Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs
View Patent ↗This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.
1. A cell comprising an orthogonal tRNA synthetase (ORS) and an orthogonal tRNA (OtRNA), as a pair having the following provisios:
(a) said ORS preferentially aminoacylates said OtRNA, wherein preferential aminoacylation is defined in that said ORS aminoacylates endogenous tRNAS of the cell with a reduced efficiency as compared to endogenous tRNA synthetase aminoacylation of said endogenous tRNAS of the cell;
(b) said ORS preferentially aminoacylates said OtRNA with an unnatural amino acid, as compared to any natural amino acid;
(c) said OtRNA is aminoacylated by the endogenous tRNA synthetases of the cell with reduced efficiency as compared to aminoacylation of the endogenous tRNAS by said endogenous tRNA synthetases;
(d) said OtRNA recognizes a selector codon of an mRNA in said cell; and wherein kcat/Km for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than kcat/Km for aminoacylation of the OtRNA by the ORS with the natural amino acid, or wherein said unnatural amino acid is incorporated into a growing polypeptide in the cell with a fidelity of greater than 75% in response to the selector codon.
2. The cell of claim 1 , wherein said unnatural amino acid is selected from a group consisting of: O-methyl-L-tyrosine, L-3-(2-naphthyl)-alanine, p-benzoyl-L-phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, p-amino-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, and isopropyl-L-phenylalanine.
3. The cell of claim 1 , wherein the unnatural amino acid is p-azido-L-phenylalanine.
4. The cell of claim 1 , wherein the unnatural amino acid is p-acetyl-L-phenylalanine.
5. The cell of claim 1 , wherein the unnatural amino acid is m-acetyl-L-phenylalanine.
6. The cell of claim 1 , wherein the unnatural amino acid is 4-(2-oxo-propoxy)-L-phenylalanine.
7. The cell of claim 1 , wherein said cell is a prokaryotic cell.
8. The cell of claim 7 wherein said cell is a bacterium.
9. The cell of claim 7 wherein said cell is an Escherichia coli cell.
10. The cell of claim 1 , wherein said selector codon is an amber codon or a four base codon.
11. The cell of claim 1 , wherein the orthogonal tRNA synthetase comprises an amino acid sequence as set forth in SEQ ID No.: 62.
12. The cell of claim 1 , wherein the orthogonal tRNA is encoded by a nucleic acid comprising a sequence as set forth in SEQ ID No.: 1.