Methods of synthesizing oligonucleotides using tethered nucleotides
Disclosed herein are methods and compositions comprising a polymerase and a phosphorylated nucleoside, wherein the polymerase and the nucleoside are covalently linked by a cleavable linker at the terminal phosphate group. Further disclosed herein are enzymatic polynucleotide synthesis using polymerase and nucleotide conjugation strategies.
1. A composition comprising a complex according to the following formula:
wherein the enzyme comprises TdT.
2. The composition of claim 1 , wherein the linker comprises at least one phosphate.
3. The composition of claim 1 , wherein the enzyme is attached to the linker via a cysteine.
4. The composition of claim 1 , wherein the linker is an acid labile linker, a base labile linker, a pH-sensitive linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, a photo-cleavable linker, orthonitrobenzyl-based linker, phenacyl linker, alkoxybenzoin linker, chromium arene complex linker, NpSSMpact linker, pivaloylglycol linker, irradiation-cleavable linker, and/or an enzymatically-cleavable linker.
5. The composition of claim 4 , wherein the linker is an enzymatically-cleavable linker that is capable of being cleaved using a peptidase or an esterase.
6. The composition of claim 1 , wherein the linker is selected from the group consisting of a silyl linker, an alkyl linker, a polyether linker, a polysulfonyl linker, a polysulfoxide linker, an amine-to-thiol cross linker, a thiomaleamic acid linker, and any combination thereof.
7. The composition of claim 1 , wherein the enzyme comprises a cysteine mutation.
8. The composition of claim 1 , wherein the template independent polymerase comprises at least one amino acid mutation to a surface-accessible amino acid residue.
9. The composition of claim 1 , wherein the base is selected from an adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or a modified base thereof.
10. The composition of claim 1 , further comprising a reversible terminator.
11. A method of synthesizing a polynucleotide, comprising:
(a) contacting a polynucleotide with a composition of claim 1 ;
(b) extending the polynucleotide by covalent addition of an extension nucleotide onto a 3′ hydroxyl of the polynucleotide by the enzyme; and
(c) cleaving the enzyme from the polynucleotide.
12. The method of claim 11 , further comprising repeating (a)-(c) to produce an extended polynucleotide.
13. The method of claim 11 , wherein the enzyme is cleaved using a peptidase or an esterase.
14. A composition comprising a complex according to the following formula:
A-L-B
wherein:
A comprises an enzyme including TdT;
B comprises a nucleotide; and
L comprises a chemical linker that covalently links the enzyme to a terminal phosphate group of the nucleotide, wherein the enzyme is configured to catalyze covalent addition of the nucleotide onto a 3′ hydroxyl of a polynucleotide, and subsequent extension of the polynucleotide.
15. The composition of claim 14 , wherein the linker comprises at least one phosphate.
16. The composition of claim 14 , wherein the linker includes an acid labile linker, a base labile linker, a pH-sensitive linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, an orthonitrobenzyl-based linker, a phenacyl linker, an alkoxybenzoin linker, a chromium arene complex linker, an NpSSMpact linker, a pivaloylglycol linker, a photo-cleavable linker, an irradiation-cleavable linker, a silyl linker, an alkyl linker, a polyether linker, a polysulfonyl linker, a polysulfoxide linker, an amine-to-thiol crosslinker, a thiomaleamic acid linker, and/or an enzymatically-cleavable linker.
17. The composition of claim 15 , wherein the linker is an enzymatically-cleavable linker that is capable of being cleaved using a peptidase or an esterase.
18. The composition of claim 13 , wherein the enzyme comprises at least one amino acid mutation to a surface-accessible amino acid residue.
19. The composition of claim 13 , wherein the complex further comprises a reversible terminator.
20. The composition of claim 13 , wherein the base is includes an adenine, a cytosine, a guanine, a thymine, a uracil, a modified adenine, a modified cytosine, a modified thymine, and/or a modified uracil.