High-affinity RNA ligands to human alpha-thrombin.
Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to isolate from a population of 10(13) RNA molecules two classes of high affinity RNAs that bind specifically to human alpha-thrombin. Class I RNAs are represented by a 24-nucleotide RNA (RNA 16.24), and class II RNAs are represented by a 33-nucleotide RNA (RNA 27.33). RNA 16.24 inhibits thrombin-catalyzed fibrin clot formation in vitro. Secondary structures are proposed for these RNAs, revealing a novel stem-loop structure for RNA 16.24, comprised of an unusually large 16-nucleotide loop. Mutants of RNA 16.24 were generated to investigate structural features critical to high-affinity binding. Phosphate modification with ethylnitrosourea identified regions of the RNAs necessary for electrostatic interactions. Competition with heparin suggests that these RNAs bind the electropositive heparin-binding site of thrombin. These ligands represent a novel class of thrombin inhibitors that may be suitable for therapeutic or diagnostic applications.
Kubik
M F
MF
NeXagen, Inc., Boulder, CO 80301.
Stephens
A W
AW
Schneider
D
D
Marlar
R A
RA
Tasset
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D005075:Biological Evolution; D002384:Catalysis; D004247:DNA; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D012313:RNA; D004247:DNA; D013917:Thrombin