Incorporation of unnatural nucleotides and methods thereof

Abstract

Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

What is claimed is: 
     
         1 . A cell comprising:
 (a) a nucleoside triphosphate transporter from  Phaeodactylum tricornutum;      (b) a tRNA from  Methanosarcina mazei ; and   (c) a pyrrolysine aminoacyl tRNA synthetase from  Methanosarcina barkeri.      
     
     
         2 . The cell of  claim 1 , wherein the cell comprises an oligonucleotide encoding the tRNA. 
     
     
         3 . The cell of  claim 1 , wherein the cell further comprises an oligonucleotide encoding the pyrrolysine aminoacyl tRNA synthetase. 
     
     
         4 . The cell of  claim 1 , wherein the cell further comprises a single oligonucleotide encoding the tRNA and the pyrrolysine aminoacyl tRNA synthetase. 
     
     
         5 . The cell of  claim 4 , wherein the cell further comprises an oligonucleotide comprising one or more unnatural nucleobases. 
     
     
         6 . The cell of  claim 5 , wherein the one or more unnatural nucleobases comprises a structure selected from and 
       
         
           
           
               
               
           
         
       
     
     
         7 . The cell of  claim 5 , wherein the oligonucleotide comprises a first unnatural nucleobase having a structure 
       
         
           
           
               
               
           
         
       
       and a second unnatural nucleobase having a structure 
       
         
           
           
               
               
           
         
       
       and wherein the first unnatural nucleobase and the second unnatural nucleobase form a base pair in the oligonucleotide. 
     
     
         8 . The cell of  claim 1 , wherein the tRNA from  Methanosarcina mazei  comprises one or more anti-codons selected from GYT and GYC, wherein Y is an unnatural nucleobase. 
     
     
         9 . The cell of  claim 8 , wherein Y is selected from 
       
         
           
           
               
               
           
         
       
     
     
         10 . The cell of  claim 1 , wherein the cell is  E. coli.    
     
     
         11 . A method of producing a protein in a cell, wherein the protein comprises an unnatural amino acid, the method comprising:
 (a) providing a cell comprising:
 (i) a nucleoside triphosphate transporter from  Phaeodactylum tricornutum  that transports a nucleotide into the cell; 
 (ii) a tRNA from  Methanosarcina mazei;    
 (iii) a pyrrolysine aminoacyl tRNA synthetase from  Methanosarcina barkeri ; and 
 (iv) an unnatural amino acid; and 
   (b) synthesizing a protein in the cell by a process of:
 (i) transporting a nucleotide into the cell by the nucleoside triphosphate transporter; 
 (ii) incorporating the nucleotide into a double-stranded oligonucleotide; 
 (iii) translating the protein from the double-stranded oligonucleotide utilizing the tRNA, the tRNA synthetase, and the unnatural amino acid. 
   
     
     
         12 . The method of  claim 11 , further comprising expressing the tRNA from a plasmid in the cell. 
     
     
         13 . The method of  claim 11 , further comprising expressing the pyrrolysine aminoacyl tRNA synthetase from a plasmid in the cell. 
     
     
         14 . The method of  claim 11 , further comprising expressing the tRNA and the pyrrolysine aminoacyl tRNA synthetase from a single plasmid in the cell. 
     
     
         15 . The method of  claim 11 , wherein the nucleotide comprises an unnatural nucleobase. 
     
     
         16 . The method of  claim 15 , wherein the unnatural nucleobase is 
       
         
           
           
               
               
           
         
       
     
     
         17 . The method of  claim 11 , further comprising:
 (a) providing a first nucleotide comprising a nucleobase having a structure   
       
         
           
           
               
               
           
         
       
       and a second nucleotide comprising a nucleobase having a structure 
       
         
           
           
               
               
           
         
       
       and
 (b) forming an unnatural base pair in the cell with the first nucleotide and the second nucleotide in the double-stranded oligonucleotide. 
 
     
     
         18 . The method of  claim 11 , wherein the tRNA from  Methanosarcina mazei  comprises one or more anti-codons selected from GYT and GYC, wherein Y comprises an unnatural nucleobase. 
     
     
         19 . The method of  claim 18 , wherein Y is selected from 
       
         
           
           
               
               
           
         
       
     
     
         20 . The method of  claim 11 , wherein the cell is  E. coli.