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1. WO2020160122 - INITIATEURS RÉUTILISABLES POUR LA SYNTHÈSE D'ACIDES NUCLÉIQUES

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CLAIMS

1. A composition comprising a solid support and an oligonucleotide, wherein the 5' end of the oligonucleotide is coupled to the solid support, and wherein the oligonucleotide comprises a sequence-specific cleavage element.

2. The composition of claim 1 wherein the oligonucleotide comprises a 5'-amine, 5'-hydroxyl, 5'-phosphate, 5'-sulfhydryl, or 5'-benzaldehyde group coupled to the solid support comprising formyl, chloromethyl, epoxide, amine, thiol, alkene, or terminal C-F bonds.

3. The composition of claim 1 wherein the oligonucleotide is irreversibly coupled to the solid support.

4. The composition of claim 1 wherein the sequence-specific cleavage element is incorporated into the oligonucleotide after the 5' end has been irreversibly coupled to the solid support.

5. The composition of claim 4, wherein the sequence-specific cleavage element is incorporated enzymatically.

6. The composition of claim 1, wherein the sequence-specific cleavage element comprises a poly-U tract.

7. The composition of claim 1, wherein the support-coupled oligonucleotide comprises an index element to act as a template during oligonucleotide strand synthesis from a 3’ end of the support-coupled oligonucleotide.

8. 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.

9. The method of claim 8 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.

10. The method of claim 8 wherein the nucleic acid initiator is irreversibly attached to the solid support.

11. The method of claim 8 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.

12. The method of claim 11 wherein the sequence-specific cleavage element is incorporated enzymatically.

13. The method of claim 8 wherein the sequence-specific cleavage element comprises a poly-U tract.

14. The method of claim 8 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 appropriate conditions to permit hairpin formation;

extending the first oligonucleotide after hairpin formation using the index element as a template.

15. The method of claim 14 wherein the 3’ sequence of the nucleic acid initiator is complimentary to the sequence-specific cleavage element.

16. The method of claim 14, further comprising

incorporating spacer nucleotides into the first oligonucleotide sufficient to create a single stranded 3’ end thereof subsequent to the hairpin.

17. The method of claim 8, 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.

18. The method of claim 8, wherein the polymerase is a nucleotidyl transferase or a modified nucleotidyl transferase.

19. The method of claim 8, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT.

20. The method of claim 8, 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.