Enzymatic synthesis of oligonucleotides
Provided are compositions and methods directed to using a short template and a polymerase or ligase to synthesize an oligonucleotide. The template is designed such that only one nucleotide is added to the primer in each round of synthesis.
1 . A method for synthesizing an oligonucleotide, comprising:
(a) providing a plurality of immobilized primers, wherein the 5′ terminus of each primer is attached to a solid support;
(b) combining the plurality of immobilized primers and
(i) a plurality of templates comprising between 2 and 100 nucleotides, wherein each template hybridizes to a primer generating a single overhang nucleotide at the 5′ terminus of the template;
(ii) a plurality of unincorporated nucleotides; and
(iii) a polymerase;
wherein the plurality of the immobilized primers are extended at the 3′ terminus by the polymerase-mediated incorporation of a single unincorporated nucleotide, wherein the single incorporated nucleotide is complementary to the single overhang nucleotide of the template hybridized to the primer; and then
(c) removing the hybridized templates and excess unincorporated nucleotides; and
(d) repeating steps (b) and (c) multiple times until the synthesis of the oligonucleotide is complete.
2 . The method of claim 1 , wherein the nucleotides in the plurality of nucleotides are 3′ protected nucleotides.
3 . The method of claim 2 , wherein the protective group of the 3′ protected nucleotides is selected from the group consisting of 3′-O-azidomethyl, 3′-O-allyl, 3′-O-methoxymethyl, 3′-O-nitrobenzyl, 3′-O-azidomethylene, and 3′-O-aminoalkoxyl.
4 . The method of claim 1 , wherein a solution comprising Ca 2+ is added prior to step (c) to prevent further elongation.
5 . The method of claim 4 , wherein the solution comprising Ca 2+ is removed in step (c) together with the plurality of templates and the excess nucleotides.
6 . The method of claim 5 , wherein a solution comprising Mg 2+ is added prior to step (d) to promote incorporation of the nucleotide to the 3′ terminus of the primer.
7 . The method of claim 1 , wherein the templates in the plurality of templates comprise between 5 and 50 nucleotides.
8 . The method of claim 1 , wherein the templates in the plurality of templates comprise between 5 and 20 nucleotides.
9 . The method of claim 1 , wherein the primers in the plurality of primers have the same sequence.
10 . The method of claim 9 , wherein the templates in the plurality of templates have the same sequence.
11 . The method of claim 1 , wherein at least two primers in the plurality of primers have different sequences.
12 . The method of claim 11 , wherein at least two templates in the plurality of templates have different sequences.
13 . A method for synthesizing an oligonucleotide, comprising:
(a) providing a plurality of immobilized primers, wherein the 5′ terminus of each primer is attached to a solid support;
(b) combining the plurality of immobilized primers and
(i) a plurality of hairpin templates comprising between 10 and 100 nucleotides, wherein each hairpin template comprises a phosphorothioate linkage between the two nucleotides at the 5′ terminus of each hairpin template, each hairpin template hybridizes to a primer, and the nucleotide at the 5′ terminus of the hairpin template is placed adjacent to the nucleotide at the 3′ terminus of the primer;
(ii) a ligase;
wherein the plurality of the immobilized primers are extended at the 3′ terminus by the ligase-mediated incorporation of the nucleotide at the 5′ terminus of the hairpin template; and then
(c) cleaving the phosphorothioate linkage;
(d) removing the plurality of hairpin templates;
(e) repeating steps (b) to (d) until the synthesis of the oligonucleotide is complete.
14 . The method of claim 13 , wherein cleaving the phosphorothioate linkage is performed by adding a solution of Ag + or Hg + .
15 . The method of claim 13 , wherein each hairpin template comprises at least one non-natural nucleotides.
16 . The method of claim 13 , wherein the primers in the plurality of primers have the same sequence.
17 . The method of claim 16 , wherein the hairpin templates in the plurality of hairpin templates have the same sequence.
18 . The method of claim 13 , wherein at least two primers in the plurality of primers have different sequences.
19 . The method of claim 18 , wherein at least two hairpin templates in the plurality of hairpin templates have different sequences.