Folding of the thrombin aptamer into a G-quadruplex with Sr²⁺: Stability, heat, and hydration

It has been shown that the DNA aptamer d(G₂T₂G₂TGTG₂T₂G ₂) 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.