Development of Sensitive FRET Sensors and Methods of Using the Same

Abstract

Intramolecular biosensors are disclosed, including PBP-based biosensors, comprising a ligand binding domain fused to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon binding ligand. At least one of the donor and fluorescent moieties may be internally fused to the biosensor such that both ends of the internally fused fluorophore are fixed. In addition, methods of improving the sensitivity of terminally fused biosensors are provided. The biosensors of the invention are useful for the detection and quantification of ligands in vivo and in culture.

1 .- 98 . (canceled) 
     
     
         99 . An isolated nucleic acid comprising a polynucleotide sequence encoding a periplasmic binding protein (PBP) that specifically binds a ligand of interest, and at least one of a donor fluorophore protein fused to the PBP or an acceptor fluorophore protein fused to the PBP, wherein the coding region of the donor fluorophore protein or the coding region of the acceptor fluorophore protein is inserted at an internal site of the coding region of the PBP. 
     
     
         100 . The isolated nucleic acid of  claim 99 , wherein the coding region of the donor fluorophore protein is fused to an internal site of the PBP. 
     
     
         101 . The isolated nucleic acid of  claim 99 , wherein the coding region of the acceptor fluorophore protein is fused to an internal site of the PBP. 
     
     
         102 . The isolated nucleic acid of  claim 99 , wherein the coding region of the donor fluorescent protein and the coding region of the acceptor fluorescent protein are fused to the same lobe of the PBP. 
     
     
         103 . The isolated nucleic acid of  claim 99 , wherein the PBP specifically binds an amino acid. 
     
     
         104 . The isolated nucleic acid of  claim 99 , wherein the PBP specifically binds a sugar. 
     
     
         105 . The isolated nucleic acid of  claim 99 , wherein the coding region of the acceptor fluorophore protein is inserted at the C-terminus of the PBP. 
     
     
         106 . The isolated nucleic acid of  claim 99 , wherein the donor fluorophore protein is selected from the group consisting of a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy) and monomeric CoralHue Kusabira-Orange (mKO). 
     
     
         107 . The isolated nucleic acid of  claim 106 , wherein the donor fluorophore protein is eCFP. 
     
     
         108 . The isolated nucleic acid of  claim 106 , wherein the donor fluorophore protein is YFP VENUS. 
     
     
         109 . The isolated nucleic acid of  claim 99 , wherein the acceptor fluorophore protein is selected from the group consisting of a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy) and monomeric CoralHue Kusabira-Orange (mKO). 
     
     
         110 . The isolated nucleic acid of  claim 100 , further comprising at least one peptide linker that links the internal site of the PBP to the at least one donor fluorophore protein. 
     
     
         111 . The isolated nucleic acid of  claim 101 , further comprising at least one peptide linker that links the internal site of the PBP to the at least one acceptor fluorophore protein. 
     
     
         112 . The isolated nucleic acid of  claim 99 , wherein the PBP comprises a glucose-galactose binding protein (GBP) or a glutamate-aspartate receptor (YbeJ). 
     
     
         113 . The isolated nucleic acid of  claim 99 , wherein the polynucleotide sequence encoding the periplasmic binding protein (PBP) comprises one or more substitution mutations that modify the affinity of the PBP to its ligand. 
     
     
         114 . An expression vector comprising the nucleic acid of  claim 99 . 
     
     
         115 . A host cell comprising the vector of  claim 114 . 
     
     
         116 . A ligand binding fluorescent indicator encoded by the nucleic acid of  claim 99 . 
     
     
         117 . The isolated nucleic acid of  claim 100 , wherein the nucleic acid further comprises a coding region of an acceptor fluorophore protein that is fused to the coding region of the PBP. 
     
     
         118 . The isolated nucleic acid of  claim 101 , wherein the nucleic acid further encodes a coding region of a donor fluorophore protein that is fused to the coding region of the PBP.