Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

• Published in: Journal of molecular biology 1997-10, Vol.272 (5), p.688-698
• Main Authors: Tasset, Diane M, Kubik, Mark F, Steiner, Walter
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide; Base Sequence; Binding Sites; Binding, Competitive; Blood Coagulation - physiology; Cross-Linking Reagents; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; DNA, Single-Stranded - pharmacology; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Epitopes - metabolism; Fibrinogen - metabolism; G-quadruplex; Gene Library; Humans; Idoxuridine - metabolism; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - genetics; Oligodeoxyribonucleotides - metabolism; Oligodeoxyribonucleotides - pharmacology; oligonucleotides; Oligonucleotides - chemistry; Oligonucleotides - pharmacology; photocrosslinking; Protein Binding; SELEX; thrombin; Thrombin - antagonists & inhibitors; Thrombin - chemistry; Thrombin - immunology; Thrombin - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1997.1275

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a K d of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide “core” sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.