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Nucleic Acids Research, 1994, Vol. 22, No. 13 2619-2626
© 1994

RNA

High-affinity RNA ligands to human {alpha}-thrombin

Mark F. Kubik, Andrew W. Stephens, Dan Schneider1, Richard A. Marlar2 and Diane Tasset*

NeXagen, Inc. 2860 Wilderness PL, Boulder, CO 80301 1Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, CO 80309 2laboratory Services, Denver, VA Medical Center and University of Colorado Health Sciences Center USA

*To whom correspondence should be addressed

Received March 14, 1994. Revised May 31, 1994. Accepted May 31, 1994.

Systematic Evolution of Ligands by Exponential enrichment (SELEX) was used to isolate from a population of 1013 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 heparinbinding site of thrombin. These ligands represent a novel class of thrombin inhibitors that may be suitable for therapeutic or diagnostic applications.


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