Modified template-independent DNA polymerase

Patent metadata

Field	Value
Publication number	US-12227775-B2
Application number	US-201816614819-A
Country	US
Kind code	B2
Filing date	May 22, 2018
Priority date	May 22, 2017
Publication date	Feb 18, 2025
Grant date	Feb 18, 2025

Abstract

Described herein are genetically engineered template-independent DNA polymerases, specifically terminal deoxynucleotidyl transferases, and methods of using these polymerases to control DNA synthesis by adding a single nucleotide (mononucleotide) at a time to the 3′ end of a growing single-stranded DNA polynucleotide.

First claim

What is claimed is:

1. A genetically-engineered terminal deoxynucleotidyl transferase (TdT), wherein a wild-type TdT has been mutated at a single amino acid residue site to incorporate an azide or cyclooctene non-naturally occurring amino acid selected from the group consisting of: 4-Azido-L-phenylalanine (AzF), N-Propargyl-Lysine (PrK), Cyclooctene-L-Lysine (TCO-K) or Cyclooctyne-Lysine (SCO-K), wherein the non-naturally occurring amino acid is modified with a bifunctional azobenzene derivative comprising two orthogonal functional domains, wherein the first functional domain comprises a click reactive group for attachment to an affinity tag peptide for purification, and the second functional domain comprises a click reactive group whereby the bifunctional azobenzene derivative is attached to the non-naturally occurring amino acid, resulting in a TdT modified with the bifunctional azobenzene derivative capable of a reversible conformational change for controlled addition of a mononucleotide to the 3′ end of a single-stranded polynucleotide.

2. The genetically-engineered TdT of claim 1 , wherein the wild-type TdT comprises the amino acid sequence SEQ ID NO: 1, SEQ ID NO:5, or a homologous TdT comprising at least about 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO:5.

3. The genetically-engineered TdT of claim 1 , wherein the genetically-engineered TdT is photoisomerizable.

4. The genetically-engineered TdT of claim 1 wherein the bifunctional azobenzene derivative is a photoswitchable moiety.

5. The genetically-engineered TdT of claim 1 , wherein the bifunctional azobenzene derivative regulates entry or binding of a mononucleotide to the active site of TdT.

6. The genetically-engineered TdT of claim 1 , wherein the single amino acid residue site is exposed on the surface of the TdT protein.

7. The genetically-engineered TdT of claim 6 , wherein the single amino acid residue site in the wild-type TdT is occupied by a lysine.

8. The genetically-engineered TdT of claim 7 , wherein the lysine is selected from a position corresponding to position 199, 238, 247, 250, 276, 338 or 419 of the amino acid sequence SEQ ID NO: 1, or a position corresponding to a position 199, 238, 247, 250, 276, 338 or 419 in an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1.

9. The genetically-engineered TdT of claim 1 , wherein the bifunctional azobenzene derivative regulates the translocation, or ratcheting, of the TdT along the single-stranded polynucleotide thereby inhibiting the addition of a mononucleotide to the polynucleotide.

10. The genetically-engineered TdT of claim 1 , wherein the non-naturally occurring amino acid residue is incorporated into a site located in the loop of the TdT protein associated with DNA ratcheting function.

11. The genetically-engineered TdT of claim 1 , wherein the non-naturally occurring amino acid residue is incorporated into a site located at a position corresponding to a position selected from the group consisting of: Q166, N242, K250, E279, F385, M339, F405, K419or Q423 of SEQ ID NO:1, and a position corresponding to position Q166, N242, K250, E279, F385, M339, F405, K419 or Q423 of an amino acid sequence with at least 95% identity to SEQ ID NO: 1.

12. The genetically-engineered TdT of claim 1 , wherein the click reactive group of the first functional domain and/or the click reactive group of the second functional domain of the bifunctional azobenzene derivative attaches to an attachment site that comprises an amine or alcohol.

13. The genetically-engineered TdT of claim 12 , wherein the attachment site for the click reactive group of the first functional domain and/or the click reactive group of the second functional domain is an alcohol, and the alcohol is converted to be a ketone, aldehyde, or carboxcylic acid.

14. The genetically-engineered TdT of claim 12 , wherein
(i) the click reactive group of the first functional domain and its attachment site on the affinity tag peptide are selected from a pair of clickable orthogonal groups, the pair comprising:

an azide-alkyne groups; tetrazine-norbomene groups; or tetrazine-trans-cyclooctene groups, and/or
(ii) the click reactive group of the second functional domain and its attachment site on the non-naturally occurring amino acid are selected from a pair of clickable orthogonal groups, the pair comprising: an azide-alkyne groups; tetrazine-norbomene groups; or tetrazine-trans-cyclooctene groups.

15. The genetically-engineered TdT of claim 1 , wherein, the bifunctional azobenzene derivative comprises the structure of:

wherein, R 1 is selected from the group consisting of alkyl, substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl; and optionally R 1 contains a bioconjugation moiety.

16. The genetically-engineered TdT of claim 1 , wherein the bifunctional azobenzene derivative comprises the structure of:

wherein, R 1 and R 2 are independently selected from the group consisting of alkyl, substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl; and optionally R 1 and R 2 contain bioconjugation moieties.

17. The genetically-engineered TdT of claim 1 , wherein the bifunctional azobenzene derivative comprises the structure of:

18. The genetically engineered TdT of claim 1 , wherein the affinity tag peptide is selected from the group consisting of: HIS- 6 , Glutathione, c-Myc, HA, V5, Xpress, Biotin acceptor domain (BAD), VSVG, protein c, S-tag and FLAG.

19. The genetically engineered TdT of claim 1 , wherein the click reactive group of the second functional domain of the bifunctional azobenzene derivative comprises a tetrazine.

20. The genetically-engineered TdT of claim 18 , wherein the affinity tag peptide is FLAG.

21. A method of template-independent polynucleotide synthesis comprising the steps of contacting mononucleotide with the genetically-engineered TdT of claim 1 , wherein the genetically-engineered TdT is immobilized on a solid support and is capable of attaching the mononucleotide to the 3′ end of a single-stranded polynucleotide under conditions suitable for the incorporation of a mononucleotide to the 3′ end of the single-stranded polynucleotide.

22. The method of claim 21 , wherein the genetically-engineered TdT is photoisomerizable.

23. The method of claim 22 , wherein the bifunctional azobenzene derivative of the genetically-engineered TdT is a photoswitchable moiety.

24. The method of claim 23 , wherein the bifunctional azobenzene derivative regulates entry or binding of a mononucleotide to the active site of TdT.

25. The method of claim 21 , wherein the mononucleotide contains a cleavable fluorescent label.

26. The method of claim 25 , wherein the mononucleotide contains a phosphate coupled fluorophore that is cleaved upon attachment to the 3′ end of the single-stranded polynucleotide.

27. A kit comprising reagents for template-independent

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Magyar Andrew P
• Cavanagh Peter
• Rogers Miles
• Mcfarland Kirsty A
• Sprachman Melissa M
• Billings-Siuti Amanda Nicole
• Raustad Nicole E

Classifications

• C07C245/08

  with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene · CPC title

• C07C247/04

  being saturated · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12Y207/07031

  DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title