Claims
- 1. A method of regulating production of a product in a cell, comprising:
inserting into a gene that produces said product or regulates the production of said product in said cell a regulatable catalytically active nucleic acid (RCANA), comprising a catalytic domain, which modifies a transcript to alter its coding potential, and a regulatory domain which recognizes an effector that alters the function of the catalytic domain; contacting said regulatory domain with an effector thereby regulating production of said product.
- 2. The method of claim 1, wherein the concentration of the effector modulates the activity of the catalytic domain of said RCANA.
- 3. The method of claim 1, wherein the production of said product is fully inhibited.
- 4. The method of claim 1, wherein the production of said product is increased compared to a normal control level.
- 5. The method of claim 1, wherein the production of said product production is partially inhibited according to the concentration of the effector.
- 6. The method of claim 1, wherein the effector is the product.
- 7. The method of claim 1, wherein the effector is a feedback inhibitor of said gene.
- 8. The method of claim 1, wherein said product is produced in a metabolic pathway that is being regulated.
- 9. The method of clain 1, wherein said effector is an intermediate in a metabolic pathway.
- 10. The method of claim 1, wherein said RCANA blocks expression of said gene.
- 11. The method of claim 1, wherein said RCANA activates expression of said gene.
- 12. The method of claim 1, wherein said product is an intermediate of a metabolic pathway.
- 13. The method of claim 1, wherein said biological pathway is a metabolic pathway.
- 14. The method of claim 1, wherein the effector is endogenous to said cell.
- 15. The method of claim 1, wherein the effector is exogenous to said cell.
- 16. The method of claim 1, wherein the effector is selected from the group consisting of a protein, an enzyme, a protein pharmaceutical, a metabolite, a drug, a dye, a vitamin, a food additive, a chemical additive, a pesticide, an insecticide, a feed compound, and a waste product.
- 17. The method of claim 16 wherein the drug is selected from the group consisting of antibiotics, anticancer drugs, antifungals, cholesterol-lowering drugs, and immunosuppressants,
- 18. The method of claim 1, wherein the product is selected from the group consisting of a protein, an enzyme, a protein pharmaceutical, a metabolite, a drug, a dye, a vitamin, a food additive, a chemical additive, a pesticide, an insecticide, and a feed compound.
- 19. The method of claim 1, wherein said effector is an endproduct of a biosynthetic process.
- 20. A method of regulating a biological pathway in a cell, comprising:
inserting into a first gene that produces a first product or regulates the production of said first product in said biological pathway in a cell a first regulatable catalytically active nucleic acid (RCANA), comprising a catalytic domain, which catalyzes cleavage of the RCANA or excision of the RCANA from gene in which it is inserted followed by ligation ofthc gene at 5′ and 3′ ends of cleavage site, and a regulatory domain which recognizes an effector that activates a function of the catalytic domain; inserting into a second gene that produces a second product or regulates the production of said second product in said biological pathway in said cell a second regulatable catalytically active nucleic acid (RCANA), comprising a catalytic domain, which catalyzes cleavage of the RCANA or excision of the RCANA from gene in which it is inserted followed by ligation of the gene at 5′ and 3′ ends of cleavage site, and a regulatory domain which recognizes an effector that activates a function of the catalytic domain; contacting said first regulatory domain with a first effector thereby regulating production of said first product, and contacting said second regulatory domain with a second effector thereby regulating production of said second product.
- 21. The method of claim 20, wherein the combination of said first and second effectors control the flux of metabolites through the biological pathway.
- 22. The method of claim 20, wherein said biological pathway is a biosynthetic pathway.
- 23. The method of claim 20, wherein said biological pathway is a metabolic pathway.
- 24. The method of claim 20, wherein the biological pathway is fully inhibited.
- 25. The method of claim 20, wherein the biological pathway is partially inhibited according to the concentration of said first and second effectors.
- 26. The method of claim 20, wherein said first product is the second effector.
- 27. The method of claim 20, further comprising inserting into a third gene that produces a third product or regulates the production of said third product in said biological pathway in said cell a third regulatable catalytically active nucleic acid (RCANA), comprising a catalytic domain, which catalyzes cleavage of the RCANA, or excision of the RCANA from gene in which it is inserted followed by ligation of the gene at 5′ and 3′ ends of cleavage site, and a regulatory domain which recognizes an effector that activates a function of the catalytic domain.
- 28. The method of claim 20, wherein said first and second RCANAs block expression of said first and second gene.
- 29. The method of claim 20, wherein said first and second RCANAs activate expression of said first and second gene.
- 30. A method of screening a population of cells for a cell that produces a bioproduct, comprising:
inserting a regulatable catalytically active nucleic acid into a reporter gene in said population of cells, such that the regulatable catalytically active nucleic acid is regulated by said bioproduct; wherein expression of said reporter gene indicates the production of said bioproduct a cell.
- 31. The method of claim 30, further comprising isolating said cell that produces said bioproduct.
- 32. The method of claim 30, wherein said reporter gene is green fluorescent protein, thymidylate synthase, or beta lactamase.
- 33. A polynucleotide that is regulated by a peptide comprising:
a regulatable, catalytically active polynucleotide, wherein the peptide interacts with the polynucleotide to affect its catalytic activity
- 34. A nucleic acid that is regulated by an effector comprising:
a regulatable, catalytically active nucleic acid, generated by the modification of at least one catalytic residue.
- 35. A nucleic acid comprising:
a gene; a regulatable, catalytically active nucleic acid inserted within the gene; wherein the presence of an effector causes the nucleic acid to catalyze a reaction.
- 36. A nucleic acid segment comprising:
a regulatable, catalytically active nucleic acid, selected from a pool of nucleic acids in which at least one of the catalytic residues has been randomized.
- 37. A regulatable, catalytically active nucleic acid segment comprising:
an effector domain; and a nucleic, acid catalyst domain in which one or more critical catalytic residues of the nucleic acid catalyst have been randomized; wherein the kinetic parameters of the catalytic domain are regulated by an effector that interacts with the effector domain.
- 38. A method of isolating a regulatable, catalytically active nucleic acid, comprising the steps of-randomizing at least one nucleotide in the catalytic domain of a catalytically active nucleic acid to create a nucleic acid pool; removing from the nucleic acid pool those nucleic acids that interact with the catalytic target of the catalytic domain; adding an effector molecule to the nucleic acids; and isolating those nucleic acids that interact with the catalytic target of the catalytic domain.
- 39. A method of isolating a regulatable, catalytically active nucleic acid having a catalytic and an effector domain, comprising the steps of-randomiziig at least one nucleotide in the catalytic domain of the nucleic acid to create a nucleic acid pool; removing from the nucleic acid pool those randomized nucleic acids that interact with the catalytic target of the catalytic domain; adding an effector to the nucleic acids; and isolating the nucleic acids that interact with the catalytic target of the catalytic domain.
- 40. A method of detection of a target using a regulatable, catalytically active nucleic acid comprising the steps of:
contacting the a regulatable, catalytically active nucleic acid with the target; and measuring the effect of the interaction between the a regulatable, catalytically active nucleic acid and the target.
- 41. A method of modifying a target using a regulatable, catalytically active nucleic acid comprising the steps of-providing a regulatable, catalytically active nucleic acid capable of target specific modification; and modifying the target under conditions that cause a regulatable, catalytically active nucleic acid-specific activity.
- 42. A method of selecting a regulatable, catalytically active nucleic acid, comprising the steps of-contacting a pool of nucleic acids, the nucleic acids having a catalytic and an effector domain, wherein at least one nucleotide in the catalytic domain of the nucleic acids has been randomized; removing from the nucleic acid pool those nucleic acids that interact with the catalytic target of the catalytic domain; adding an effector to the remaining nucleic acids; and isolating those nucleic acids that interact with the catalytic target of the catalytic domain; introducing the nucleic acids into a host cell; and measuring the catalytic activity of the nucleic acid upon exposure of the host cell to the effector.
- 43. A method of detecting a regulatable, catalytically active nucleic acid, comprising the steps of:
isolating a regulatable, catalytically active nucleic acid; creating a construct in which the nucleic acid is in position to regulate the expression of a reporter gene; introducing the construct into a host cell; and measuring the catalytic activity of the nucleic acid upon exposure of the host cell to an effector.
- 44. A method of modulating expression of a nucleic acid, the method comprising providing a polynucleotide that is regulated by a peptide, the polynucleotide comprising a regulatable, catalytically active polynucleotide, wherein the peptide interacts with the polynucleotide to affect its catalytic activity; and contacting the polynucleotide with the peptide, thereby modulating expression of a nucleic acid.
- 45. The method of claim 44, wherein the polynucleotide is provided in a cell.
- 46. The method of claim 44, wherein the cell is provided in vitro.
- 47. The method of claim 44, wherein the cell is provided in vivo.
- 48. The method of claim 44, wherein the cell is a prokaryotic cell.
- 49. The method of claim 44, wherein the cell is a eukaryotic cell.
- 50. A method of modulating expression of a nucleic acid, the method comprising the steps of-providing a nucleic acid that is regulated by an effector, the nucleic, acid comprising: a regulatable, catalytically active nucleic acid, wherein the regulatable, catalytically active nucleic acid molecule includes at least one modified catalytic residue; and contacting the nucleic acid with the effector, thereby modulating expression of a nucleic acid.
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No. 60/324,715, filed Sep. 24, 2001; and is a continuation in part of U.S. Ser. No. 09/661,658, filed Sep. 14, 2000, which claims the benefit of U.S. Ser. No. 60/212,097, filed Jun. 15, 2000; and is a continuation in part of U.S. Ser. No. 09/666,870, filed Sep. 20, 2000, which claims the benefit of U.S. Ser. No. 60/212,097, filed Jun. 15, 2000; and is a continuation in part of U.S. Ser. No. 09/883,119, filed Jun. 14, 2001, which claims the benefit of U.S. Ser. No. 60/212,097, filed Jun. 15, 2000, each of which is incorporated herein by reference in its entirety.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60324715 |
Sep 2001 |
US |
|
60212097 |
Jun 2000 |
US |
|
60212097 |
Jun 2000 |
US |
Continuation in Parts (3)
|
Number |
Date |
Country |
Parent |
09661658 |
Sep 2000 |
US |
Child |
10254568 |
Sep 2002 |
US |
Parent |
09883119 |
Jun 2001 |
US |
Child |
10254568 |
Sep 2002 |
US |
Parent |
09666870 |
Sep 2000 |
US |
Child |
10254568 |
Sep 2002 |
US |