Non-natural amino acid tRNA synthetases for pyridyl tetrazine

Patent metadata

Field	Value
Publication number	US-9988619-B2
Application number	US-201415028379-A
Country	US
Kind code	B2
Filing date	Oct 10, 2014
Priority date	Oct 11, 2013
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

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.

First claim

What is claimed is:

1. 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.

2. The composition of claim 1 , wherein the RS:
a) has the amino acid substitution L65A or L65V
b) has a sequence identity of at least least 90% of SEQ ID NO: 1, or
c) is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

3. A polynucleotide encoding the aminoacyl-tRNA synthetase (RS) of claim 1 .

4. The polynucleotide of claim 3 encoding the RS, wherein the RS
a) has the amino acid substitution L65A or L162V;
b) has a sequence identity of at least least 90% of SEQ ID NO: 1; or
c) is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

5. A cell free protein synthesis system 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 system comprising:
a) a cell free extract of bacteria having biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis;
b) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
c) the compound of Formula II in a concentration sufficient to permit selective incorporation of the compound of Formula II into the protein of interest;
d) a tRNA able to be charged with the compound of formula II and complementary to the suppression codon of the protein of interest; and
e) the aminoacyl-tRNA synthetase (RS) of claim 1 .

6. The cell free protein synthesis system of claim 5 , wherein the cell free extract has an active oxidative phosphorylation system.

7. The cell free protein synthesis system of claim 5 , wherein the protein of interest is an antibody or antibody fragment.

8. 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.

9. The method of claim 8 , further comprising the step of conjugating a biologically active adduct to the compound of Formula II within the protein of interest.

10. The method of claim 9 , wherein the conjugation is by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a trans-cyclooctene on a biologically active adduct or by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a methylcyclopropene on a biologically active adduct.

Assignees

• Sutro Biopharma Inc

Inventors

• Zimmerman Erik S
• Thanos Christopher

Classifications

• C12Y601/01001

  Tyrosine-tRNA ligase (6.1.1.1) · CPC title

• C12Y601/01

  Ligases forming aminoacyl-tRNA and related compounds (6.1.1) · CPC title

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title