Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.
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.
Tasset
D M
DM
NeXstar Pharmaceuticals, Inc., Boulder, CO 80301, USA.
Kubik
M F
MF
Steiner
W
W
D052157:Aptamers, Nucleotide; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000502:physiology; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D000939:Epitopes / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007065:Idoxuridine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D052157:Aptamers, Nucleotide; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; C081676:thrombin aptamer; D005340:Fibrinogen; D013917:Thrombin; D007065:Idoxuridine