Methods of incorporating an amino acid comprising a bcn group into a polypeptide using an orthogonal codon encoding it and an orthorgonal pylrs synthase

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 . A polypeptide comprising an amino acid having a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group. 
     
     
         2 . A polypeptide according to  claim 1  wherein said BCN group is present as a residue of a lysine amino acid. 
     
     
         3 . 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. 
     
     
         4 . A method according to  claim 3  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; 
 (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said amino acid having a BCN group into the polypeptide chain. 
 
     
     
         5 . A method according to  claim 3  wherein said amino acid comprising a BCN group is a BCN lysine. 
     
     
         6 . A method according to  claim 4  wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtRNA CUA , said amino acid having a BCN group comprises a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine and said tRNA synthetase comprises a PylRS synthetase having the mutations Y271M, L274G and C313A (BCNRS). 
     
     
         7 . A polypeptide according to  claim 1 , or a method according to any of  claims 3  to  6 , wherein said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide. 
     
     
         8 . A polypeptide according to  claim 1  which comprises a single BCN group. 
     
     
         9 . A polypeptide according to  claim 1  wherein said BCN group is joined to a tetrazine group. 
     
     
         10 . A polypeptide according to  claim 9  wherein said tetrazine group is further joined to a fluorophore. 
     
     
         11 . A polypeptide according to  claim 10  wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY). 
     
     
         12 . An amino acid comprising bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN). 
     
     
         13 . An amino acid according to  claim 12  which is bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) lysine. 
     
     
         14 . BCN lysine according to  claim 13  having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         15 . A method of producing a polypeptide comprising a tetrazine group, said method comprising providing a polypeptide according to  claim 1 , contacting said polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine to the BCN group by an inverse electron demand Diels-Alder cycloaddition reaction. 
     
     
         16 . A method according to  claim 15  wherein the tetrazine is selected from 6 to 17 of  FIG. 1 . 
     
     
         17 . A method according to  claim 15  wherein the tetrazine is selected from 6, 7, 8 and 9 of  FIG. 1  and the pseudo first order rate constant for the reaction is at least 80 M −1  s −1 . 
     
     
         18 . A method according to  claim 15  wherein said reaction is allowed to proceed for 10 minutes or less. 
     
     
         19 . A method according to  claim 18  wherein said reaction is allowed to proceed for 1 minute or less. 
     
     
         20 . A method according to  claim 19  wherein said reaction is allowed to proceed for 30 seconds or less. 
     
     
         21 . A method according to  claim 15  wherein said tetrazine compound is a tetrazine compound selected from the group consisting of 11 and 17 of  FIG. 1 . 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled)