Nucleic acid synthesis and sequencing using tethered nucleoside triphosphates
Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.
1. A conjugate comprising a nucleotide, a linker, and a template-independent polymerase capable of catalyzing the covalent addition of the nucleotide onto the 3′ end of a nucleic acid, wherein the linker tethers the nucleotide to the template-independent polymerase.
2. The conjugate of claim 1 , wherein the linker is selectively cleavable.
3. The conjugate of claim 2 , wherein cleavage of the linker releases the template-independent polymerase from the nucleotide.
4. The conjugate of claim 3 , wherein cleavage of the linker leaves a scar on the nucleobase of the nucleotide, and wherein the scar comprises a part of the linker retained on the nucleobase after cleavage of the linker.
5. The conjugate of claim 4 , wherein the scar on the nucleobase is selectively removable.
6. The conjugate of claim 5 , wherein removal of the scar leaves a naturally occurring nucleobase.
7. The conjugate of claim 6 , wherein the scar is selectively removable by chemical cleavage, enzymatic cleavage, or photocleavage.
8. The conjugate of claim 3 , wherein cleavage of the linker does not produce a scar on the nucleotide.
9. The conjugate of claim 1 , wherein the nucleotide is a nucleoside triphosphate.
10. The conjugate of claim 1 , wherein the nucleotide comprises a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.
11. The conjugate of claim 1 , wherein the linker is bound to the nucleotide at the nucleobase, sugar, or the α-phosphate of the nucleotide.
12. The conjugate of claim 1 , wherein the polynucleotide is tethered to a solid support.
13. The conjugate of claim 1 , wherein the template-independent polymerase is engineered to comprise a predetermined target site to which the nucleotide is specifically tethered.
14. The conjugate of claim 13 , wherein the predetermined target site comprises a modified amino acid.
15. The conjugate of claim 14 , wherein the modified amino acid comprises p-propargyloxyphenylalanine or p-azidophenylalanine.
16. The conjugate of claim 1 , wherein the template-independent polymerase comprises one or more cysteine substitutions.
17. The conjugate of claim 1 , wherein the linker is specifically attached at a predetermined target site that is a cysteine residue of the template-independent polymerase; a lysine residue of the template-independent polymerase; an unnatural amino acid residue of the template-independent polymerase; a peptide tag of the template-independent polymerase; a labeling domain fused to the template-independent polymerase; or an aldehyde specifically generated within the template-independent polymerase.
18. The conjugate of claim 1 , wherein the linker is specifically attached at a predetermined target site that is a cysteine residue of the template-independent polymerase, wherein the linker is attached via a sulfhydryl-reactive chemistry.
19. The conjugate of claim 1 , wherein the template-independent polymerase is a terminal deoxynucleotidyl transferase (TdT), or a variant thereof.
20. The conjugate of claim 1 , further comprising a fluorescent label.
21. The conjugate of claim 1 , wherein the linker has a length of 4-100 Å.
22. The conjugate of claim 1 , wherein the linker comprises a chemically-cleavable linkage, an enzyme-cleavable linkage, or a photo-cleavable linkage.
23. The conjugate of claim 22 , wherein the chemically-cleavable linkage is selected from the group consisting of: a base-cleavable linkage, an acid-cleavable linkage, and a nucleophile-cleavable linkage.
24. The conjugate of claim 23 , wherein the base-cleavable linkage is selected from the group consisting of: an ester, a quaternary ammonium salt, and a urethane.
25. The conjugate of claim 23 , wherein the acid-cleavable linkage is selected from the group consisting of: a benzyl alcohol derivative, a teicoplanin aglycone, an acetal, a thioacetal, a thioether and a sulfonyl.
26. The conjugate of claim 23 , wherein the nucleophile-cleavable linkage is selected from the group consisting of: a phthalimide, an ester, and a Weinreb amide.
27. The conjugate of claim 22 , wherein the chemically-cleavable linkage is selected from the group consisting of: a disulfide, a phosphorothioate, a diol, and an azobenzene.
28. The conjugate of claim 22 , wherein the enzyme-cleavable linkage comprises an ester.
29. A method of nucleic acid synthesis, comprising:
providing a conjugate comprising a nucleotide, a linker, and a template-independent polymerase capable of catalyzing the covalent addition of the nucleotide onto the 3′ end of a nucleic acid, wherein the linker tethers the nucleotide to the template-independent polymerase; and contacting a sample comprising the nucleic acid with the conjugate.
30. A method of synthesizing a polynucleotide having a defined sequence, comprising steps:
(a) contacting a substrate comprising a nucleic acid with a conjugate comprising nucleotide, a linker, and a template-independent polymerase capable of catalyzing the covalent addition of the nucleotide onto the 3′ end of the nucleic acid, wherein the linker tethers the nucleotide to the template-independent polymerase;
(b) exposing the substrate to conditions sufficient to cleave the linker to separate the template-independent polymerase from the nucleotide; and
(c) repeating steps (a) and (b) one or more times to synthesize the polynucleotide having a defined sequence.
31. The conjugate of claim 3 , wherein cleavage of the linker does not produce a scar on the nucleobase.
32. The conjugate of claim 3 , wherein cleavage of the linker leaves a naturally occurring nucleobase.