NOVEL FLUORESCENT, CELL-PERMEANT EPITOPE TAGGANTS

University Of California San Diego, La Jolla CA

Investigators

Abstract

This proposal describes the design, synthesis and evaluation of several new fluorescent probes to be tested as membrane-permeant, selective epitope tags, compatible with living cells. These agents have been designed to form high affinity complexes with a short epitope sequences that contain four cysteine residues which are located in pairs separated by two turns of an alpha-helix. Means for optimizing fluorophore-epitope binding affinity and selectivity are described, including the preparation of one-bead one-compound epitope libraries, and phage display techniques. Finally, experiments are described to demonstrate "proof of concept" by visualizing fluorescence resonance energy transfer within living cells, using the above-mentioned probes and recombinant proteins that incorporate suitable epitope sequences. Given the broad implications of protein-protein binding in nature, the availability of this method could provide an important new tool for the non-invasive study of numerous biological processes.