Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

A eukaryotic 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;
• and,
• (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.

A eukaryotic 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;
• and,
• (d.) said OtRNA recognizes a selector codon of an mRNA in said cell;
• and 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.