Synthetic Fluorescent Protein Biosensors and Use Thereof in Drug Screening Methods
AssigneeThe Regents of the University of Colorado, a body corporate
InventorsXuedong Liu, Douglas A. Chapnick, William Old, Tristan D. Mcclure-Begley, Eric Bunker
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Abstract
Provided are FRET-based biosensor constructs, and multiplexed platforms or arrays of these biosensor constructs useful for screening candidate drug molecules for efficacy and/or specificity of drug activity. Optionally the biosensor constructs may be located on an inner membrane within a cell or engineered to be located on the cell's surface. The cells or cell lines displaying the biosensors on a cell surface may be arranged as an array of cells for high throughput evaluation of the efficacy and/or specificity of drug candidates, such as a library of candidate drug compounds.
Claims20
A method of evaluating drug activity comprising:
- a) detecting whether the drug has activity in modulating ligand binding or proteolytic activity by contacting a biosensor with the drug, illuminating the sample, and detecting fluorescence resulting from a FRET energy transfer pair;
- and
- c) determining the drug activity by comparing the fluorescence resulting from drug contact with the biosensor construct with a reference correlation of fluorescence and known or standard drug activity;
- wherein the biosensor comprises from N-terminus to C-terminus:
- 1) a FRET acceptor or donor;
- 2) a proteolytic cleavage site;
- 3) a ligand;
- 4) a FRET acceptor or donor that completes a FRET energy transfer pair with the first position;
- 5) a ligand binding domain and/or a protease specific for the proteolytic cleavage site.
A method of evaluating drug activity comprising:
- a) detecting whether the drug has activity by contacting at least one biosensor with the drug, illuminating the sample, and detecting fluorescence resulting from the FRET energy transfer pair;
- and
- c) determining the drug activity in the sample by comparing the fluorescence resulting from drug contact with the biosensor construct with a reference correlation of fluorescence and known or standard drug activity;
- wherein the at least one biosensor comprises from N-terminus to C-terminus:
- 1) a FRET acceptor or donor;
- 2) a sensing domain comprising DNA;
- and
- 3) a FRET acceptor or donor that completes a FRET energy transfer pair with the first position.
A method of evaluating drug activity from a library of biosensor constructs comprising:
- generating at least two polynucleotides comprising different nucleic acid sequences;
- inserting each of the at least two polynucleotides into separate biosensor constructs comprising, from N-terminus to C-terminus:
- 1) a FRET acceptor or donor;
- 2) a sensing domain comprising one of the at least two polynucleotides;
- and
- 3) a FRET acceptor or donor that completes a FRET energy transfer pair with the first position;
- to form a library of biosensor constructs comprising sensing domains comprising different nucleic acid sequences.
Dependencies are inferred from the claim text.