Investigating the Role of T7 and T12 Residues on the Biological Properties of Thrombin-Binding Aptamer

Thrombin is a sodium-activated allosteric enzyme playing a key role in blood coagulation. Due to its central role in the coagulation cascade, malfunctions in the regulatory mechanism of thrombin activity cause pathological states such as hemorrhage or abnormal clot growth. Thrombosis and connected diseases are among the main causes of mortality in Western countries; thus, the discovery of molecules capable of modulating thrombin activity represents a major target for the development of anticoagulant strategies. Aptamer technology has been efficiently employed to obtain new direct thrombin inhibitors by selecting thrombin-binding oligonucleotides. The first reported consensus sequence able to inhibit thrombin activity was the 15-mer oligonucleotide GGTTGGTGTGGTTGG, usually known with the acronym TBA (Thrombin-Binding Aptamer). In the presence of thrombin and/or monovalent cations TBA folds into a monomolecular chair-like G-quadruplex, consisting of two G-tetrads connected by one TGT loop and two TT loops, that dictates its thrombin-binding affinity. The structures of TBA alone and in complex with thrombin were determined by NMR and X-ray methods, respectively. Binding of thrombin by TBA leads to the inactivation of the former and to significant prolongation of blood clotting time. In this communication we present our recent results on the synthesis and the anticoagulant property of new TBA analogues in which an acyclic pyrimidine analogue, containing a five-member cycle fused on the pyrimidine ring, was introduced at position 7 or 12 of the TBA. Characterization by 1H NMR and CD spectroscopies of the resulting aptamers showed their ability to fold into the typical antiparallel chair-like G-quadruplex structure formed by TBA. The anticoagulant activity of new aptamers was valued with PT (measured on human plasma) and fibrinogen (using human and bovine thrombin) assays. The obtained structure-activity relationships were investigated by structural and computational studies. Taken together, our results have revealed the active role of TBA residues T7 and T12 and the relevance of some amino acids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.