Guanosine- and deoxyguanosine-rich nucleic acids can form G-quadruplex structures (G4) that are stabilized by guanine tetrads (G4 tetrads). G4s find numerous applications in biotechnology. Here, we study a so called thrombin-binding aptamer (TBA), developed by SELEX procedures, that adopts a G4 conformation and inhibits clotting of thrombin. We investigate the TBA G4 and its variants with either four adenosine desoxynucleotides or four abasic sites attached either to the 5'-terminus (A4-TBA and ab4-TBA) or the 3'-terminus (TBA-ab4 and TBA-A4). These variants have been shown to exhibit differential anticlotting activities previously. The variant TBA-ab4, which was the most biological active in earlier investigations, has an exceptional stability against nuclease restriction, while all other variants show similar decay rates in mammalian serum. Biophysical characterization of the variants reveals that the structure of the aptamer remains unchanged, but that also their different thermal stabilities correlate with the anticlotting activity of TBA. Hydrogen exchange quantified by nuclear magnetic resonance spectroscopy (NMR) reveals individual G4 tetrad thermodynamics. Our data indicate that while enthalpy, entropy and free energy of base pair opening show surprisingly low variation, a hotspot for stabilization of the G4 is present at the 3', 5' terminal tetrad of TBA.
Wirmer‐Bartoschek et al. (2026) studied this question.