Guanosine- and deoxyguanosine-rich nucleic acids can form G-quadruplex structures (G4) that are stabilized by guanine tetrads (G4 tetrads). G4s find numerous application in biotechnology and we study here the so called TBA aptamer, 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). Biophysical characterization of the variants reveals that the structure of the aptamer remains unchanged but that their different thermal stabilities correlate with the anti-clotting 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 shows surprisingly low variation, a hotspot for stabilization of the G4 is present at the 3’-terminal tetrad of TBA.

NMR assignment raw data (Bruker format), NMR hydrogen exchange raw data (Bruker format), CD melting curve raw data (text-files)