Anti-thrombin aptamers are G-quadruplex-bearing oligonucleotides, which recognizes the exosites of human thrombin. The first anti-thrombin aptamer, TBA, was generated through via SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology in 1992 by L.C. Bock, J.J. Toole and colleagues. A second thrombin-binding aptamer, HD22, recognizes thrombin exosite II and was discovered in 1997 by NeXstar (now Gilead Sciences). These two aptamers have high affinity and good specificity and have been widely studied and used for the development of aptamer-based therapeutics and diagnostics. Aptamer TBA (the exosite I-binding aptamer) The aptamer TBA (also known as G15D, HTQ, HD1 or ARC183) is a 15-mer single-stranded DNA with the sequence 5-GGTTGGTGTGGTTGG-3. It interacts with the exosite I of human alpha-thrombin, which is the binding site of fibrinogen, so this aptamer acts as an anti-coagulant agent inhibiting the activation of fibrinogen as well as platelet aggregation. In addition, TBA shows good affinity and specificity against thrombin. The dissociation constant of TBA-thrombin has been reported in nano-molar range, and TBA does not interact with other plasma proteins or thrombin analogues (e.g., gamma-thrombin). As a result, TBA has been used as a short-term anti-coagulant designed for the application in the coronary artery bypass graft surgery, and its optimized form (NU172) is now under the phase II of clinical trial by ARCA Biopharma (NCT00808964). Also, due to its high affinity and specificity, a variety of sensors was coupled with TBA and developed for thrombosis diagnostics. TBA structure The tertiary structure of TBA is an anti-parallel G-quadruplex. This chair-like structure is folded through the stacking of two guanine (G)-tetrads, and four guanines interacts with one another through non Watson-Crick-like hydrogen bonds (more likely Hoogsteen-like hydrogen bonds). In the structure of TBA, G1, G6, G10 and G15 form the top layer of G-tetrad; G2, G5, G11 and G14 form the second layer. The first crystallographic images with 2.9 Å resolution (1HUT) was reported in 1993. It showed that the T7-G8-T9 loop and TT loops (T3-T4 and T12-T13) connected the narrow and the wide grooves, respectively. However, since the improved NMR (1HAO) and X-ray crystallographic images (4DIH; 4DII) were provided, another topology with the TGT loop on the wide side and the TT loops on the narrow sites has been considered as a correct structure of TBA. In addition to protein-selectivity, TBA also shows ion preference. A potassium ion helps TBA fold into a G-quadruplex structure, which results in a