Photoactive fluorophores and methods of in vivo labeling

Abstract

Provided are a photoactive fluorophore, a photoactive ligand, and a photoactive complex. The photoactive fluorophore includes a photoactivatable derivative of an azetidine-containing Janelia-Fluor dye. The photoactive ligand includes a photoactive fluorophore and a protein tag. The photoactive complex includes a photoactive ligand conjugated to a protein. Also provided are methods of in vivo labeling with and photoactivation of the photoactive fluorophore, ligand, and complex.

What is claimed is: 
     
         1 . A photoactive fluorophore comprising a photoactivatable derivative of an azetidine-containing Janelia-Fluor dye. 
     
     
         2 . The photoactive fluorophore of  claim 1 , wherein the fluorophore is decarboxylated by photoinduction. 
     
     
         3 . The photoactive fluorophore of  claim 1 , wherein the photoactivatable derivative is photoactivatable Janelia-Fluor 549. 
     
     
         4 . The photoactive fluorophore of  claim 1 , wherein the photoactivatable derivative is photoactivatable Janelia-Fluor 646. 
     
     
         5 . A method of forming a photoactive fluorophore, the method comprising caging a Janelia-Fluor dye. 
     
     
         6 . The method of  claim 5 , wherein the caging comprises:
 adding oxalyl chloride to a solution of the Janelia-Fluor dye, forming a first mixture;   adding triethylamine and (trimethylsilyl)diazomethane in succession to the first mixture, forming a second mixture;   concentrating the second mixture, forming a concentrate; and   purifying the concentrate, providing the photoactive fluorophore.   
     
     
         7 . The method of  claim 6 , wherein the Janelia-Fluor dye comprises a Si-rhodamine. 
     
     
         8 . A photoactive ligand comprising a photoactive fluorophore and a protein tag. 
     
     
         9 . The photoactive ligand of  claim 8 , wherein the photoactive fluorophore comprises a photoactivatable derivative of an azetidine-containing Janelia-Fluor dye. 
     
     
         10 . The photoactive ligand of  claim 8 , wherein the protein tag is a HaloTag ligand. 
     
     
         11 . The photoactive ligand of  claim 8 , wherein the protein tag is a SNAP-tag ligand. 
     
     
         12 . The photoactive ligand of  claim 8 , wherein conjugation of the photoactive ligand to a cognate protein increases light absorption of the photoactive ligand after photolysis. 
     
     
         13 . The photoactive ligand of  claim 8 , wherein a brightness of the photoactive fluorophore is substantially similar to the brightness of a parent fluorophore from which the photoactive fluorophore is derived. 
     
     
         14 . A photoactive complex comprising a photoactive ligand conjugated to a protein. 
     
     
         15 . The photoactive complex of  claim 14 , wherein the photoactive ligand is conjugated to the protein in vivo. 
     
     
         16 . The photoactive complex of  claim 14 , wherein the photoactive ligand comprises a photoactive fluorophore and a protein tag. 
     
     
         17 . The photoactive complex of  claim 14 , wherein the photoactive fluorophore is arranged and disposed to decarboxylate upon photoinduction. 
     
     
         18 . The photoactive complex of  claim 17 , wherein the photoactive fluorophore is arranged and disposed to form a methyl-Janelia-Fluor compound upon decarboxylation. 
     
     
         19 . The photoactive complex of  claim 17 , wherein the photoactive fluorophore is arranged and disposed to provide increased fluorescence upon decarboxylation.