Polypeptides and methods

Abstract

The invention relates to a polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group, particularly when said BCN group is present as: a residue of a lysine amino acid. The invention also relates to a method of producing a polypeptide comprising a BCN group, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide. The invention also relates to an amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), particularly and amino acid which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. In addition the invention relates to a PylRS tRNA synthetase comprising the mutations Y271M, L274G and C313A.

1 - 17 . (canceled) 
     
     
         18 . A polypeptide characterized by comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group. 
     
     
         19 . The polypeptide according to  claim 18 , wherein said BCN group is present as a residue of a lysine amino acid. 
     
     
         20 . The polypeptide according to  claim 18 , which comprises a single BCN group. 
     
     
         21 . The polypeptide according to  claim 18 , wherein said BCN group is joined to a tetrazine group. 
     
     
         22 . The polypeptide according to  claim 21 , wherein said tetrazine group is further joined to a fluorophore. 
     
     
         23 . The polypeptide according to  claim 22 , wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY). 
     
     
         24 . The polypeptide according to  claim 18 , wherein the BCN group is BCN lysine and has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         25 . An amino acid incorporated into a polypeptide, wherein the amino acid comprises bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN). 
     
     
         26 . The amino acid according to  claim 25 , characterized by the fact that it is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. 
     
     
         27 . The amino acid according to  claim 26 , wherein the BCN lysine has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         28 . A method of producing a polypeptide comprising a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group in a cell, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide in a cell, wherein said amino acid comprising a BCN group is a BCN lysine, and wherein producing the polypeptide comprises:
 (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group; and   (ii) translating said nucleic acid in the cell,
 wherein said BCN group is in the exo form, 
 wherein said BCN lysine has the structure: 
   
       
         
           
           
               
               
           
         
       
     
     
         29 . The method according to  claim 28 , wherein the nucleic acid is translated in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognizing said orthogonal codon and incorporating said amino acid having a BCN group into the polypeptide chain. 
     
     
         30 . The method according to  claim 28 , wherein said orthogonal codon comprises an amber codon (TAG) and said tRNA is mbtRNA CUA . 
     
     
         31 . The method according to  claim 28 , wherein said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide. 
     
     
         32 . The method according to  claim 28 , wherein the method produces a polypeptide comprising a single BCN group. 
     
     
         33 . The method according to  claim 28 , further comprising:
 (iii) contacting the translated polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine compound to the BCN group by an inverse electron demand Diels-Alder cycloaddition reaction.   
     
     
         34 . The method according to  claim 33 , wherein the tetrazine compound has the chemical formula of 
       
         
           
           
               
               
           
         
         a) Formula VI, wherein:
 (i) X=CH, R=BOC (Formula VI-1); 
 (ii) X=N, R=BOC (Formula VI-2); 
 (iii) X=CH, R=TAMRA-X (Formula VI-3); 
 (iv) X=N, R=TAMRA-X (Formula VI-4); 
 (v) X=CH, R=BODIPY TMR-X (Formula VI-5); or 
 (vi) X=CH, R=TAMRA (Formula VI-6), 
 
       
       
         
           
           
               
               
           
         
         b) Formula VII, wherein:
 (i) R=BOC (Formula VII-1): 
 (ii) R=TAMRA-X (Formula VII-2); or 
 (iii) R=BODIPY-FL (Formula VII-3), 
 
       
       
         
           
           
               
               
           
         
         c) Formula VIII, wherein R=BOC (Formula VIII-I), or 
       
       
         
           
           
               
               
           
         
         d) Formula IX, wherein:
 (i) R=BOC (Formula IX-I); or 
 (ii) R=5-(6)-carboxyfluorescein diacetate (CFDA) (Formula IX-2). 
 
       
     
     
         35 . The method according to  claim 34 , wherein the tetrazine compound has the chemical formula selected from the group consisting of Formula VI-1, Formula VI-2, Formula VII-1 and Formula VIII-1, and wherein the pseudo first order rate constant for the reaction is at least 80 M −1 S −1 . 
     
     
         36 . The method according to  claim 33 , wherein said reaction of step (iii) is allowed to proceed for 10 minutes or less. 
     
     
         37 . The method according to  claim 33 , wherein said reaction of step (iii) is allowed to proceed for 1 minute or less. 
     
     
         38 . The method according to  claim 33 , wherein said reaction of step (iii) is allowed to proceed for 30 seconds or less. 
     
     
         39 . The method according to  claim 34 , wherein said tetrazine compound has the chemical formula selected from the group consisting of Formula VI-3, Formula VI-4, Formula VI-5, Formula VI-6, Formula VII-2, Formula VII-3, and Formula IX-2. 
     
     
         40 . The method according to  claim 33 , wherein said tetrazine compound is further joined to a fluorophore. 
     
     
         41 . The method according to  claim 40 , wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).