Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

J Mol Biol. 1997 Oct 10;272(5):688-98. 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 Kd 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.

MeSH terms

  • 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
  • Gene Library
  • Humans
  • Idoxuridine / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / genetics
  • Oligodeoxyribonucleotides / metabolism
  • Oligodeoxyribonucleotides / pharmacology*
  • Oligonucleotides / chemistry
  • Oligonucleotides / pharmacology*
  • Protein Binding
  • Thrombin / antagonists & inhibitors*
  • Thrombin / chemistry
  • Thrombin / immunology
  • Thrombin / metabolism


  • Aptamers, Nucleotide
  • Cross-Linking Reagents
  • DNA, Single-Stranded
  • Enzyme Inhibitors
  • Epitopes
  • Oligodeoxyribonucleotides
  • Oligonucleotides
  • thrombin aptamer
  • Fibrinogen
  • Thrombin
  • Idoxuridine