Folding of the Thrombin Aptamer into a G-Quadruplex with Sr2+: Stability, Heat, and Hydration

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Abstract
It has been shown that the DNA aptamer d(G2T2G2TGTG2T2G2) adopts an intramolecular G-quadruplex structure in the presence of K+. Its affinity for trombin has been associated with the inhibition of thrombin-catalyzed fibrin clot formation. In this work, we used a combination of spectroscopy, calorimetry, density, and ultrasound techniques to determine the spectral characteristics, thermodynamics, and hydration effects for the formation of G-quadruplexes with a variety of monovalent and divalent metal ions. The formation of cation−aptamer complexes is relatively fast and highly reproducible. The comparison of their CD spectra and melting profiles as a function of strand concentration shows that K+, Rb+, NH4+, Sr2+, and Ba2+ form intramolecular cation−aptamer complexes with transition temperatures above 25 °C. However, the cations Li+, Na+, Cs+, Mg2+, and Ca2+ form weaker complexes at very low temperatures. This is consistent with the observation that metal ions with ionic radii in the range 1.3−1.5 Å fit well within the two G-quartets of the complex, while the other cations cannot. The comparison of thermodynamic unfolding profiles of the Sr2+−aptamer and K+−aptamer complexes shows that the Sr2+−aptamer complex is more stable, by ∼18 °C, and unfolds with a lower endothermic heat of 8.3 kcal/mol. This is in excellent agreement with the exothermic heats of −16.8 kcal/mol and −25.7 kcal/mol for the binding of Sr2+ and K+ to the aptamer, respectively. Furthermore, volume and compressibility parameters of cation binding show hydration effects resulting mainly from two contributions: the dehydration of both cation and guanine atomic groups and water uptake upon the folding of a single-strand into a G- quadruplex structure.