Modification of 3′ terminal ends of nucleic acids by DNA polymerase theta
The invention provides compositions and methods for modifying the 3′-terminal ends of nucleic acids using DNA polymerase θ terminal transferase activity.
1. A method of modifying a 3′ terminal end of a nucleic acid with a substrate, the method comprising:
forming a mixture comprising an A family polymerase, a substrate, a nucleic acid, and a reaction solution,
wherein the A family polymerase is Polθ or an active fragment or variant thereof,
wherein the nucleic acid is selected from the group consisting of a single stranded nucleic acid, double stranded nucleic acid possessing a 3′ single stranded overhang, double stranded nucleic acid lacking a 5′ single stranded overhang, and nucleic acid lacking a 5′ single stranded overhang; and
wherein the reaction solution comprises at least one divalent metal,
wherein the divalent metal comprises manganese (Mn 2+ );
incubating the mixture; and
isolating a 3′-terminal end modified nucleic acid.
2. The method of claim 1 , wherein the nucleic acid is selected from the group consisting of single stranded DNA (ssDNA), double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA.
3. The method of claim 1 , wherein the nucleic acid is RNA.
4. The method of claim 1 , wherein Polθ comprises the amino acid sequence of SEQ ID NO 1.
5. The method of claim 1 , wherein the substrate is selected from the group consisting of dATP, dGTP, dCTP, dUTP, ATP, CTP, UTP, GTP, a modified nucleotide, or any combination thereof.
6. The method of claim 5 , wherein the modified nucleotide is selected from cy3-dUTP, Digoxigenin-11-dUTP, Biotin-16AA-dUTP, Texas Red-5-dCTP, Cyanine 3-AA-UTP, 4-Thio-UTP, Biotin-16-AACTP, Ganciclovir Triphosphate, N6-(6-Azido)hexyl-adenosine-5′-triphosphate, and 5-Hydroxymethyl-2′-deoxyuridine-5′-Triphosphate.
7. The method of claim 1 , wherein the divalent metal further comprises cobalt (Co 2+ ).
8. The method of claim 1 , wherein the divalent metal is at a concentration of about 1 mM to about 50 mM.
9. The method of claim 8 , wherein the divalent metal is at a concentration of about 5 mM.
10. The method of claim 1 , wherein the reaction solution further comprises glycerol, a non-ionic detergent, and a buffer.
11. The method of claim 10 , wherein a concentration of the glycerol in the reaction solution is less than or equal to 20%.
12. The method of claim 11 , wherein the concentration of glycerol in the reaction solution is 10%.
13. The method of claim 10 , wherein the non-ionic detergent is NP-40.
14. The method of claim 10 , wherein a concentration of the non-ionic detergent is less than 1%.
15. The method of claim 14 , wherein the concentration of the non-ionic detergent is 0.1%.
16. The method of claim 10 , wherein the buffer is MES/TRIS and wherein MES/TRIS is at a concentration of about 20 mM to about 100 mM.
17. The method of claim 10 , wherein the pH of the buffer is 6.5-8.8.
18. The method of claim 17 , wherein the pH of the buffer is 8.2.
19. The method of claim 1 , wherein the incubating the mixture is incubating the mixture for at least 2 hours.
20. The method of claim 1 , where the incubating the mixture is incubating the mixture at 25° C.-42° C.
21. The method of claim 20 , where the incubating the mixture is incubating the mixture at 42° C.
22. A kit for modifying a 3′ terminal end of a nucleic acid with a substrate, the kit comprising an A-family polymerase and a reaction solution,
wherein the nucleic acid is selected from the group consisting of a single stranded DNA (ssDNA), double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA;
wherein the A-family polymerase is Polθ, or an active fragment or variant thereof; and
wherein the reaction solution comprises manganese (Mn2+).
23. The kit of claim 22 , the kit further comprising the substrate.
24. The kit of claim 22 , wherein the nucleic acid is a single stranded RNA or a double stranded RNA possessing a 3′ single stranded RNA overhang.
25. The kit of claim 22 , wherein the reaction solution comprises 5 mM Mn 2+ , 20 mM Tris HCl pH 8.2, 10% glycerol, 0.01% NP-40 and 0.1 mg/mL BSA.
26. A method of synthesizing sequence-specific nucleic acids, the method comprising:
forming a mixture comprising an A family polymerase, at least one nucleotide, and a reaction solution, wherein the reaction solution comprises a nucleic acid and at least one divalent metal,
wherein the nucleic acid is selected from the group consisting of a single stranded DNA (ssDNA), double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA,
wherein the A family polymerase is Polθ, or an active fragment or variant thereof; and
wherein the divalent metal comprises manganese (Mn 2+ );
incubating the mixture; and
isolating a nucleic acid with a defined sequence.
27. The method of claim 26 , wherein the nucleic acid is a single stranded RNA or a double stranded RNA possessing a 3′ single stranded RNA overhang.
28. The method of claim 26 , wherein the at least one nucleotide is selected from ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, dCTP, a modified nucleotide, and any combination thereof.
29. The method of claim 1 , wherein the 3′-terminal end modified nucleic acid is selected from the group consisting of a ssDNA, double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA.