A Crystallographic Approach for Understanding the Recognition Mechanism of Thrombin and G-quadruplex Aptamers

Human-thrombin, a serine protease that maintains blood hemostasis by balancing pro- and anti-coagulant actions is an example of protein with multiple binding sites1. In addition to the active site, the enzyme possesses two electropositive regions, in near-opposition on the protein surface, known as exosite I and exosite II, respectively. These two regions have a primary role in the regulation of enzymatic activity since they can bind molecules with diverse functions2-4. Given its central role in the clot formation, thrombin is an attractive target for the development of agents that effectively interfere with thrombogenesis. A special class of thrombin synthetic ligands is represented by nucleic acid aptamers adopting G-quadruplex structures. HD1, a 15-mer oligonucleotide recognizing exosite I5, and HD22, a 29-mer binding exosite II6, are the most studied thrombin binding aptamers and show high affinity toward their target (Kd (HD1)≈ 100 nM; Kd(HD22) ≈0.7 nM). The increased interest in the use of DNA aptamers as drugs has stimulated the search of HD1 and HD22 variants with improved properties. In particular, the bimodular oligonucleotides RE317 and NU1728, which have been obtained by addition of a duplex motif to the HD1 quadruplex module, show higher affinity for thrombin and anticoagulant activity, and a slower disappearance rate in human plasma in comparison with HD1. Here I will present the most relevant results regarding the elucidation of the interactions, which govern the recognition between thrombin and DNA G-quadruplex aptamers9-14.