Modified template-independent enzymes for polydeoxynucleotide synthesis
The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.
1. A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in the TGSR motif and capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.
2. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-138 bovine TdT and a protein tag sequence fused to the N-terminus.
3. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-151 bovine TdT and a protein tag sequence fused to the N-terminus.
4. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-160 bovine TdT and a protein tag sequence fused to the N-terminus.
5. The modified TdT of claim 1 , wherein the modified TdT exhibits an increased rate of incorporation of modified nucleotides, compared to native TdT.
6. The modified TdT of claim 1 , wherein the modified TdT is capable of adding adenine, cytosine, guanine, and thymine deoxyribonucleotides modified with a removable 3′-O-blocking moiety.
7. The modified TdT of claim 6 , wherein said nucleotides are 2′-deoxyribonucleotides.
8. The modified TdT of claim 1 , wherein the modified TdT is capable of adding adenine, cytosine, guanine, and uracil ribonucleotides modified with a removable 3′-O-blocking moiety.
9. The modified TdT of claim 1 , wherein said removable 3′-O-blocking moiety comprises a 3′-O-azidomethyl group.
10. The modified TdT of claim 1 , wherein said removable 3′-O-blocking moiety comprises a 3′-O-amino group.
11. The modified TdT of claim 1 , wherein said removable 3′-O-blocking moiety comprises a 3′-O-allyl group.
12. The modified TdT of claim 1 , wherein said removable 3′-O-blocking moiety is selected from the group consisting of O-phenoxyacetyl; O-methoxyacetyl; O-acetyl; O-(p-toluene)sulfonate; O-phosphate; O-nitrate; O-[4-methoxy]-tetrahydrothiopyranyl; O-tetrahydrothiopyranyl; O-[5-methyl]-tetrahydrofuranyl; O-[2-methyl,4-methoxy]-tetrahydropyranyl; O-[5-methyl]-tetrahydropyranyl; and O-tetrahydrothiofuranyl.
13. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating a 3′-O-blocked nucleotide 5′-triphosphate, and said removable blocking moiety comprises a group selected from esters, ethers, carbonitriles, phosphates, carbonates, carbamates, hydroxylamine, borates, nitrates, sugars, phosphoramide, phosphoramidates, phenylsulfenates, sulfates, sulfones and amino acids.
14. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a reaction temperature of about 30° C.
15. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a reaction temperature from 30° C. to 80° C.
16. The modified TdT of claim 1 , wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 1000 μM or less.
17. The modified TdT of claim 16 , wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 100 μM or less.
18. The modified TdT of claim 1 , wherein the modified TdT is expressed by an organism having a genome comprising a nucleic acid sequence being at least 90% identical to SEQ ID NOS. 2, 4, 6, or 8.
19. The modified TdT of claim 1 , wherein the TGSR motif comprises a mutation selected from G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H.