Claims
- 1. A nucleic acid molecule comprising an aptamer and a polynucleotide that encodes a transcriptional regulatory polypeptide, wherein binding of a ligand to the aptamer inhibits translation of the transcriptional regulatory polypeptide.
- 2. The nucleic acid of claim 1, wherein the ligand is a cell-permeable small organic molecule.
- 3. The nucleic acid of claim 2, wherein the ligand is Hoechst dye 33258.
- 4. The nucleic acid of claim 1, wherein the ligand is a metal ion.
- 5. The nucleic acid of claim 1, wherein the ligand is an antibiotic.
- 6. The nucleic acid of claim 1, wherein the ligand is a steroid.
- 7. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide is a repressor.
- 8. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide is a transcriptional activator.
- 9. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide is a coactivator.
- 10. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide comprises a DNA-binding domain.
- 11. The nucleic acid of claim 10, wherein the DNA-binding domain is that of a protein selected from the group consisting of E2F-1, GAL4, a STAT protein, a steroid/thyroid receptor protein, a Cys2-His2 zinc finger DNA binding motif, and a tetracycline repressor.
- 12. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide comprises a transcriptional repressor domain.
- 13. The nucleic acid of claim 12, wherein the transcriptional repressor domain is that of a protein selected from the group consisting of Rb protein, v-erbA, retinoic acid receptor alpha, thyroid hormone receptor alpha, yeast Ssn6/Tup1 protein complex, SIR1, NeP1, TSF3, SFI, WT1, Oct-2.1, E4BP4, KRAB and ZF5.
- 14. The nucleic acid of claim 12, wherein the transcriptional repressor domain is that of p53.
- 15. The nucleic acid of claim 1, wherein the transcriptional regulatory polypeptide comprises a transcriptional activation domain.
- 16. The nucleic acid of claim 1, wherein the nucleic acid is an MnRNA molecule.
- 17. The nucleic of claim 16, wherein the mRNA is bound to a ligand.
- 18. An expression cassette that comprises a promoter operably linked to a polynucleotide from which is transcribed the nucleic acid of claim 1.
- 19. An expression vector that comprises the expression cassette of claim 18.
- 20. The expression vector of claim 19, wherein the expression vector is a viral vector.
- 21. The expression vector of claim 20, wherein the viral vector is selected from the group consisting of an adenoviral vector, a retroviral vector, and an adeno-associated viral vector.
- 22. The expression vector of claim 19, wherein the expression vector is a nonviral vector.
- 23. The expression vector of claim 19, wherein the expression vector further comprises a second polynucleotide, wherein transcription of the second polynucleotide is regulated by the transcriptional regulatory polypeptide.
- 24. The expression vector of claim 23, wherein the second polynucleotide encodes a therapeutic polypeptide.
- 25. The expression vector of claim 23, wherein the second polynucleotide is operably linked to a binding site for the transcriptional regulatory polypeptide.
- 26. A cell that comprises the nucleic acid molecule of claim 1.
- 27. The cell of claim 26, wherein the cell further comprises a second polynucleotide, wherein transcription of the second polynucleotide is regulated by the transcriptional regulatory polypeptide.
- 28. The cell of claim 27, wherein the second polynucleotide is included in the nucleic acid.
- 29. The cell of claim 27, wherein transcription of the second polynucleotide yields an antisense nucleic acid.
- 30. The cell of claim 27, wherein the second polynucleotide encodes a polypeptide.
- 31. The cell of claim 30, wherein the polypeptide is a therapeutic polypeptide.
- 32. The cell of claim 31, wherein the therapeutic polypeptide is selected from the group consisting of a toxin, a cytokine, a kinase, a phosphatase, a transcriptional regulatory protein, an antibody, and a tumor suppressor.
- 33. The cell of claim 32, wherein the polypeptide is a tumor suppressor.
- 34. The cell of claim 33, wherein the tumor suppressor is p53.
- 35. The cell of claim 26, wherein the cell further comprises a ligand that binds to the aptamer.
- 36. A method of regulating expression of a gene, the method comprising:
contacting with an aptamer-binding ligand an RNA that comprises an aptamer and a polynucleotide that encodes a transcriptional regulatory polypeptide that regulates expression of the gene; wherein the ligand binds to the aptamer, thus inhibiting translation of the transcriptional regulatory polypeptide resulting in a change in the expression level of the gene.
- 37. The method of claim 36, wherein the change in the expression level of the gene is proportional to the amount of aptamer-binding ligand administered.
- 38. The method of claim 36, wherein the transcriptional regulatory polypeptide is a repressor, whereby binding of the ligand to the aptamer inhibits translation of the repressor thus causing an increase in the expression level of the gene.
- 39. The method of claim 36, wherein the transcriptional regulatory polypeptide is a transcriptional activator, whereby binding of the ligand to the aptamer inhibits translation of the transcriptional activator thus causing a decrease in the expression level of the gene.
- 40. The method of claim 36, wherein the gene comprises a binding site for the transcriptional regulatory polypeptide.
- 41. The method of claim 36, wherein the gene is included in a chromosome.
- 42. The method of claim 36, wherein the gene is extrachromosomal.
- 43. The method of claim 36, wherein the MRNA is contained in a cell.
- 44. The method of claim 43, wherein the cell is part of a multicellular organism.
- 45. The method of claim 44, wherein the contacting is accomplished by administering the ligand to the organism.
- 46. The method of claim 36, wherein the RNA is transcribed from an expression cassette that comprises a promoter operably linked to a polynucleotide from which is transcribed the RNA.
- 47. The method of claim 46, wherein the promoter is a constitutive promoter.
- 48. A method of retarding undesirable cell proliferation, the method comprising administering to undesirably proliferating cells:
a nucleic acid construct that comprises a promoter operably linked to a polynucleotide, wherein the polynucleotide is transcribed to yield an MRNA that comprises an aptamer and a polynucleotide sequence that encodes a transcriptional regulatory polypeptide that regulates expression of a gene involved in regulation of cell proliferation; an aptamer-binding ligand that binds to the aptamer; wherein the binding of the ligand to the aptamer inhibits translation of the transcriptional regulatory polypeptide, thus causing a change in the expression level of the gene, which change in expression level ameliorates the undesirable cell proliferation.
- 49. The method of claim 48, wherein the gene involved in regulation of cell proliferation is a tumor suppressor gene and the transcriptional regulatory polypeptide is a repressor, whereby binding of the ligand to the aptamer results in an increase in the tumor suppressor gene expression level.
- 50. The method of claim 48, wherein the gene involved in regulation of cell proliferation is a transgene.
- 51. The method of claim 50, wherein the gene comprises a promoter operably linked to a polynucleotide that encodes a polypeptide involved in regulation of cell proliferation.
- 52. The method of claim 51, wherein the promoter is a constitutive promoter.
- 53. The method of claim 50, wherein the gene is included on an expression vector that is administered to the undesirably proliferating cells.
- 54. The method of claim 53, wherein the expression vector further comprises the nucleic acid construct.
- 55. The method of claim 48, wherein the nucleic acid construct is included on an expression vector that is administered to the undesirably proliferating cells.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/242,106, filed Oct. 20, 2000, which application is incorporated herein by reference for all purposes.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60242106 |
Oct 2000 |
US |