Selection of single-stranded DNA molecules that bind and inhibit human thrombin.
Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.
Bock
L C
LC
Gilead Sciences Inc., Foster City, California 94404.
Griffin
L C
LC
Latham
J A
JA
Vermaas
E H
EH
Toole
J J
JJ
D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013917:Thrombin / Q000037:antagonists & inhibitors*; D013918:Thrombin Time
D004277:DNA, Single-Stranded; D013917:Thrombin