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Graduate Defense: Mark Smith

February 2 @ 2:00 pm - 4:00 pm MST

Dissertation Information

Title: New Applications for the Kinetic Exclusion Assay (Kinexa)

Program: Doctor of Philosophy in Biomolecular Sciences

Advisor: Dr. Daniel Fologea, Physics, Co-chair, and Dr. Denise Wingett, Biological Sciences, Co-chair

Committee Members: Dr. Eric Hayden, Biological Sciences

Abstract

The Kinetic Exclusion Assay (KinExA) technology is a relatively new technique intended for accurate determination of equilibrium, kinetics, and analyte concentration in a label-free solution system. This technology is widely used for characterization of bimolecular interactions between antibodies and antigens. However, the working principles of the technology may be easily expanded to a large variety of affinity and concentration measurements, which constitute the scientific objectives of my work. To achieve these goals, I focused my investigations on characterization of aptamer-target and ligand-membrane interactions. In this endeavor, I interrogated the use of KinExA technology with DNA aptamers, using a well-known and extensively studied aptamer which specifically binds thrombin. The technology proved capable of not only accurate determination of affinity but also to identifying deviations from a 1:1 binding model. The aptamer system is also shown to be useful as a means to measure concentration of thrombin in an unknown solution, in the pM to nM range. Further, I explored complex capture agents in the KinExA system, using liposomes made from purified reagents or from natural cellular membranes to capture analytes and characterize membrane-ligand interactions. The usefulness of the process is demonstrated for toxins binding to artificial membranes, antibodies binding to natural membranes, and non-specific membrane dyes. In conclusion, the KinExA technology may be used for reliable measurements of interactions and analyte concentrations in a large variety scientific, biomedical, and biotechnological applications.