Methods to generate DNA mini-circles

1. A method for generating a circular nucleic acid, comprising:

providing a nucleic acid template, wherein the nucleic acid template comprises a single recombination site;

amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence, comprising multiple recombination sites; and

generating multiple circular nucleic acids by incubating the tandem repeat nucleic acid sequence with a recombination protein in a cell-free system, wherein the recombination protein is chosen from a Cre recombinase, a bacteriophage lambda integrase, or a bacterial XerCD recombinase, and wherein each of the generated circular nucleic acids comprises a single recombination site.

2. The method of claim 1 , wherein the recombination site is a site-specific recombination site.

3. The method of claim 1 , wherein the recombination site is a loxP site.

4. The method of claim 1 , wherein the recombination protein is a Cre recombinase from bacteriophage P1.

5. The method of claim 1 , further comprising incubating the generated circular nucleic acid with an exonuclease.

6. The method of claim 5 , wherein the exonuclease is chosen from an exonuclease I, an exonuclease III, or combinations thereof.

7. The method of claim 1 , further comprising incubating the generated circular nucleic acid with a single-stranded DNA-binding protein.

8. The method of claim 1 , further comprising transforming the generated circular nucleic acid to a prokaryotic cell.

9. The method of claim 1 , further comprising transfecting the generated circular nucleic acid to a eukaryotic cell.

10. A method for generating circular nucleic acids in a cell-free system, comprising:

providing a circular nucleic acid template, wherein the circular nucleic acid template is engineered to comprise a single recombination site;

amplifying the circular nucleic acid template by rolling circle amplification to form a concatamer comprising multiple recombination sites, wherein the concatamer comprises tandem repeat units of the circular nucleic acid template sequence; and

generating multiple circular nucleic acids by incubating the concatamer with a recombination protein, wherein the recombination protein is chosen from a Cre recombinase, a bacteriophage lambda integrase, or a bacterial XerCD recombinase, and wherein each of the generated circular nucleic acids comprises a single recombination site.

11. The method of claim 10 , wherein the rolling circle amplification comprises incubating the circular nucleic acid template with a primer and a Phi29 DNA polymerase in the presence of deoxyribonucleoside triphosphates.

12. The method of claim 10 , wherein the recombination site comprises SEQ ID NO: 1.

13. The method of claim 10 , wherein the recombination protein is a Cre recombinase from bacteriophage P1.

14. The method of claim 10 , further comprising processing the circular nucleic acids to generate a nucleic acid vaccine.

15. A method for generating a nucleic acid vaccine, comprising:

providing a nucleic acid template, wherein the nucleic acid template comprises a single recombination site;

amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence, comprising multiple recombination sites; and

generating the nucleic acid vaccine by incubating the tandem repeat nucleic acid sequence with a recombination protein, wherein the recombination protein is chosen from a Cre recombinase, a bacteriophage lambda integrase, or a bacterial XerCD recombinase, wherein each of the generated nucleic acid vaccine comprises a single recombination site.

16. The method of claim 15 , wherein the nucleic acid template further comprises an expression cassette.