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US 2026/0126438 A1Published application
Systems and method for target engagement
AssigneePromega Corporation
InventorsCesear CORONA, Ming CHEN, Wenhui ZHOU
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Abstract
Provided herein are systems and methods for the detection and quantification of a target protein via bioluminescence and/or bioluminescent resonance energy transfer (BRET). In particular, provided herein are assay methods using a multi-component biochemical system that assembles into a detectable in vitro chemical complex at a protein target of interest.
Claims29
A system comprising:
- (a) a bioluminescent protein or a bioluminescent complex tethered to a target protein;
- (b) fluorophore tethered to a reference ligand for the target protein;
- (c) a test ligand for the target protein;
- and
- (d) a substrate for the bioluminescent protein or complex;
- wherein the bioluminescent protein or complex emits a bioluminescent signal at a first wavelength in the presence of the substrate;
- wherein the fluorophore absorbs light at the first wavelength and emits a fluorescent signal at the second wavelength;
- wherein when the reference ligand is bound to the target protein the fluorophore is in proximity to the bioluminescent protein or complex to allow bioluminescence resonance energy transfer (BRET) from the bioluminescent protein or complex;
- and
- wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the fluorescent signal resulting from the BRET.
7 more elements
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A system comprising:
- (a) a first component of a bioluminescent complex tethered to a target protein;
- (b) a second component of the bioluminescent complex tethered to a reference ligand;
- (c) a test ligand for the target protein;
- and
- (d) a substrate for the bioluminescent protein or complex;
- wherein the bioluminescent protein or complex emits a bioluminescent signal in the presence of the substrate;
- wherein when the reference ligand is bound to the target protein the second component of the bioluminescent complex is in proximity to the first component of the bioluminescent complex to facilitate formation the bioluminescent complex and generation of the bioluminescent signal in the presence of the substrate;
- and
- wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the bioluminescent signal from the bioluminescent complex.
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26-41. (canceled)
A method comprising:
- (a) contacting within a biochemical sample:
- (i) a bioluminescent protein or a bioluminescent complex tethered to a target protein;
- (ii) fluorophore tethered to a reference ligand for the target protein;
- and
- (iii) a substrate for the bioluminescent protein or complex;
- wherein the bioluminescent protein or complex emits a bioluminescent signal at a first wavelength in the presence of the substrate;
- wherein the fluorophore absorbs light at the first wavelength and emits a fluorescent signal at the second wavelength;
- wherein when the reference ligand is bound to the target protein the fluorophore is in proximity to the bioluminescent protein or complex to allow bioluminescence resonance energy transfer (BRET) from the bioluminescent protein or complex;
- (b) detecting bioluminescence from the bioluminescent protein or complex;
- (c) contacting the sample with a test ligand for the target protein;
- and
- (d) detecting bioluminescence from the bioluminescent protein or complex;
- wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the fluorescent signal resulting from the BRET.
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42-65. (canceled)
A method comprising:
- (a) a first component of a bioluminescent complex tethered to a target protein;
- (b) a second component of the bioluminescent complex tethered to a reference ligand;
- (c) a test ligand for the target protein;
- and
- (d) a substrate for the bioluminescent protein or complex;
- wherein the bioluminescent protein or complex emits a bioluminescent signal in the presence of the substrate;
- wherein when the reference ligand is bound to the target protein the second component of the bioluminescent complex is in proximity to the first component of the bioluminescent complex to facilitate formation the bioluminescent complex and generation of the bioluminescent signal in the presence of the substrate;
- and
- wherein if the test ligand is capable of binding to the target protein the test ligand competes with the reference ligand for binding to the target protein, thereby decreasing the bioluminescent signal from the bioluminescent complex.
6 more elements
67-82. (canceled)
Dependencies are inferred from the claim text.