High-sensitive fluorescent energy transfer assay using fluorescent amino acids and fluorescent proteins

Abstract

The disclosure provides method and composition utilizing fluorescent amino acids and fluorescent proteins comprising a moiety capable of undergoing FRET. The methods and compositions of the disclosure are useful in analyzing protein structure and function, and screening molecular inhibitors.

1 - 24 . (canceled) 
     
     
         25 . A method of performing Förster resonance energy transfer (FRET)-based high throughput screening (HTS), the method comprising:
 a) providing a sample in a plurality of wells of a multi-well plate, wherein the sample comprises a first polypeptide comprising a CyPet moiety and a second polypeptide comprising a YPet moiety; 
 b) contacting the sample with a library of candidate agents; 
 c) exposing the plurality of wells to a first wavelength of light, wherein the first wavelength of light is suitable for exciting the CyPet moiety; 
 d) measuring a second wavelength of light in the plurality of wells, wherein the second wavelength of light is emitted from the YPet moiety; and 
 e) calculating the ratiometric FRET signal between the CyPet moiety and the YPet moiety in each of the plurality of wells. 
 
     
     
         26 . The method of  claim 25 , further comprising f) comparing the ratiometric FRET signal calculated in step e) to a standard. 
     
     
         27 . The method of  claim 25 , further comprising f) comparing the ratiometric FRET signal calculated in step e) to a control ratiometric FRET signal obtained by a method comprising:
 i) providing a second sample in one or more wells of a multi-well plate, wherein the second sample comprises the first polypeptide comprising the CyPet moiety and the second polypeptide comprising the YPet moiety;   ii) exposing the one or more wells to a first wavelength of light, wherein the first wavelength of light is suitable for exciting the CyPet moiety;   iii) measuring a second wavelength of light in the one or more wells, wherein the second wavelength of light is emitted from the YPet moiety; and   iv) calculating the control ratiometric FRET signal between the CyPet moiety and the YPet moiety in the one or more wells.   
     
     
         28 . The method of  claim 25 , wherein the first wavelength of light is about 414 nm. 
     
     
         29 . The method of  claim 25 , wherein the second wavelength of light is about 530 nm. 
     
     
         30 . The method of  claim 25 , wherein the first polypeptide further comprises a SUMO1 polypeptide. 
     
     
         31 . The method of  claim 30 , wherein the SUMO1 polypeptide is fused to the CyPet moiety. 
     
     
         32 . The method of  claim 25 , wherein the second polypeptide further comprises an additional polypeptide selected from the group consisting of a SUMO ligase, a SUMO-conjugating enzyme, and a SUMO-specific peptidase. 
     
     
         33 . The method of  claim 32 , wherein the additional polypeptide is fused to the YPet moiety. 
     
     
         34 . The method of  claim 25 , wherein at least one of the candidate agents in the library decreases the ratiometric FRET signal between the first and second polypeptides. 
     
     
         35 . The method of  claim 25 , wherein the multi-well plate is a 96-well plate or 384-well plate. 
     
     
         36 . The method of  claim 25 , wherein the sample comprises a mammalian cell expressing the first polypeptide and the second polypeptide. 
     
     
         37 . The method of  claim 25 , wherein at least one of the candidate agents in the library is an inhibitor. 
     
     
         38 . The method of  claim 37 , wherein the at least one candidate agent disrupts the interaction between the first and second polypeptides. 
     
     
         39 . The method of  claim 25 , wherein the library of candidate agents is a small molecule library. 
     
     
         40 . The method of  claim 25 , wherein the library of candidate agents is selected from the group consisting of a library of drugs, a chemical library, and a library of biologics. 
     
     
         41 . A kit comprising:
 a) a multi-well plate;   b) a first polypeptide comprising a CyPet moiety; and   c) a second polypeptide comprising a YPet moiety and a polypeptide selected from the group consisting of a SUMO ligase, a SUMO-conjugating enzyme, and a SUMO-specific peptidase.   
     
     
         42 . The kit of  claim 41 , further comprising d) one or more candidate agents selected from the group consisting of a drug, a chemical, and a biologic. 
     
     
         43 . The kit of  claim 41 , wherein the first polypeptide further comprises a SUMO1 polypeptide. 
     
     
         44 . The kit of  claim 41 , wherein the multi-well plate is a 96-well plate or 384-well plate.