Back to results
US 9,988,619 B2Granted
Non-natural amino acid tRNA synthetases for pyridyl tetrazine
AssigneeSutro Biopharma, Inc.
InventorsErik S. Zimmerman, Christopher Thanos
Life of this patent
Estimating the term…
Abstract
The invention provides methods and compositions for a mutein aminoacyl-tRNA synthetase that preferentially charges a tRNA with a non-natural amino acid. Also provided are methods for incorporating the non-natural amino acids, pyridinyl-amino tetrazine amino acids, into a protein, and further conjugating a biologically active adduct to the pyridinyl-amino tetrazine.
Claims10
A composition comprising an aminoacyl-tRNA synthetase (RS) wherein the RS:
- i) preferentially aminoacylates to a degree of greater than 90% a tRNA with (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
- compared to the 20 common naturally occurring amino acids;
- ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO:
- 1;
- and
- iii) using SEQ ID NO:
- 1 as a reference sequence, has:
- a) at position Y32 amino acid:
- G;
- b) at position L65 amino acid:
- V or A;
- c) at position H70 amino acid:
- A or H
- d) at position F108 amino acid:
- P, F or W;
- e) at position Q109 amino acid:
- Q, H or S;
- f) at position D158 amino acid:
- G;
- g) at position 1159 amino acid:
- I or A;
- and
- h) at position L162 amino acid:
- S or G.
21 more elements
A method for selectively incorporating (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
- into a protein of interest, the method comprising the steps of:
- a) combining a cell free extract of bacteria having containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis with the following reagents:
- i) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
- ii) the compound of Formula II in a concentration sufficient to permit selective incorporation of the compound of Formula II into the protein of interest;
- iii) a tRNA able to be charged with the compound of formula II and complementary to the suppression codon of the protein of interest;
- and
- iv) an aminoacyl-tRNA synthetase (RS) wherein the RS:
- preferentially aminoacylates to a degree of greater than 90% a tRNA with the compound of Formula II compared to the 20 common naturally occurring amino acids;
- has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO:
- 1;
- and
- using SEQ ID NO:
- 1 as a reference sequence, has:
- a) at position Y32 amino acid:
- G;
- b) at position L65 amino acid:
- V or A;
- c) at postion H70 amino acid:
- A or H
- d) at position F108 amino acid:
- P, F or W;
- e) at position Q109 amino acid:
- Q, H or S;
- f) at position D158 amino acid:
- G;
- g) at position 1159 amino acid:
- I or A;
- and
- h) at position L162 amino acid:
- S or G;
- and
- b) incubating the combination of step (a) under conditions permitting selective incorporation of the compound of Formula II into the protein of interest.
29 more elements
Dependencies are inferred from the claim text.