Characterization of Thrombin-Aptamer Binding Using Fluorescence Anisotropy

Science Center, Bent Corridor

Without a general screening test for ovarian cancer, patients are most often diagnosed at advanced stages of the disease, where survival is the bleakest. Aptamers are potential alternatives to antibodies, which are currently used to probe for ovarian cancer, and may provide the basis for this general screening test. Development of these tests require a well characterized model system. Thrombin, a blood clotting enzyme, has two well-studied aptamers that often act as this model system. Literature values for their dissociation constants (KD—the measure of binding affinity) vary widely, making the thrombin-aptamer systems difficult to use accurately as models. Because of the literature KD’s wide variability, the Whelan lab characterized the thrombin-aptamer systems’ binding affinity in different buffer/ion environments using a fluorescence anisotropy assay. In FA, polarized light is shined onto fluorescently-labeled molecules, which then rotate and emit polarized light in various directions. Larger molecules, like proteinaptamer complexes, rotate more slowly and emit light in fewer directions resulting in higher anisotropy values. Aptamer concentration was held constant while thrombin concentration varied. Anisotropy values were gathered at each thrombin concentration and put into a Hill plot, from which the KD was derived. With these assays, the Whelan lab is measuring the strength of thrombin-aptamer binding while observing the effects of ions. By establishing criteria for “normal” thrombin-aptamer binding and ionic effects on this binding, the thrombin-aptamer system can be better utilized as a model system. As a model system, the thrombin-aptamer system can be used to test new target-aptamer assays and potential diagnostic tests for ovarian cancer and much more.