Adding photoregulated amino acids to the genetic code

A method for selecting an active orthogonal aminoacyl-tRNA synthetase (O-RS) that charges an α-aminocaprylic acid, o-nitrobenzyl cysteine, or azobenzyl-Phe on an orthogonal tRNA (O-tRNA), the method comprising:

• subjecting a population of cells to selection, wherein the cells collectively comprise:
• the O-tRNA, wherein the O-tRNA is orthogonal to members of the population of cells that comprise the O-tRNA;
• a plurality of O-RS that comprises one or more active O-RS members that load the O-tRNA with an α-aminocaprylic acid, o-nitrobenzyl cysteine, or azobenzyl-Phe in one or more cells of the population;
• a polynucleotide that encodes a selectable marker, wherein the polynucleotide comprises at least one selector codon that is recognized by the O-tRNA;
• and,
• α-aminocaprylic acid, o-nitrobenzyl cysteine, or azobenzyl-Phe;
• wherein a target cell in the population that comprises the active O-RS is identified by an enhanced suppression efficiency of the selectable marker as compared to a suppression efficiency of a control cell lacking the plurality of RS but comprising the O-tRNA;
• and, selecting the target cell, thereby selecting the active O-RS.

A method of producing a protein in a cell, which protein comprises one or more α-aminocaprylic acid, o-nitrobenzyl cysteine, azobenzyl-Phe, photoregulated serine, photoregulated serine analogue, fluorophore, spin labeled amino acid, or an amino acid comprising a dansyl side chain. at one or more specified position, the method comprising:

• growing the cell in an appropriate medium, which cell comprises a nucleic acid that comprises at least one selector codon and that encodes a protein;
• and,
• providing α-aminocaprylic acid, o-nitrobenzyl cysteine, azobenzyl-Phe, photoregulated serine, a photoregulated serine analogue, a fluorophore, a spin labeled amino acid, or an amino acid comprising a dansyl side chain;
• which cell further comprises:
• an orthogonal tRNA (O-tRNA) that recognizes the selector codon;
• and,
• an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the α-aminocaprylic acid, o-nitrobenzyl cysteine, azobenzyl-Phe, photoregulated serine, a photoregulated serine analogue, a fluorophore, a spin labeled amino acid, or an amino acid comprising a dansyl side chain.;
• and,
• incorporating the α-aminocaprylic acid, o-nitrobenzyl cysteine, azobenzyl-Phe, photoregulated serine, a photoregulated serine analogue, a fluorophore, a spin labeled amino acid, or an amino acid comprising a dansyl side chain into the specified position in response to the selector codon, thereby producing the protein.

A library of polynucleotide members useful for the identification of an orthogonal aminoacyl-tRNA synthetase (O-RS) that functions in a host cell, wherein said polynucleotide members encode variants of an amino acid sequence selected from:

• (i) an amino acid sequence set forth in SEQ ID NO:
• 4, said polynucleotide members comprising randomized nucleotide positions in codons encoding Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162 in SEQ ID NO:
• 4;
• or
• (ii) an amino acid sequence of an Archaea aminoacyl-tRNA synthetase other than the amino acid sequence set forth in SEQ ID NO:
• 4, said polynucleotide members comprising randomized nucleotide positions in codons whose corresponding amino acid positions spatially correspond to Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162 in SEQ ID NO:
• 4.

A method for identifying a desired orthogonal aminoacyl-tRNA synthetase (O-RS), the method comprising:

• a) providing
• (i) a library of polynucleotide members encoding variants of an amino acid sequence set forth in SEQ ID NO:
• 4, said polynucleotide members comprising randomized nucleotide positions in codons encoding Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162 in SEQ ID NO:
• 4;
• and
• (ii) a host cell;
• and
• b) detecting a polynucleotide member from said library that encodes a polypeptide that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid in said host cell, thereby identifying a desired O-RS.

A library of polynucleotide members useful for the identification of an orthogonal aminoacyl-tRNA synthetase (O-RS) that functions in a host cell, wherein said polynucleotide members encode variants of an amino acid sequence selected from:

• (i) an amino acid sequence set forth in SEQ ID NO:
• 3, said polynucleotide members comprising randomized nucleotide positions in codons encoding Met40, Leu41, Tyr499, Tyr527, and His537 in SEQ ID NO:
• 3;
• or
• (ii) an amino acid sequence of an Eubacterial aminoacyl-tRNA synthetase other than the amino acid sequence set forth in SEQ ID NO:
• 3, said polynucleotide members comprising randomized nucleotide positions in codons whose corresponding amino acid positions spatially correspond to Met40, Leu4, Tyr499, Tyr527, and His537 in SEQ ID NO:
• 3.

A method for identifying a desired orthogonal aminoacyl-tRNA synthetase (O-RS), the method comprising:

• a) providing
• (i) a library of polynucleotide members encoding variants of an amino acid sequence set forth in SEQ ID NO:
• 3, said polynucleotide members comprising randomized nucleotide positions in codons encoding Met Leu41, Tyr499, Tyr527, and His537 in SEQ ID NO:
• 3;
• and
• (ii) a host cell;
• and
• b) detecting a polynucleotide member from said library that encodes a polypeptide that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid in said host cell, thereby identifying a desired O-RS.