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 c lass II RNAs are represented by a 33-nucleotide RNA (RNA 27.33). RNA 1 6.24 inhibits thrombin-catalyzed fibrin clot formation in vitro. Secon dary structures are proposed for these RNAs, revealing a novel stem-lo op structure for RNA 16.24, comprised of an unusually large 16-nucleot ide loop. Mutants of RNA 16.24 were generated to investigate structura l features critical to high-affinity binding. Phosphate modification w ith ethylnitrosourea identified regions of the RNAs necessary for elec trostatic interactions. Competition with heparin suggests that these R NAs bind the electropositive heparin-binding site of thrombin. These l igands represent a novel class of thrombin inhibitors that may be suit able for therapeutic or diagnostic applications.