic characterization of G4-ligand interactions has shown to be a powerful adjunct to structural information in the rational design and optimization of potent G-quadruplex ligands for use in therapeutics, diagnostics, or other technological a
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Introduction Over the past years significant effort has been devoted to the search for G-quadruplex (G4) targeting ligands. The plethora of studies on G4-ligand interactions mostly derives from the realization that G4-forming DNA tracts are widely distributed in the human genome and frequently occur in regulatory genomic regions associated with uncontrolled cell proliferation and oncogenesis [1]. Consequently, these tetra-stranded nucleic acid structures have been identified as attractive drug targets for novel anticancer strategies [2–4]. On the other hand, various DNAzymes and nucleic acid aptamers that are based on the G-quadruplex scaffold rely on specific intermolecular interactions with other binding partners for their ever-increasing use in bio- and nanotechnological applications [5–7]. For a rational drug design, the binding event should ideally be described in terms of both the resulting complex structure and the thermodynamics of complex formation. Whereas the three-
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Andrea Funke and Klaus Weisz
Fig. 1 (a) Schematic diagram of an ITC instrument. Sample and reference cell in an adiabatic jacket are kept at the same temperature by providing a constant thermal power to the reference cell and a feedback controlled power to the sample cell. If aliquots of the ligand in the syringe are injected to the receptor in the sample cell, the heat released or consumed upon exothermic or endothermic binding will result in a temperature change of the sample that is compensated by power adjustments of the feedback heater to restore identical temperatures in the cells. (b) The additional compensating thermal power recorded as a function of time results in a differential power signal due to binding events after each injection. As binding sites become saturated, peaks become smaller and eventually only heats of dilution contribute to the recorded signal
dimensional structure of a complex may reveal specific interactions at the interface between components, insight into the contribution of molecular forces that drive the association are hardly provided by structural details. Studying the thermodynamics of a molecular interaction involves quantification of energy changes when going from free to complexed species. In contrast to spectroscopy-based methodologies, isothermal titration calorimeters (Fig. 1) allow for a very accurate direct measurement of the heat associated with complex formation without any chemical modification or immobilization of the interacting components [8, 9]. As an additional benefit, molar enthalpies of association can directly be derived without resorting to van’t Hoff-based analyses and their inherent limitations. Thus, with the availability of high-sensitivity calorimeters, ITC is expected to become increasingly important for the future development
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