Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer

Abstract

Provided herein are compositions and methods for detection and analysis of intracellular binding of a bioactive agent to a cellular target. In particular, bioactive agents tethered to fluorophores, cellular targets fused to bioluminescent reporters, and methods of detecting and analyzing the interaction of bioactive agents with cellular targets therewith.

1 . A system comprising:
 (a) a bioactive agent conjugated to a fluorophore;   (b) a polynucleotide for expression of a protein fusion in a cell, said polynucleotide comprising: (i) a protein of interest and (ii) a luciferase, wherein the bioactive agent is capable of binding non-covalently to the protein of interest upon interaction therewith, wherein the emission spectrum of the luciferase overlaps with the excitation spectrum of the fluorophore; and   (c) a substrate for the luciferase.   
     
     
         2 . The system of  claim 1 , wherein the bioactive agent is a small molecule, a peptide or a nucleic acid. 
     
     
         3 . The system of  claim 3 , wherein the nucleic acid is RNA. 
     
     
         4 . The system of  claim 3 , wherein the nucleic acid is DNA. 
     
     
         5 . The system of  claim 1 , wherein the fluorophore is a small molecule fluorophore. 
     
     
         6 . The system of  claim 5 , wherein the small molecule fluorophore is a carboxy rhodamine analog. 
     
     
         7 . The system of  claim 1 , wherein the luciferase comprises a polypeptide with at least 70% sequence identity with SEQ ID NO.: 1. 
     
     
         8 . The system of  claim 1 , wherein the substrate for the luciferase is coelenterazine or a coelenterazine derivative. 
     
     
         9 . The system of  claim 8 , wherein the substrate is 2-furanylmethyl-deoxy-coelenterazine. 
     
     
         10 . The system of  claim 1 , wherein the conjugate of the bioactive agent and fluorophore is added extracellularly and enters the cell 
     
     
         11 . The system of  claim 1 , wherein the bioactive agent conjugated to the fluorophore is cell permeable. 
     
     
         12 . The system of  claim 11 , further comprising a permeabilization agent to potentiate entry of the bioactive agent conjugated to the fluorophore into the cell. 
     
     
         13 . The system of  claim 1 , wherein the protein fusion is one of a plurality of different proteins of interest fused to luciferase. 
     
     
         14 . The system of  claim 1 , wherein the bioactive agent is produced by non-natural chemical synthesis. 
     
     
         15 . The system of  claim 1 , wherein:
 (i) said luciferase has a first emission spectrum with a first peak emission;   (ii) said fluorophore has an excitation spectrum that overlaps said first emission spectrum; and   (iii) said fluorophore has a second emission spectrum with a second peak emission, said second peak emission being separated from said first peak emission.   
     
     
         16 . The system of  claim 15 , wherein said second peak emission is separated from said first peak emission by at least 80 nm. 
     
     
         17 . The system of  claim 1 , wherein upon binding of the bioactive agent to the protein of interest, conversion of the substrate to a reaction product by the luciferase results in excitation of the fluorophore by bioluminescence resonance energy transfer (BRET and fluorescence emission from the fluorophore. 
     
     
         18 . The system of  claim 1 , wherein the polynucleotide is expressed from a vector. 
     
     
         19 . The system of  claim 18 , wherein the vector is a plasmid or viral vector.