IP Library › Granted Patent US 12,227,775
Granted Patent B2
US 12,227,775 · App. 16/614,819 · Granted Feb 18, 2025

Modified template-independent DNA polymerase

Inventors: Andrew P. Magyar (Arlington, MA); Peter Cavanagh (Stanford, CA); Miles Rogers (Cambridge, MA); Kirsty A. McFarland (Melrose, MA); Melissa M. Sprachman (Somerville, MA); Amanda Nicole Billings-Siuti (Framingham, MA); Nicole E. Raustad (Charlestown, MA)
Assignee: The Charles Stark Draper Laboratory, Inc.
C12N9/1264C07C245/08C07C247/04C12P19/34C12Q1/68C12Y207/07031
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Quick Facts
Patent No.
US 12,227,775
App. No.
16/614,819
Granted
Feb 18, 2025
Kind
B2
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.

Claims (36)

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 polynucleotide synthesis and the genetically-engineered TdT of claim 1 , wherein the genetically-engineered TdT is capable of attaching a 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.

28. The kit of claim 27 , wherein the bifunctional azobenzene derivative of the genetically-engineered TdT is a photoswitchable moiety.

29. The kit of claim 27 , wherein the reagents include deoxynucleotide triphosphates (dNTPs), or analogs thereof.

30. The kit of claim 29 , wherein the dNTPs contain a fluorescent label.

31. The kit of claim 30 , wherein the fluorescent label is a cleavable fluorescent label.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 31, 2022
From: MAGYAR, ANDREW P.; CAVANAGH, PETER; ROGERS, MILES; MCFARLAND, KIRSTY A.; SPRACHMAN, MELISSA M.; BILLINGS-SIUTI, AMANDA NICOLE
To: THE CHARLES STARK DRAPER LABORATORY, INC.
Reel/Frame 060956/0591 →
Continuity (3)
Provisional Application 62542410 · Aug 8, 2017
Provisional Application 62509549 · May 22, 2017
Related Publication 20200263152A1 · Aug 20, 2020
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