Reusable initiators for synthesizing nucleic acids
The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.
1. A method for synthesizing oligonucleotides on a reusable solid support, the method comprising:
exposing a nucleic acid initiator attached to a solid support to nucleotide analogs in the presence of a polymerase to create a first oligonucleotide, wherein the nucleic acid initiator comprises a sequence-specific cleavage element;
contacting the nucleic acid initiator with a releasing agent to cleave the sequence-specific cleavage element and release the first oligonucleotide from the nucleic acid initiator;
dephosphorylating a 3′-end of the solid support-attached nucleic acid initiator; and
enzymatically renewing the sequence-specific cleavage element.
2. The method of claim 1 wherein the nucleic acid initiator comprises a 5′-amine, 5′-hydroxyl, 5′-phosphate, 5′-sulfhydryl, or 5′-benzaldehyde group attached to the solid support comprising formyl, chloromethyl, epoxide, amine, thiol, alkene, or terminal C-F bonds.
3. The method of claim 1 wherein the nucleic acid initiator is irreversibly attached to the solid support.
4. The method of claim 1 wherein the sequence-specific cleavage element is incorporated into the nucleic acid initiator after a 5′ end of the nucleic acid initiator has been irreversibly coupled to the solid support.
5. The method of claim 4 wherein the sequence-specific cleavage element is incorporated enzymatically.
6. The method of claim 1 wherein the sequence-specific cleavage element comprises a poly-U tract.
7. The method of claim 1 wherein the nucleic acid initiator comprises an index element, the method further comprising:
extending a 3′ end of the nucleic acid initiator with complementary nucleotide analogs to a 3′ sequence of the nucleic acid initiator to synthesize the first oligonucleotide under to permit hairpin formation;
extending the first oligonucleotide after hairpin formation using the index element as a template.
8. The method of claim 7 wherein the 3′ sequence of the nucleic acid initiator is complimentary to the sequence-specific cleavage element.
9. The method of claim 7 , further comprising incorporating a homopolymer tract into the first oligonucleotide to create a single stranded 3′ end thereof subsequent to the hairpin.
10. The method of claim 1 , further comprising:
before contacting the nucleic acid initiator with a releasing agent, incorporating a second sequence-specific cleavage element on a 3′ end of the first oligonucleotide to create a second nucleic acid initiator and exposing the second nucleic acid initiator, attached to the first oligonucleotide, to nucleotide analogs in the presence of a polymerase to create a second oligonucleotide,
wherein contacting the nucleic acid initiator and the second nucleic acid initiator with the releasing agent cleaves the sequence-specific cleavage element and the second sequence-specific cleavage element and release the first oligonucleotide and the second oligonucleotide from the nucleic acid initiator and the second nucleic acid initiator.
11. The method of claim 1 , wherein the polymerase is a nucleotidyl transferase or a modified nucleotidyl transferase.
12. The method of claim 1 , wherein the polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT.
13. The method of claim 1 , wherein the nucleotide analogs have an unmodified 3′-OH and a cleavable terminating group, and wherein the cleavable terminating group blocks subsequent nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage of the terminating group.