Use of Fluorescent Aptamers as Probes to Sense Changes in Protein Conformation.

摘要

A bioanalytical tool was developed using fluorescent aptamers as probes to sense changes in protein conformation for a set of clinically relevant proteins. The affinity of an aptamer for its target protein is dependent on non-covalent interactions including base-pair stacking, hydrogen bonding, and molecular shape complementarity between residues on the protein surface and its aptamer. A conformational change is hypothesized to alter the protein surface chemistry, affecting aptamer binding to its specific target. Protein-aptamer binding affinities were determined to quantitatively characterize conformational changes. Protein-aptamer complexes were separated from free aptamers by Capillary Electrophoresis and detected by Laser-Induced Fluorescence (CE/LIF). Protein conformational changes were induced using heat, aptamers were subsequently introduced, and samples equilibrated prior to CE analysis. Binding affinity proportions were determined as a function of temperature. Parallel thermal studies were performed on human a-thrombin using an enzyme activity assay and circular dichroism spectroscopy, providing further support for the technique.