Circular DNA vectors for synthesis of RNA and DNA
The present invention provides methods for synthesis and therapeutic use of DNA and RNA oligonucleotides and analogs. RNA oligonucleotides are synthesized using a small, circular DNA template which lacks an RNA polymerase promoter sequence. The RNA synthesis is performed by combining a circular single-stranded oligonucleotide template with an effective RNA polymerase and at least two types of ribonucleotide triphosphate to form an RNA oligonucleotide multimer comprising multiple copies of the desired RNA oligonucleotide sequence. Preferably, the RNA oligonucleotide multimer is cleaved to produce RNA oligonucleotides having well-defined ends. Preferred RNA oligonucleotide multimers contain ribozymes capable of both cis (autolytic) and trans cleavage.
1. A method for synthesizing an RNA oligonucleotide inside a cell comprising introducing into a cell a single-stranded circular oligonucleotide template comprising at least one copy of a nucleotide sequence complementary to the sequence of the RNA oligonucleotide, such that the circular oligonucleotide is processed intracellularly to yield an RNA oligonucleotide multimer comprising multiple copies of the RNA oligonucleotide.
2. The method of claim 1 wherein the circular oligonucleotide has about 15-1500 nucleotides.
3. The method of claim 1 wherein the cell is a plant cell or an animal cell.
4. The method of claim 1 wherein the cell is a bacterial cell.
5. The method of claim 1 wherein the cell is a mammalian cell.
6. The method of claim 1 further comprising cleaving the RNA oligonucleotide multimer to yield multiple copies of the RNA oligonucleotide.
7. The method of claim 6 wherein the cleavage is autolytic.
8. The method of claim 6 wherein the RNA oligonucleotide is linear.
9. The method of claim 6 wherein the RNA oligonucleotide is circular.
10. The method of claim 6 wherein the RNA oligonucleotide is biologically active.
11. The method of claim 10 wherein the biologically active RNA oligonucleotide comprises a catalytic RNA, an antisense RNA, or a decoy RNA.
12. The method of claim 10 wherein the biologically active RNA oligonucleotide has endonuclease, exonuclease, polymerase, ligase, phosphorylase, dephosphorylase, or protease activity.
13. The method of claim 10 wherein the biologically active RNA oligonucleotide is capable of intramolecular ligation.
14. The method of claim 10 wherein the biologically active oligonucleotide comprises a ribozyme.
15. The method of claim 14 wherein the ribozyme is a hairpin, hammerhead-motif, or hepatitis delta catalytic ribozyme.
16. The method of claim 14 wherein the ribozyme is capable of trans cleavage.
17. The method of claim 14 wherein the ribozyme cleaves a target disease-associated RNA.
18. The method of claim 10 wherein the biologically active RNA oligonucleotide modifies the structure or the function of a target disease-associated DNA, RNA, or protein.
19. The method of claim 1 wherein a gene encoding an effective RNA polymerase operably linked to a promoter is co-introduced into the cell.
20. The method of claim 19 wherein the RNA polymerase is T7 or E. coli polymerase.
21. The method of claim 1 wherein the circular oligonucleotide template is introduced into the cell using direct injection, electroporation, heat shock, calcium phosphate treatment, lipid-mediated delivery, or cation-mediated delivery.
22. The method of claim 1 further comprising implanting the cell into a plant or animal after introducing the single-stranded circular oligonucleotide template into the cell.
23. The method of claim 1 performed in a cell explanted from a plant or animal.
24. The method of claim 23 further comprising implanting the cell into a plant or animal after introducing the single-stranded circular oligonucleotide template into the cell.
25. The method of claim 24 wherein the cell is reimplanted into the plant or animal from which it was explanted.
26. The method of claim 23 wherein the animal is a mammal.
27. The method of claim 1 performed in cell culture.
28. The method of claim 1 performed in situ in a living organism.
29. The method of claim 28 wherein the circular oligonucleotide is administered to the organism using direct injection, inhalation, intranasal administration, ocular administration, site-specific incubation or infusion.