Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Regulatable, catalytically active nucleic acids

 

Compositions and methods are provided to make, isolate, characterize and use regulatable, catalytically active nucleic acids (RCANA). The present invention is directed to RCANA that transduce molecular recognition into catalysis. Also, RCANAs according to the invention can be used as regulatory elements to control the expression of one or more genes in a metabolic pathway. RCANAs can also be used as regulated selectable markers to create a selective pressure favoring (or disfavoring) production of a targeted bioproduct.

What is claimed is:

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.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of catalytic nucleic acids and in particular to regulatable, catalytically active nucleic acids that modulate their kinetic parameters in response to the presence of an effector.

BACKGROUND OF THE INVENTION

[0003] Dyes, vitamins, food/chemical additives, enzymes, protein pharmaceuticals, pesticides, insecticides, and feed compounds for industrial chemical processes are but a few categories of compounds (bioproducts) derived from biological hosts. Many compounds in use or under development as therapeutics are synthesized entirely or partially in biological hosts, e.g., drugs such as antibiotics, anticancer drugs, antiftingals, cholesterol-lowering drugs, and immunosuppressants. In addition, a range of waste products (e.g., heavy metals) are actively accumulated within biological hosts in some bioremediation applications. Biological hosts used to synthesize/accumulate bioproducts include bacteria, eukaryotic microorganisms (e.g., Saccharomyces cerevisiae), plants, animal cells (e.g., transformed insect cells growing in culture), and animals.

[0004] The production of bioproducts can be high because many are generated at relatively low levels in host expression systems. To optimize expression of desired compounds that are synthesized or accumulated in biological host cells/organisms, careful control over the biosynthetic process is often required. Expression levels of a natural product can vary between related strains of a biological host.

[0005] Ribozymes are oligonucleotides of RNA that can act like enzymes and are sometimes called RNA enzymes. Generally, they have the ability to behave like an endoribonuclease, catalyzing the cleavage of RNA molecules. The location of the cleavage site is highly sequence specific, approaching the sequence specificity of DNA restriction endonuclcases. By varying conditions, ribozymes can also act as polymerases or dephosphoryl ases.

[0006] Ribozymes were first described in connection with Tetrahymena thermophilia. The Tetrahymena rRNA was shown to contain an intervening sequence (IVS) capable of excising itself out of a large ribosomal RNA precursor. The IVS is a catalytic RNA molecule that mediates self-splicing out of a precursor, whereupon it converts itself into a circular form. The Tetrahymena IVS is more commonly known now as the Group I Intron. A subclass of ribozymes are the catalytically active, regulatable nucleic acids (RCANAs). The catalytic activity of the RCANAs can be regulated by an effector. Effector-sensitive RCANAs have been described, wherein the activity of the RCANA is regulated by a ligand-binding moiety. Upon binding the ligand, the RCANA activity on a target RNA is changed. Such RCANAs have only been described for small molecule ligands such as organic or inorganic molecules. Effector-sensitive RCANAs can also be controlled by proteins, peptides, or other macro-molecules.

[0007] The invention makes it possible to rapidly optimize the production of useful bioproducts by identifying optimal conditions for production, by engineering the metabolic pathways of host cells/organisms used in biosynthesis/bioremediation, and by identifying genetic variants with improved biosynthetic/bioremediative properties.

SUMMARY OF THE INVENTION

[0008] The present invention includes RCANAs wherein the catalytic activity of the RCANA is regulated by an effector. The RCANA of the present invention are, therefore, regulatable in that their activity is under the control of a second portion of the RCANA. Just as allosteric protein enzymes undergo a change in their kinetic parameters or of their enzymatic activity in response to interactions with an effector, the catalytic abilities of the RCANA may similarly be modulated by the effector(s). Thus, the present invention is directed to RCANA that transduce molecular recognition into catalysis. Also, RCANAs according to the invention can be used as regulatory elements to control the expression of one or more genes in a metabolic pathway. RCANAs can also be used as regulated selectable markers to create a selective pressure favoring (or disfavoring) production of a targeted bioproduct.

[0009] As will become apparent below, RCANA are more robust than allosteric protein enzymes in several ways: (1) they can be selected in vitro, which facilitates the engineering of particular constructs; (2) the levels of catalytic modulation are much greater for RCANA than for protein enzymes; and (3) since RCANA are nucleic acids, they can potentially interact with the genetic machinery in ways that protein molecules may not.

[0010] It should be noted that the methods described herein may include any type of nucleic acid. For example, these methods are not limited to RNA-based RCANA, but also encompass DNA RCANA and RNA or DNA RCANA. Furthermore, the methods can be applied to any catalytic activity the ribozymes are capable of carrying out. For example, the methods are not limited to ligases or splicing reactions, but could also encompass other ribozyme classes. The methods are also not limited to protein or peptide ligands, but also include-other molecular species, such as ions, small molecules, organic molecules, metabolites, sugars and carbohydrates, lipids and nucleic acids. The methods may also be extended to effectors that are not molecules, such as heat or light or electromagnetic fields. Furthermore, the methods are not limited to ligand-induced conformational. changes, but could also take into account chimeric catalysts in which residues essential for chemical reactivity were provided by both the nucleic acid and the ligand, in concert.

[0011] The effector may be a peptide, a polypeptide, a polypeptide complex, or a modified polypeptide or peptide. The effector may even be, e.g., an enzyme or even light (such as visible light) or even a magnet. The effector may be activated by a second effector that acts on the first effector (also referred to herein as an effector-effector), which may be an inorganic or an organic molecule. The polypeptide, peptide or polypeptide complex can be either endogenous, i.e., derived from the same cell type as the polynucleotide, or exogenous, i.e., derived from a cell type different than the cell from which the polynucleotide is derived.

[0012] The polypeptide or peptide may be phosphorylated or dephosphorylated. Alternatively, the effector may include a pharmaceutical agent. In some embodiments, the nucleic acid catalyzes a reaction that causes the expression of a target gene to be upregulated. In other embodiments, the nucleic acid catalyzes a reaction that causes the expression of a target gene to be down-regulated. If desired, the nucleic acid may be used to detect at least one exogenous effector from a library of candidate exogenous effector molecules. In some embodiments, the nucleic acid and the effector form a nucleic acid-effector complex.

[0013] In some embodiments, the kinetic parameters of nucleic acid catalysis are altered in the presence of a supermolecular structure, e.g., a viral particle or a cell wall. The nucleic acid may further include a regulatory element that can recognize a target molecule of interest. The nucleic acid may in addition include a transducer element that transmits information from the regulatory element to the catalytically active region of the nucleic acid.

[0014] Modification of Catalytic Residiues of RCANA. In one embodiment of this invention, the RCANA is generated by the modification of at least one catalytic residue. One of the unique features of the present selection protocol relative to others that have previously been published is that the present invention randomizes not only a domain that is pendant on the catalytic core, but a portion of the catalytic core itself. Thus, the selection for ligand-dependence not only yields species that bind to ancillary regions of the RCANA, but that may help stabilize the catalytic core of the RCANA.

[0015] Also provided by the invention is a method of isolating a regulatable, catalytically active nucleic, acid created by randomizing at least one nucleotide in the catalytic domain of a catalytically active nucleic acid to create a nucleic acid pool. The nucleic acid pool whose nucleic, acids interact with the catalytic target of the catalytic domain are removed. The method further may also include the step of adding an effector to the remaining pool of nucleic acids. In some embodiments, the method may also include the step of adding 4 an effector to the remaining nucleic acids, wherein the effector acts on the nucleic acids to alter the catalytic activities of the nucleic acids. The method may include optionally the step of purifying the isolated nucleic acid, and, if desired the step of sequencing the isolated nucleic acid. In various embodiments, the step of removing the nucleic acids is under high stringency conditions, moderate stringency conditions, or low stringency conditions.

[0016] In vitro sensing (or detection) applications. The current invention also provides for the use of RCANA for detection of a wide variety molecular species in vitro. For example, RCANA may be anchored to a chip, such as wells in a multi-well plate. Mixtures of analytes and fluorescently tagged substrates are added to each well. Where cognate effectors are present, the RCANA will covalently attach the fluorescent tags to themselves. Where RCANA have not been activated by effectors, the tagged substrates are washed out of the well. After reaction and washing, the presence and amounts of co-immobilized fluorescent tags are indicative of amounts of ligands that were present during the reaction. The reporter may be a fluorescent tag, but it may also be an enzyme, a magnetic particle, or any number of detectible particles. Additionally, the RCANA may be immobilized on beads, but they could also be directly attached to a solid support via covalent bonds.

[0017] One advantage of this embodiment is that covalent immobilization of reporters (as opposed to non covalent immobilization, as in EL1SA assays) allows stringent wash steps to be employed. Additionally, ribozyme ligases have the unique property of being able to transduce effectors into templates that may be amplified, affording an additional boost in signal prior to detection.

[0018] Modified nucleotides may be introduced into the RCANA that substantially stabilize them from degradation in environments such as sera or urine. The analytical methods of the present invention do not rely on binding per se, but only on transient interactions. The present invention requires mere recognition rather that actual binding, providing a potential advantage of RCANA over antibodies. That is, even low affinities are sufficient for activation and subsequent detection, especially if individual immobilized RCANA are examined (i.e., by ligand-dependent immobilization of a quantum dot).

[0019] Expression of RCANA in cells. The RCANAs of the present invention may also be expressed inside cells. The RCANAs of the present invention that are expressed inside a cell are not only responsive to a given effector, but are also able to participate in genetic regulation or responsiveness. In particular, self-splicing introns can splice themselves out of genes in response to exogenous or endogenous effector molecules.

[0020] The present invention includes RCANA constructs that may be inserted into a gene of interest, e.g, a gene targeting expression vector. The RCANA sequence provides gene specific recognition as well as modulation of the RCANA's kinetic parameters. The kinetic parameters of the RCANA vary in response to an effector. Specifically, in the case of RCANA that performs self splicing in the presence of the effector, the RCANA may splices itself out of the gene in response to the effector to regulate expression of the gene. RCANAs according to the invention can be used as regulatory elements to control the expression of one or more genes in a metabolic pathway. RCANAs can also be used as regulated selectable markers to create a selective pressure favoring (or disfavoring) production of a targeted bioproduct.

[0021] In another aspect, the invention includes a method of modulating expression of a nucleic acid by providing a polynucleotide that is regulated by a peptide. The polynucleotide may be a regulatable, catalytically active polynucleotide, in which the peptide interacts with the polynucleotide to affect its catalytic activity. The polynucleotide is contacted with the peptide, thereby modulating expression of a nucleic acid. The polynucleotide may be provided in a cell, and the cell may be, e.g, provided in vitro or in vivo and may be a prokaryotic cell or a even a eukaryotic cell.

[0022] The present invention also includes an RCANA construct with a regulatable oligonucleotide sequence having a regulatory domain, such that the kinetic parameters of the RCANA on a target gene vary in response to the interaction of an effector with the regulatory domain.

[0023] In vivo sensing (or detection) applications. It is possible to activate or repress a reporter gene (e.g., luciferase) containing an engineered intron in response to an endogenous activator. In this way, luciferase-engineered intron constructs may be used to monitor intracellular levels of proteins or small molecules such as cyclic AMP. This method may be used for in vivo measurements in both cellular systems, such as cell culture, and in whole organisms, such as animal models. Such applications may be used for high-throughput screening. If a particular intracellular signal (e.g, the production of a tumor repressor) was desired, compound libraries for pharmacophores that induce the signal (the tumor repressor) are screened for activation of the reporter gene. Thus, the information desired is changed or morphed into the detection of glowing cells.

[0024] Gene therapy applications. Similarly, a gene can be activated or repressed in response to an exogenously introduced effector (drug) for gene therapy. The RCANA may be used for gene expression up regulation (increasing production of the gene product) or down regulation (decreasing the production of the gene product). The construct of one embodiment of the present invention provides a DNA oligonucleotide coding for a catalytic domain and effector binding domain. The advantages of the nucleic acid-based technology of the present invention include, e.g., the ability to continually modulate gene expression with a high degree of sensitivity without additional gene therapy interventions.

[0025] In another aspect, the invention includes a method of modulating expression of a nucleic acid in a cell by providing a polynucleotide that is regulated by an effector, e.g., a peptide. The polynucleotide may be a regulatable, catalytically active polynucleotide, in which the peptide interacts with the polynucleotide to affect its catalytic activity. The polynucleotide is contacted with the peptide, thereby modulating expression of a nucleic acid. The polynucleotide may be provided in a cell, and the cell may be, e.g., provided in vitro or in vivo and may be a prokaryotic cell or a even a eukaryotic cell.

[0026] Biosynthelic application of RCANAs. This invention describes methods by which effector-sensitive RCANAs can be used to facilitate industrial biosynthesis and bioremediation. For example, provided are methods in which effector-dependent ribozymes can be used to (1) control production of a natural product in a biological host, (2) to identify environmental conditions which increase biosynthetic yields, and (3) to isolate strains of a biological host with improved product yields and/or properties.

[0027] As sensors, effector-sensitive RCANAs can be used to accurately monitor the concentration of a natural product as it is produced, either directly in vivo or ex vivo (e.g. following lysis). A wide range of environmental conditions and variant strains can be tested for synthesis of a natural product and the riboreporter used to define the best conditions/variants to use for production. Sensor applications include both in vivo and in vitro applications.

[0028] As regulatory elements, effector-sensitive RCANAs can be used to control the expression of one or more genes involved in a metabolic pathway. By coordinating gene expression, it is possible to optimize production of a targeted metabolite, staging production of the intermediates required to generate it or timing induction of bioproduct synthesis with respect to host growth.

[0029] As regulated selectable markers, effector-sensitive RCANAs can be used to create a selective pressure favoring (or disfavoring) production of targeted bioproduct. Host strains carrying the RCANA-selectable marker construct can be subjected to mutagenesis or transformed with a vector library in ways that change the concentration of bioproduct generated by the host. When subjected to appropriate selection conditions, variants with the highest (or lowest) internal concentrations of the targeted bioproduct are favored for survival.

[0030] The present invention includes, methods of regulating (e.g., increase or decrease) production of a product in a cell. Production is regulated by inserting into a gene that produces the product or alternatively regulates the production of the product a RCANA. An RCANA includes a catalytic domain and a regulatory (or effector) domain. The regulatory domain is contacted with an effector such as to alter (i.e., activate) a function of the catalytic domain.

[0031] Also provided by the invention are methods of regulating a biological pathway in a cell, by inserting into two or more genes that produce a product or regulates the production of the product in the biological pathway in a cell a RCANA. The regulatory domain of the RCANA is contacted with an effector such as to alter (i.e., activate) a function of the catalytic domain. For example, a biological pathway is regulated in a cell, by inserting into a first gene that produces a first product or regulates the production of the first product in the biological pathway in a cell a first RCANA. Following insertion of the first RCANA, an RCANA is inserted into a second gene that produces a second product or regulates the production of the second product in the biological pathway in the cell. The first effector activating the first RCANA is then contacted with the first regulatory domain thereby regulating production of the first product and the second regulatory domain is contacted with the second effector thereby regulating production of the second product. The first product can be the second effector.

[0032] The first and second RCANAs can block expression of the first and second gene. Alternatively, the first and second RCANAs can activate expression of the first and second gene. Accordingly, the combination of the first and second effectors can control the flux of metabolites through a biological pathway.

[0033] In various embodiments, the above method can be further elaborated by inserting an RCANA into a third gene that produces a third product or regulates the production of a third product. RCANAs can be inserted into additional genes (e.g., four, five six or more genes) to further regulate the biological pathway.

[0034] The biological pathway can be for example a biosynthetic pathway. The biological pathway can a metabolic pathway. The biological pathway can be either fully inhibited or partially inhibited according to the concentration of the first and second effectors.

[0035] By catalytic domain is meant that the domain modifies a transcript to alter its coding potential, i.e., the ability to be transcribed or translated to yield the encoded polypeptide. Modification includes splicing, e.g., self splicing and rescission and ligation, and endonucleoltyic cleavage. Modification can be non-covalent, e.g., structural or conformational alteration or a covalent modification.

[0036] By regulatory domain is meant the region of effector binding on an RCANA.

[0037] Function of the catalytic domain include, endonucleolytic and ligase activity. For example the catalytic domain catalyzes cleavage of the RCANA. Alternatively the catalytic domain catalyzes the excision of the RCANA from the gene in which it is inserted followed by ligation of the gene at the 5' and 3' ends of the cleavage site.

[0038] The product is the endproduct of a biosynthetic/metabolic process. For example, the product is 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, or a feed compound.

[0039] The effector is a substance other than the target product or it can be the target product. The effector is 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, or a waste product. Where the effector is a drug, the effector can be an antibiotic, anticancer drug, antifungal, cholesterol-lowering drug, or immunosuppressant.

[0040] For example, where the effector is the product, it can also act as a feedback inhibitor of the gene in which the RCANA is inserted or be an intermediate in a biosynthetic/metabolic pathway. The effector is either exogenous or endogenous to the cell. Contacting the regulatory domain with the effector(s) may either increase or decrease production of the targeted product compared to the level of target product observed in the absence of effector(s) (i.e., normal control level). The effector(s) may mediate RCANA activity in a concentration-dependent manner.

[0041] The RCANA blocks or activates the expression of the gene. By blocks expression is meant that the RCANA interrupts the transcription of the gene or the translation of the protein. By activates expression is meant that the effector binding of the RCANA leads to enhanced transcription of the gene or the translation of the protein.

[0042] The effector may act via a feedback mechanism to regulate the activity of the target gene. That is, an effector-activated RCANA can act in a negative feedback loop to inhibit the target gene. Alternatively, an effector-activated RCANA can act in a positive feeback loop to increase expression of the target gene.

[0043] The cell is for example a prokaryotic eukaryotic, bacterial, or insect cell. In yet another aspect, the present invention includes, methods of screening a population of cells for a cell that produces a bioproduct. This screening method includes inserting an RCANA into a reporter gene, e.g., green fluorescent protein, thymidylate synthase, or beta lactamase in a population of cells, such that the RCANA is regulated by the bioproduct. The product of the reporter gene provides a growth advantage to host cells expressing the bioproduct. Cells producing the bioproduct can be isolated by measuring the expression of the reporter gene which indicates the production of the bioproduct in the cell. Cell are isolated by methods know in the art such a fluorescent activated cell sorting.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0045] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures in which corresponding numerals in different figures refer to corresponding parts and in which:

[0047] FIG. 1 is a depiction of the secondary structure of the Group I theophylline-dependent (td) intron of bacteriophage T4 (wild type).

[0048] FIG. 2a is a photograph of a gel showing activation of the GpITh1P6.131 aptamer construct, together with a graphical representation of the gel, of one embodiment of the present invention; FIG. 2b is a photograph of a gel showing activation of GpITh2P6.133 aptamer construct, together with a graphical representation of the gel of one embodiment of the present invention.

[0049] FIG. 3 is a schematic depiction of an in vivo assay system for group I introns of one embodiment of the present invention.

[0050] FIG. 4a is a depiction of a portion of the P6 region of the Group I ribozyme joined to the anti-theophylline aptamer by a short randomized region to generate a pool of aptazymes of the present invention.

[0051] FIG. 4b is a schematic depiction of a selection protocol for the Group I P6 Aptazyme Pool of FIG. 4a.

[0052] FIG. 5 is a diagram of one embodiment of the present invention depicting exogenous or endogenous activation of Group I intron splicing;

[0053] FIG. 6 is a diagram of another embodiment of the present invention depicting a strategy for screening libraries of exogenous activators;

[0054] FIG. 7 is a diagram of an alternative embodiment of the present invention for screening libraries of exogenous activators;

[0055] FIG. 8 is a diagram of yet another alternative embodiment of the present invention for screening libraries of exogenous activators;

[0056] FIG. 9 is a diagram of an embodiment of the present invention for screening for endogenous activators;

[0057] FIG. 10 is a diagram of an alternative to the embodiment of FIG. 9 of the present invention to screen for endogenous activators;

[0058] FIG. 11 is a diagram of another embodiment of the present invention to screen for compounds that perturb cellular metabolism;

[0059] FIG. 12 is a diagram of a further embodiment of the present invention that provides a non-invasive readout of metabolic states;

[0060] FIG. 13 is a diagram of yet a further embodiment of the present invention wherein endogenous suppressors provide a non-invasive readout of multiple metabolic states;

[0061] FIG. 14 is a schematic depiction of an example of a work surface for automatic selection procedures of one embodiment of the invention;

[0062] FIG. 15a is an illustration of the L1 ligase aptazyme construct of one embodiment of the present invention; FIG. 15b is an illustration of a modified L1 ligase aptazyme construct of FIG. 15a of the present invention;

[0063] FIG. 15c is a schematic diagram of a selection protocol of one embodiment of the present invention;

[0064] FIG. 16(a-c) is a schematic diagram of a method to anchor an aptazyme construct of the present invention to a solid support in one embodiment of the present invention;

[0065] FIGS. 17(a-c) is a schematic showing the L1 ligase was the starting point for pool design;

[0066] FIG. 18(a-d) are charts and graphs showing the progression of the L1-N50 selections;

[0067] FIG. 19(a & b) is a schematic showing protein-dependent regulatable, catalytically active nucleic acid sequences and structures;

[0068] FIG. 20 is a graph demonstrating the ribozyme activity with inactivated protein samples;

[0069] FIG. 21 is a graph demonstrating an aptamer competition assays;

[0070] FIG. 22 is a flow chart of a method for negative and positive selection of RCANA;

[0071] FIG. 23 is a flow chart of a method for negative and positive selection of RCANA;

[0072] FIG. 24 is a graph showing the progress of the L1-N50 Rev selection;

[0073] FIG. 25(a,b,c) schematic showing the theophylline-dependent td group I intron constructs of the present invention;

[0074] FIG. 26 is a schematic showing the design of an FMN-dependent td nucleic acid intron splicing construct;

[0075] FIGS. 27(a-c) is a graph showing the relative growth curves of theophylline-dependent in vivo growth;

[0076] FIG. 28 is a graph showing 3-Methylxanthine dependent in vivo growth;

[0077] FIG. 29(a & b) is a schematic of a ribozyme ligase array;

[0078] FIG. 30 is an image showing the results of a rcgulatable, catalytically active ligase array;

[0079] FIG. 31 is a graph showing the titrations of individual allosteric ribozyme ligases;

[0080] FIG. 32 is a schematic showing RCANA-mediated control of gene expression in a biochemical metabolic pathway using a single exogenous effector;

[0081] FIG. 33 is a schematic showing RCANA-mediated control of gene expression in a biochemical metabolic pathway using multiple exogenous effectors;

[0082] FIG. 34 schematic showing shows the usc of metabolite-sensitive RCANAs to maintain constant levels of end-product in a biochemical metabolic pathway;

[0083] FIG. 35 is a schematic showing the use of RCANA-mediated control of gene expression;

[0084] FIG. 36 is a schematic showing RCANA-based cell selection;

[0085] FIG. 37 is a schematic showing RCANA-based cell selection;

[0086] FIG. 38 is a schematic showing RCANA- based in vivo sensors;

[0087] FIG. 39 is a schematic showing synthesis of an RCANA-based in vivo sensor.

DETAILED DESCRIPTION OF THE INVENTION

[0088] While the making and using of various embodiments of the present invention are discussed in detail below, the present invention provides many applicable inventive concepts that may be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0089] The present invention includes compositions or matter, methods and automation that permit the creation, isolation, identification, characterization and optimization of regulatable catalytically active nucleic acids. Furthermore, it includes methods to use RCANA for in vitro sensing (or detection), in vivo sensing (or detection), and gene therapy. Regulatable, catalytically active nucleic, acids selected by the method of the present invention also have advantages over other biopolymers that might be used for sensing or gene regulation. Regulatable, catalytically active nucleic acids are more robust than allosteric protein enzymes in several ways: (1) they can be selected in vitro (facilitating the engineering of particular constructs); (2) the levels of catalytic modulation arc much greater than those typically observed with protein enzymes; and (3) since regulatable, catalytically active micleic acids are nucleic acids, they can potentially interact with the genetic machinery in ways that protein molecules may not.

[0090] The method is not limited to RNA pools, but may also encompass DNA pools or modified RNA pools. Modified nucleotides may be introduced into the regulatable, catalytically active nucleic acids that substantially stabilize them from degradation in environments such as sera or urine. The method is not limited to ligascs, but could also encompass other ribozyme classes. The method is not limited to protein-induced conformational changes, but could also take into account chimeric catalysts in which residues essential for chemical reactivity were provided by both the nucleic acid and the protein in concert. Initially, many protein targets may prove refractive to selection. Many derivatives of the base method can be developed, however, to deal with novel targets or target classes.

[0091] A. Protein Dependent RCANA

[0092] Effector-dependent ribozymes have been shown to be responsive to small organic compounds, such as ATP and theophylline. The present inventors recognized the need for effector-dependent ribozymes, or as used herein, "regulatable, catalytically active nucleic acids" that are responsive to larger molecules, such as, e.g., peptides or proteins. The peptides, proteins or other large molecules may be provided from endogenous sources (e.g., expressed within a cell or cell extract), or exogenous sources (added or expressed in a cell or cell extract).

[0093] In order to understand the present invention, it is important to understand that previous attempts to make catalytically active nucleic acids that interact and respond to a large effector, by the inventors and others, have failed. Initially, attempts were made to generate protein-dependent ribozymes by the addition of aptamers (known binding sequences) to ribozymes (catalytically active domains). This design and method was unsuccessful in providing regulatable nucleic acids. Next, attempts were made to generate protein-dependent ribozymes by adding random sequence regions between an aptamer (binding) and a ribozyme (catalytic) and selecting for effector-dependence. These attempts were also unsuccessful. Next, the inventors attempted to generate protein-dependent ribozymes by adding a large random sequence region to the catalytic cores of ribozymes and selecting for effector-dependence. These attempts were also unsuccessful. In other words, all previously detailed methods for the generation of ribozymes that were dependent on small organic compounds were unsuccessful for generating ribozymes that were dependent on proteins.

[0094] To date, the present inventors have selected a number of protein-and peptide dependent ribozyme ligases. One example is the isolation of a protein-dependent, regulatable, catalytically active nucleic acid with an activity that was increased in a standard assay by 75,000-fold in the presence of its cognate protein effector, tyrosyl tRNA synthetase from Neurospora mitochondria (Cyt18). The Cyt18-dependent ribozyme was not activated by non-cognate proteins, including other tRNA synthetases.

[0095] A protein-dependent regulatable, catalytically active nucleic acid was also created and selected with an activity that was increased by 3,500-fold in the presence of its cognate protein effector, hen egg white lysozyme. The lysozyme-dependent ribozyme was not activated by most non-cognate proteins, including T4 lysozyme, but was activated by a very closely related protein, turkey egg white lysozyme. Moreover, the protein-dependent ribozyme was inhibited by a RNA binding species that specifically bound to lysozyme. In other words, the activation of these protein-dependent ribozymes was highly specific.

[0096] A peptide-dependent, regulatable, catalytically active nucleic acids was also created and isolated with activity was increased by 18,000-fold in the presence of its cognate peptide effector, the arginine-rich motif (ARM) from the HIV-I Rev protein. The Rev-dependent nucleic acid was not activated by other ARMs from other viral proteins, such as UTLV-I Rex. Using the present invention, regulatable, catalytically active nucleic acids may be developed that are regulated by any of a vast number of effectors.

[0097] As will be clear from the continued description, protein dependent RCANAs are useful in a variety of applications. For example, protein-regulated catalytically active nucleic acids can be used (1) for the acquisition of data about whole proteomes, (2) as in vitro diagnostic reagents to detect proteins specific to disease states, such as prostate specific antigen or viral proteins, (3) as sentinels for the detection of biological warfare agents, or (4) as regulatory elements in gene therapies.

B. Modification of Residues in Catalytic Domain

[0098] In one embodiment, the present invention randomizes a portion of the catalytic core itself, not necessarily a domain that is pendant on the catalytic core. One example for selection using the present invention was using the L1 ligase. The catalytic core of the L1 ligase has been mapped by deletion analysis and by partial randomization and re-selection. FIG. 15a depicts the L1 ligase that was the starting point for pool design. Stems A, B, and C are indicated. The shaded region contains the catalytic core and ligation junction. Primer binding sites are shown in lower case, an oligonucleotide effector required for activity is shown in italics, and the ligation substrate is bolded. The `tag` on the ligation substrate can be varied, but was biotin in the exemplary selection described herein. The L1 pool contains random sequence positions and overlaps with a portion of the ribozyme core. In FIG. 15b, Stem B was reduced in size and terminated with a stable GNRA tetraloop, but stem A was unchanged.

[0099] A pool was synthesized in which the random sequence region spanned the catalytic core. Protein-dependent ribozymes were selected from this random sequence pool by selecting for the ability to ligate an oligonucleotide tag in the presence of a protein effector followed by capturing the oligonucleotide tag on an affinity matrix, followed by amplification in vitro or in vivo. Because the catalytic core has been randomized, the selection for protein-dependence not only yields species that may bind to ancillary regions of the ribozyme, but species in which the protein effector actually helps to organize the catalytic core of the ribozyme.

[0100] Selection for protein-dependence from a pool in which at least a portion of the catalytic core of the ribozyme is randomized differs from selection for protein dependence from a pool in which the catalytic core is not randomized. For example, the catalytic core of the protein-dependent ribozymes that was selected differed substantially from the catalytic core of the original ribozyme and the catalytic core of other, nonprotein-dependent ribozymes selected based on the original ribozyme.

[0101] FIG. 15a depicts the L1 ligase that was the starting point for pool design in the Cyt18 RCANA selection, as an example of a protein-activated regulated, catalytically active nucleic acid. Stems A, B, and C are indicated. The shaded region contains the catalytic core and ligation junction. Primer binding sites are shown in lower case, an oligonucleotide effector required for activity is shown in italics, and the ligation substrate 14 is bold font. The `tag` on the ligation substrate can be varied, but was biotin in the exemplary selection described herein. The LI pool contains 50 random sequence positions and overlaps with a portion of the ribozyme core. In FIG. 15b, Stem B was reduced in size and terminated with a stable GNRA tetraloop, but stem A was unchanged.

[0102] Because one or more residues in the catalytic core have been randomized, the effectors may add essential catalytic residues for a given reaction. That is, both the effector molecule and the regulatable, catalytically active nucleic acids contribute a portion of the active site of the ribozyme. For example, using the method of the present invention a ribozyme and an effector molecule that would only carry-out poorly an enzymatic function independently may perform that enzymatic function upon interaction with one another. As such, a regulatable, catalytically active nucleic acid that contributes a guanosine and an adenosine and a protein effector that contributes a histidine together form a complex that has greater activity than either of the individual compounds. Using the methods disclosed herein it is possible to identify a chimeric effector:ribozyme (e.g., a protein:RNA complex) active site that would lead to catalysis. The invention describes ribozymes that have a detectable, basal chemical reactivity, and that the presence of the effector modulates this basal chemical reactivity. It is for this reason that the present invention differs significantly from other inventions which have claimed protein:RNA complexes in which no basal catalytic activity exists in the ribozyme or protein alone.

C. Selection of RCANA

[0103] FIG. 15c schematically shows the following selection scheme: the RNA pool was incubated with a biotinylated tag and reactive variants were removed from the population. The remaining species were again incubated with a biotinylated tag in the presence of the target (for example the protein Cyt18). Reactive variants were removed from the population and preferentially amplified by reverse transcription, PCR, and in vitro transcription.

[0104] Ligand-dependent, regulatable, catalytically active nucleic acids selected by this method differ from functional nucleic acids selected from random sequence pools. Selection for ligand-dependence requires a selection for catalytic activity as opposed to a selection for binding. Therefore, protein-dependent, regulatable, catalytically active nucleic acids are not aptamers. The composition of matter of a selected protein dependent ribozyme will be different than the composition of matter of a selected aptamer. For example, the sequence of the lysozyme-dependent ribozyme is different from the sequence of anti-lysozyme aptamers. An important feature of the present invention is that the regulatable, catalytically active nucleic acids disclosed herein only required recognition rather than selected or enhanced binding ability. For example, the affinity of lysozyme for the naive, unselected RNA pool is identical to the affinity of lysozyme for the selected, regulatable, catalytically active nucleic acid. The only difference is that the way in which lysozyme is recognized by the regulatable catalytically active nucleic acids leads to activation, while for the pool as a whole non-specific binding does not lead to activation. In other words, binding is a concomitant but secondary function of selection for regulatability; that is, the regulatable ribozymes disclosed herein may bind the effector or target very poorly, but upon interaction the activity of the ribozyme may nonetheless be modulated.

D. Automated Selection of RCANA

[0105] Robotic workstations have become essential to high-throughput manipulations of biomolecules, such as in high-throughput screening for drugs with a particular mechanism of action. The invention also includes the automation of in vitro selection procedures, and a mechanized system that executes both common and modified in vitro selection procedures. Automation facilitates the execution of this procedure, accomplishing in h to days what once necessitated weeks to month. In particular, the present inventors have adapted a robotic workstation to the selection of aptamers and ribozymes. However, the automation methods are generalizable to a number of different types of selections, including selections with DNA or modified RNA, selections for ribozymes, and selections for phage-displayed or cell-surface proteins.

[0106] In short, in vitfro selection involves several components: generation of a random sequence pool, sieving the random sequence pool for nucleic acid species that bind a given target or catalyze a given reaction, amplification of the sieved species by a combination of reverse transcription, the polymerase chain reaction, and in vitro transcription. Beyond the generation of the random sequence pool, each of these steps can potentially be carried out by a robotic workstation. The pool can be pipetted together 16 with a target molecule. If the target is immobilized on a magnetic bead, then the nucleic acid:target complex can be sieved from solution using an integrated magnetic bead collector. Finally, selected nucleic acid species can be eluted from the complex and amplified via a series of enzymatic steps that include the polymerase chain reaction carried out via an integrated thermal cycler.

[0107] There are many potential ways in which binding species can be sieved fiom a random sequence population. However, not all of these methods are amenable to automated selection. For example, to select aptamers, others have suggested that targets can be immobilized onto microtitre plates and binding species can be sieved by panning. The present inventors have had little success with this method, likely because panning is a relatively inefficient, low stringency method for sieving. Instead, the present inventors have discovered that when targets are immobilized on beads and mixed with a random sequence pool, binding species can be efficiently sieved from non-binding species by filtration of the beads. Beads can be readily manipulated by pipetting, allowing for the facile recovery and elution of the binding species, which are then amplified and carried into subsequent rounds of selection. This method differs from the magnetic bead capture method, and can be carried out with much higher stringency. This method is novel, and has not previously been used for in vitro selection experiments.

[0108] FIG. 14 depicts schematically an exemplary work surface for yet another embodiment of the present invention: automated selection. See, J. C. Cox, et al., Automated RNA Selection Biotechnol Prog., 14, 845 850, 1998.

[0109] Base protocol. Automated selection involves several, sequential automated steps.

[0110] Several modules are placed on the robotic work surface, including a magnetic bead separator, and enzyme cooler, and a thermal cycler. After manually preparing reagents and preloading those reagents (including random pool RNA, buffers, enzymes, streptavidin magnetic beads, and biotinylated target) and tips onto the robot, a program is run. The selection process, automated by the robot, goes as follows: RNA pool is incubated in the presence, of biotinylated target conjugated to streptavidin magnetic beads. After incubation, the magnets on the magnetic bead separator are raised, and the beads (now bound by pool RNA-the selected nucleic acids) are pulled out of solution. Thus, the beads can be washed, leaving only RNA bound to targets attached to beads. These RNA molecules are reverse transcribed, reamplified. via PCR, and the PCR DNA is in vitro transcribed into RNA to be used in iterative rounds of selection.

[0111] The Bioworks method for in vitro selection. This scripted programming method contains all movements necessary in order to facilitate automated selection. This includes all physical movements to be coordinated, and also communication statements. For instance, five rounds of automated selection against a single target requires over 5,000 separate movements in x, y, z, t coordinate space. Additionally, the method also holds all relevant measurements, offsets, and integrated equipment data necessary to prevent physical collisions and permit concerted communication between devices.

[0112] "Beads on filler" selections. While the vast majority of manual selections have been performed on nitrocellulose-based filters, a small few have also been performed on solid surfaces, such as beads. A novel selection scheme was developed whereby selection is performed on magnetic beads that are placed on nitrocellulose filters, and washed as the bead is the selection target itself. This method allows for much greater specificity of selection, thereby promoting `winning` molecules to amplify in greater number, and thus reduce the overall amount of rounds necessary to complete the selection procedure. Manual selection does not involve a combination of surfaces to enhance selection. An alternative method is to take the magnetic beads, or nucleic acids attached to beads using methods other than beads, and running buffer over the beads and through a filter. It has been found that a complete filter washing step provides improved performance in the selection due to decreased background. One example of the automation of such a methods would be to remove, e.g., nucleic acids attached to the beads by placing the beads in a 96-well plate with a filtered bottom, the beads washed with buffer followed by subsequent elution of the target nucleic acids.

[0113] Cross-contamination avoidance. The introduction of contaminating species of nucleic acid strands in a manual selection may be commonplace. This is especially true if selection is done against multiple targets in parallel, and also when a researcher reuses the same pool for different selection tools. Contaminating species have been shown in the past to interfere with a manual selection such that it could not be completed. Automated in vitro selection takes steps to minimizing and/or eliminate the possibility of cross-contamination between pools and targets. Movement of the mechanical pod along the 18 work surface is unidirectional when carrying potentially contaminating material. This movement away from .degree.Clean' things and only towards items that have already been exposed to replicons greatly diminishes the possibility of cross-contaminating reactions. The only circumstance in which the pod reverses its direction is to acquire a new, clean pipette tip. Additionally, the reagent trays were sealed with aluminum foil for a physical barrier between the environment and unexposed reagents. See FIG. 14, a layout of the robotic work surface that reduces cross-contamination.

[0114] Using this method the present inventors have successfully selected aptamers against a number of protein targets, including Cyt18, lysozyme, the signaling kinase MEKI, Rho from a thermophilic bacteria, and the Herpes virus US11 protein. The robot can perform 6 rounds of selection/day versus individual protein targets, and selections are typically completed within 12-18 rounds. In each instance, selected populations showed a substantially greater affinity for their cognate proteins than naive populations. In addition, when selected populations were sequenced one or more sequence families typically predominated. Sequence families are a hallmark of a successful selection, and indicate that the robotic method faithfully recapitulates manual selection methods.

[0115] The use of beads for target immobilization allows automated selection to be generalized to virtually any target class. For example, small organic molecules could be directly conjugated to beads. Similarly, antibodies could be conjugated to beads and in turn could be used to capture macromolecular structures, such as viruses or cells.

[0116] In another embodiment, the robotic workstation can be used for the selection of nucleic acid catalysts. For example, a DNA library was incubated that contained an iodine leaving group at its 5' end with a DNA oligonucleotide substrate containing a 3' phosphorothioate nucleophile and a 5' biotin. The biotin can be captured on beads bearing streptavidin, and the beads can in turn be captured either by magnetic separation or by filtration. Any molecules in the DNA pool that ligate themselves to the biotinylated substrate are co-immobilized with that substrate. Immobilized species can be directly amplified following transfer to the integrated thermal cycler. The inclusion of a biotin on one of the primers used for amplification allows single-stranded DNA to be prepared by denaturation of the non-biotinylated strand in base, followed by neutralization of the solution. While this method has proved successful for the selection of deoxyribozyme 19 ligases, variations could also have been attempted. For example, the biotinylated DNA oligonucleotide substrate could have been pre-immobilized on beads, and the DNA pool incubated with the beads. In this instance, any molecules in the DNA pool that ligate themselves to the substrate will also be directly captured on the beads.

[0117] The use of beads for catalyst immobilization immediately suggests other selection protocols. For example, nucleic acid cleavases could be selected by first immobilizing a pool on the beads, then selecting for those species that cleave themselves away from the beads. Similarly, nucleic acid Diels-Alder synthetases may be selected by first immobilizing a diene on the beads, creating a nucleic acid pool that terminates in a dienophile, and selecting for those species that most efficiently con ugate the diene and dienophile.

[0118] This method can be applied to the selection of RCANAs. The ability to use a robotic workstation to select for ligases demonstrates that it is possible to select for regulatable ribozymes. For example, the selection protocols described in this invention can be altered so that ligases that immobilized themselves in the absence of a protein effector are removed from the random sequence population, while ligases that subsequently immobilized themselves once a protein effector were added are transferred to the integrated thermal cycler, amplified, and used for additional rounds of selection. This automated selection methods for regulatable ribozymes can readily be extended to other classes or catalysts than ligases, such as cleavases or Diels Alder synthetases by those skilled in the art.

[0119] Automating selection greatly diminishes human error in the actual pipetting and biological manipulations. While programming the robot is often not a trivial task, and can be time-consuming, automated selection is far faster and more efficient than manual selection. The scicntist's time is thus put to better use preparing samples and analyzing data, rather than performing the actual selection. Additionally, automated selection may include real-time monitoring methods (e.g., molecular beacons, TaqMan) and software that can make intelligent decisions based on real-time monitoring.

E. Chip-based RCANA for in vitro Detection Applications

[0120] Regulatable catalytically active nucleic acids are especially useful for biosensor applications. For example, different protein-regulated catalytically active nucleic acids may be anchored to a surface, such as wells in a multi-well plate. Mixtures of analytes and fluorescently tagged substrates are added to each well. Where cognate effectors are present, the protein-regulated catalytically active nucleic acids will covalently attach the fluorescent tags to themselves. Where protein-regulated catalytically active nucleic acids have not been activated by effectors, the tagged substrates will be washed out of the well. After reaction and washing, the presence and amounts of co-immobilized fluorescent tags are indicative of amounts of ligands that were present during the reaction.

[0121] In one embodiment of the invention, the reporter may be a fluorescent tag, but it may also be an enzyme, a magnetic particle, or any number of detectable particles. Additionally, the protein-regulated catalytically active nucleic acids may be attached to beads or non-covalently linked to a surface rather than covalently linked to a surface.

[0122] One advantage of this method is that covalent immobilization of reporters (as opposed to non-covalent immobilization, as in ELISA assays) allows stringent wash steps to be employed. Additionally, ribozyme ligases have the unique property of being able to transduce effectors into nucleic acid templates that can be amplified, affording an additional boost in signal prior to detection.

[0123] Another advantage is that the analytical methods of the present invention do not rely on binding per se, but only on transient interactions. The present invention requires mere recognition rather than a binding event that must be physically isolated, providing a potential advantage of protein-regulated catalytically active nucleic acids over antibodies. That is, even low affinities are sufficient for activation and subsequent detection, especially if individual, immobilized protein-regulated catalytically active nucleic acids are examined (i.e., by ligand-dependent immobilization of a quantum dot).

[0124] FIG. 16 schematically depicts one way to anchor aptazymes to a chip for a particular embodiment of the present invention. In this schematic, different ribozyme ligases (indicated by different colored allosteric sites) are shown immobilized on beads in wells, and mixtures of analytes (differentiated by shape and color) and fluorescently tagged substrates have been added to each well. In the middle panel of this figure, where 21 cognate effectors are present (same color analyte and allosteric site), the aptazymes will covalently attach the fluorescent tags to themselves. Where RCANA have not been activated by effectors, the tagged substrates are washed out of the well. In the last panel of FIG. 16, after reaction and washing, the presence and amounts of co-immobilized fluorescent tags are indicative of amounts of ligands that were present during the reaction.

[0125] In the embodiment of FIG. 16, the reporter may be a fluorescent tag, but it may also be an enzyme, a magnetic particle, or any number of detectible particles. Additionally, the RCANA could be immobilized on beads, but they could also be directly attached to a solid support via covalent bonds.

[0126] One advantage of this embodiment is that covalent immobilization of reporters allows stringent wash steps to be employed. This can be distinguished from to non covalent immobilization assays such as ELISA assays where stringent washing may destroy the signal. An additional advantage is that ribozyme ligases have the unique property of being able to transduce effectors into templates that can be amplified, affording an additional boost the in signal prior to detection.

[0127] Additionally, the method of the present invention contemplates that the RCANA construct may be amplified by polymerase chain reaction. Finally, the method contemplates that the RCANA oligonucleotide sequence of the construct may include RNA nucleotides, so that the method further includes reverse transcription of the RNA using reverse transcriptase to produce a DNA complementary to the RNA template.

[0128] Modified nucleotides may be introduced into the RCANA that substantially stabilize them from degradation in environments such as sera or urine. The analytical methods of the present invention do not rely on binding per se, but only on transient interactions. The present invention requires mere recognition rather that actual binding, thus providing a potential advantage of RCANA over antibodies. That is, even low affinities are sufficient for activation and subsequent detection, especially if individual immobilized RCANA are examined (i.e., by ligand-dependent immobilization of a quantum dot).

F. In vitro Engineering and Selection of RCANAs for in vivo Applications

[0129] The above discussion has disclosed methods for the in vitro creation of RCANAs, and has disclosed some of their in vitro applications. In the following section we describe the design, engineering, and in vitro selection of RCANAs for in vivo applications.

[0130] This invention utilizes ribozymes that can alter the level of mRNAs in a cellular system. In one embodiment, the ribozyme can be a self splicing intron, such as the group I intron. This ribozyme can be inserted into a gene. If the ribozyme is active, it will catalyze the a self-splicing reaction that removes itself from the gene, allowing accurate expression of the gene. In another embodiment, the ribozyme may be one that acts in trans to cleave a mRNA. Again, changing the activity of the ribozyme will lead to a change in the level of the mRNA in the cell, thereby altering the level of the protein coded by that gene. Those skilled in the art will recognize that other ribozyme activities may be used. For the purpose of illustration, the invention is now described in detail with the use of the self splicing intron.

[0131] The intron is first modified to function as an RCANA. Briefly, the methods described above can be used generate RCANA introns. A pool of potential RCANA introns is created by randomizing one or more regions of the intron. The randomized region optionally includes one or more residues from the catalytic core. A selection protocol is then developed that allows the active RCANA introns to be partitioned from the inactive ones. For example, the active RCANA introns can be partitioned from the inactive RCANA intron based on the mobility in gel electrophoresis. Other methods will be clear to those skilled in the art. Based on this partitioning method, an iterative procedure of partitioning and subsequent amplification of the RCANA introns is used to select RCANAs that are regulated by an effector. With the exception of the partitioning method, this procedure is essentially identical to the selection described about for RCANA ligases.

[0132] As an alternative to the selection of RCANA introns, it is also possible to engineer RCANA introns. For example, one of the stem-loop structures of the intron can be replaced by an aptamer for the desired effector. Interaction of the effector-with this engineered RCANA intron-will result in a modulation of the RCANA intron activity. Because an aptamer is different from an regulatory element (as was detailed above), the 23 present method will, in general, lead to RCANAs that are regulated by the effector. however, as will be shown in an example below, an important aspect of the current invention is that this level of regulation can be adequate for in vivo applications.

G. In vivo Selection and Optimization of RCANAs.

[0133] Here we disclose methods to generate RCANAs by using in vivo selection. FIG. 4b shows a selection protocol for the Group I P6 RCANA Pool of FIG. 4a. Positive and negative selections are made in vitro to select Group I RCANA that are dependent on activator. The selections are described above in Example 2 for a specific embodiment of the present invention--a theophylline dependent RCANA. In vivo screens and selections are used to select Group I RCANA that exhibit strong theophylline-dependence. The selected RCANA are mixed at various ratios with mutant Group I ribozymes that splice at a low but continuous level to determine the level at which RCANA can be selected against background. Because activation domains are often in the form of a stem-loop, the mutations can be concentrated in a single stem loop structure of the RCANA intron. In an alternate embodiment, the mutations can include catalytic residues. In yet another embodiment, the mutations are randomly dispersed in the intron. Finally, the best theophylline-dependent Group I aptazymes that have been derived by any of the methods described herein may undergo further selection by partially randomizing their sequences and selecting for improved in vivo performance.

[0134] Strategies similar to those depicted in FIGS. 4a and 4b may be used to develop RCANA on any desired effector. Positive and negative in vitro selection such as depicted in FIG. 4b are described above in Example 2 for a specific embodiment of the present invention.

[0135] From 10.sup.6 to 10.sup.10 variants can be efficiently transformed as described herein, sufficient to encompass most variants in the populations discussed so far. This efficiency of transformation, however, is likely to be insufficient to encompass a significant fraction of a completely random pool. Nonetheless, sequences have been selected from completely random expressed pools that can protect bacteria from bacteriophage infection.

[0136] The above procedure described how to select in vivo RCANAs. A similar procedure can be used to optimize engineered RCANAs. Residues in the RCANA that might include the ligand binding region, structural stem-loops, or even catalytic residues can be mutated. The selection procedure described above is then used to select for optimized RCANAs.

[0137] Since the rules that govern Group I intron splicing in different gene contexts are well known to those skilled in the art, the skilled artisan can remove RCANA introns from one context and modularly insert them into other genes. Should the efficiency or effector-dependence of intron splicing be compromised in the new gene, the intron may be reaccommodated to its new genetic environment by a selectable marker to the interrupted gene of interest and selecting for an effector-dependent phenotype.

[0138] Similarly, modulation of genes by cleavage is also well known to those skilled in the art. The skilled artisan can engineer endonucleolytic RCANAs that, upon activation, cause endonucleolytic cleavage of target nucleic acid. This endonucleolytic cleavage strategy may be applied to either upregulate or downregulate target polypeptide synthesis.

[0139] To the extent that aptazymes are self-sufficient, they should also function in eukaryotic cells, including human cells. Selecting for effector-dependence may also be performed in eukaryotic cells. Selection in eukaryotic systems may be performed, e.g., by fusing the gene of interest to a reporter gene such as GFP or luciferase. Variants of the RCANA that promote effector-dependent protein production may then be selected using a FACS. A pool of 10.sup.6 to 10.sup.8 variants may be screened by this procedure, a range comparable to the bacterial system previously described.

H. In vivo Detection Applications

[0140] Using the present invention, it is possible to activate or repress a reporter gene (e.g., liciferase or GFP) containing an engineered RCANA in response to an endogenous protein activator, or a post-translationally modified form of an endogenous protein activator (e.g., protein kinases such as ERK 1 and phosphorylated ERK 1). It is also possible to activate or repress a reporter gene (e.g., luciferase or GFP) containing an engineered RCANA in response to small molecule effectors (e.g., cyclic AMP, glucose, bioactive peptides, bioactive nucleic acids, or low molecular weight drugs such as antibiotics, antineoplastics or the like.). Thus, reporter gene-engineered RCANA constructs may be used to monitor intracellular levels of proteins, post-translationally modified forms of proteins or small molecules such as cyclic AMP and the like. Such applications may be used for high-throughput cell-based assays and screens for drug leads or for drug optimization and development.

[0141] Bacterial strains such as Escherichia coli (E. coli), and Bacillils subtilis (B. subtilis), or yeast strains such as Saccharomyces cerevisiae (S. cerevisiae), and Schizosaccharomyces pombe (S. pombe) are transformed with an expression vector encoding a reporter gene regulated by a RCANA, and these engineered microbial cell lines are used for cell-based assays and tests for drug discovery and development. Similarly, standard mammalian cell lines such as CHO, NIH3T3, 293, and 293T are transfected with appropriate vectors (e.g., pCDNA, pCMV, or retrovirus), that are engineered to contain RCANA-regulatable reporter genes, and these re-engineered cell lines may be used subsequently for cell-based assays and tests. In another use of the RCANA reporter gene technology, tumorigenic cell lines such as LNCaP, MCF-7, IMAMB-435, SK-Mel, DLI, PC3, T47D and the like, may be transfected in vitro with appropriate vectors encoding an RCANA-regulatable reporter gene. These re-engineered tumorigenic cell lines may be used in cell-based screens for the discovery and development anti-neoplastic drugs.

[0142] In another in vivo application, reporter gene-RCANA constructs (e g., luciferase or GFP) may be used to generate live animal models for use in drug development. In one embodiment the RCANA construct may be used in an engineered tumorigenic cell line to indicate the levels of a target molecule used to generate a tumor xenograft in nude mice. Mice bearing the tumors derived from the engineered cell line may then be used to screen for drugs that alter the level of the target molecule. For example, a transfected MDA-MB-435 line engineered to express a GFP gene under regulatable control by intron response to the protein activator phospho-ERK 1 is used to screen for drugs which both inhibit tumor growth and block formation of phospho-ERK. In another embodiment of the RCANA intron invention, transgenic mouse models may be generated in which tissue or cell type specific expression of the reporter gene is controlled by the effector activated RCANA intron. For example transgenic mice expressing a phospho-VEGF receptor tyrosine kinase (RTK) specific RCANA regulated GFP gene under control of the MMTV (mouse mammary tumor virus) promoter would show expression of GFP in mouse mammary tissue in a phospho-VEGF RTK dependent manner. Furthermore, these mice may be used to screen compounds for anti-VEGF RTK activity.

[0143] FIG. 5 is a diagrammatic representation of another embodiment of the present invention. Exogenous or endogenous activation of Group I intron splicing is depicted. A reporter gene such as Luciferase or beta-Gal is fused to the gene of interest which also contains the group I intron (td). Splicing-out of the Group I intron is induced by an effector, shown in the diagram as a protein, in this case Cyt18, by the shaded oval. Activation of the RCANA and auto-excision of the intron results in expression of the reporter gene to detect the desired reaction. The use of a reporter gene of this embodiment may be suitable for use in eukaryotic systems.

[0144] FIG. 6 is a diagram of another embodiment of the present invention. Libraries of candidate exogenous activators (E.sub.1-n) may be generated from a randomized RCANA pool indicated by the triangle. As in the embodiment of FIG. 5, a reporter gene is expressed in cells where the exogenous activator activates the RCANA to release the intron from the gene. As will be known to those of skill in the art any number of current and future libraries may be used with the present invention.

[0145] FIG. 7 depicts an alternative embodiment for screening libraries of exogenous activators. In the embodiment of the present invention of FIG. 7, Group I introns are induced into trans-splicing. Extracted and amplified introns are used to transform cells.

[0146] FIG. 8 shows yet another alternative embodiment for screening libraries of exogenous activators of the present invention. In the embodiment of FIG. 8, the effector (shaded oval), shown in this illustration as protein Cyt18, is mutagenized (triangle) to form an effector library. A second effector (E.sub.1-n) interacts with and activates one or more members of the effector library. The effector-effector complex is exposed to the gene containing both the Group I intron and a reporter gene. Cell sorting reveals the cells that express the reporter gene to indicate successful activation of the RCANA by the effector-effector complex.

[0147] FIG. 9 is a diagram of an embodiment of the present invention for screening for endogenous activators. In this embodiment, an endogenous effector, in this illustration shown as a protein activator from endogenous or transformed origin (shaded oval), activates self-splicing of the Group I intron. Cell sorting is used to reveal the expression of the reporter gene. To protect against spontaneous auto-excision of the intron, the gene 27 may be transferred into a different background system such as yeast or E. Coli, for example.

[0148] FIG. 10 depicts an alternative to the embodiment of FIG. 9 to screen for endogenous activators of the present invention. In FIG. 10, the activator that is being screened for may include, inter alia, a phosphorylated protein, a product of ubiquitination, or a protein-protein complex. For example, a protein activator, shown as the small shaded oval, may phosphorylate an effector such as Cyt18, shown as a large shaded oval with the phosphorylation indicated by the shaded rectangle. The phosphorylated effector activates intron self-splicing with concomitant expression of the reporter gene, shown here for illustration as Luciferase or beta-Galactosidase.

[0149] FIG. 11 shows yet another embodiment of the present invention to monitor compounds that perturb cellular metabolism. In this embodiment, a ribozyme similar to described in FIG. 6, and designated in this diagram by a line with a triangle is activated by a protein effector, shown as a shaded oval in FIG. 11. The protein effector may be a phosphoprotein, an induced protein, or a protein complex, for example. One or more second effectors, designated as a series of circles, alters the level of or degree of modification of the protein effector. The source of the second effectors may be endogenous or the effectors may be the product of a transformation construct used to transform a cell. Alteration of the level or modification of the protein effector results in an alteration in the expression of the reporter gene (shown as a dark circle with "lightning bolts"). The functioning of the gene of interest may thereby be perturbed, providing information about the function and/or regulation of the gene or gene product. FIG. 1 describes a method for taking the products of the screen described in FIGS. 8 and 10 and using them to monitor cellular or metabolic states.

[0150] FIG. 12 shows a further embodiment of the present invention that provides a non-invasive readout of metabolic states. An RCANA construct of the present invention may be introduced to a gene of interest. A protein suppressor from either an endogenous source from the product of cell transformation activates self-splicing of the RCANA, leading to expression of the endogenous gene, shown here again as a dark circle with lightning bolts. Whether or not the gene of interest is expressed upon release of the RCANA intron from the gene provides information about the metabolic state of the gene 28 of interest. The embodiment of the present invention of FIG. 12 thus provides a noninvasive means to determine the metabolic state of an organism with regard to a gene of interest.

[0151] FIG. 13 depicts a further embodiment of the present invention wherein endogenous suppressors provide a non-invasive readout of multiple metabolic states. Multiple protein activators (endogenous or transformed) arc exposed to a pool of Group I introns of the present invention. The pool comprises introns with length polymorphisms that are depicted in FIG. 13 by a discontinuity or break in the line representing the Group I intron (thick line) residing in a gene of interest (thin line). Activation of the RCANA leads to trans-splicing among the various polymorphisms. The products of trans-splicing may be extracted and amplified. Separation of the trans-splicing products by gel clectrophoresis provides a read out of the protein function or the metabolic pathway. The readout may even be digitized for analysis.

[0152] In vivo Uses of RCANAs for Gene Therapy

[0153] One important feature of using RCANAs and the method of the present invention for gene therapies is that regulated introns may be used to control gene expression, for any of a variety of genes, since the introns may be inserted into and be engineered to accommodate virtually any gene. Moreover, since the RCANAs may be engineered to respond to any of a variety of effectors, the characteristics of the effector (oral availability, synthetic accessibility, pharmacokinetic properties) may be chosen in advance. The drug is chosen prior to engineering the target of the drug. In part because of these extraordinary capabilities, RCANA provide perhaps the only viable route to medically successful and practical gene therapies. Drugs may be given throughout the treatment (or lifetime) of a patient who had undergone a single initial gene therapy. In addition, by making the gene therapy regulatable, the amount of a gene product may be easily increased or decreased in different individuals at different times during the treatment by increasing or decreasing the doses of effectors.

[0154] The present method also includes transforming a cell with the RCANA construct so that the construct is inserted into a gene of interest. An effector is provided to activate the RCANA so that administering to the cell an effective amount of the effector induces the RCANA to splice itself out of the gene to regulate expression of the gene.

[0155] The method of the present invention contemplates that the RCANA construct may be a plasmid. The method then further includes transforming the cell with the plasmid. The method of the present invention also contemplates ligating the RCANA construct into a vector and transforming the cell with the vector.

DEFlNITIONS

[0156] As used herein, the term "regulatable, catalytically active nucleic acid" or "RCANA" means a ribozyme or nucleic acid enzyme that is regulated by an effector.

[0157] As used herein, the term "regulatory domain" and "effector domain" are interchangeable terms meaning the region of effector binding on an RCANA.

[0158] The kinetic parameters of the RCANA may be varied in response to the amount of an effector, which may be an allosteric effector molecule. just as allosteric, protein enzymes undergo a change in their kinetic parameters or of their enzymatic activity in response to interactions with an effector molecule, the catalytic abilities of RCANAs may be similarly modulated by effectors. As demonstrated herein, the effectors may be small molecules, proteins, peptides or molecules that interact with proteins, peptides or other molecules. RCANAs transduce molecular recognition into catalysis upon interaction with an effector that interacts with a portion of the RCANA.

[0159] As used herein, the term "effector," "effector molecule," "allosteric effector" or "allosteric effector molecule" means a molecule or process that changes the kinetic parameters or catalytic activity of an RCANA.

[0160] As used herein, the term "catalytic residue" refers to residues that when mutated decrease the activity of the RCANA. Mutating a residue that affects the catalytic activity of a ribozyme following the selection of the RCANA, may cause different residues to become sensitive to mutation than in the original ribozyme. The relative mutational sensitivity of a given "catalytic residue" may change before and after the selection of the RCANA. These secondary mutations are also encompassed by the present invention.

[0161] As used herein, the term "aptamer" refers to a nucleic acid that has been specifically selected to optimally bind to a target ligand. As described above, it is important to recognize that an aptamer is fundamentally different than an RCANA.

[0162] As used herein, the term "kinetic parameters" refers to any aspect of the catalytic activity of the nucleic acid. Changes in the kinetic parameters of a catalytic RCANA produce changes in the catalytic activity of the RCANA such as a change in the rate of reaction or a change in substrate specificity. For example, self-splicing of an intron RCANA out of a gene environment may result from a change in the kinetic parameters of the RCANA. Similarly, cleavage of an endonucleolytic RCANA in a gene environment also may result fiom a change in the kinetic parameters of the RCANA.

[0163] As used herein, the term "catalytic" or "catalytic activity" refers to the ability of a substance to affect a change in itself or of a substrate under permissive conditions. As used herein, the term "protein-protein complex" or "protein complex" refers to an association of more than one protein. The proteins that make up a protein complex may be associated by functional, stereochemical, conformational, biochemical, or electrostatic mechanisms. It is intended that the term encompass associations of any number of proteins.

[0164] As used herein, the term "in vivo" refers to cellular systems and organisms, e.g., cultured cells, yeast, bacteria, plants and/or animals.

[0165] As used herein the terms "protein", "polypeptide" or "peptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. As used herein, the term "endogenous" refers to a substance the source of which is from within a cell, cell extract or reaction system. Endogenous substances are produced by the metabolic activity of, e.g., a cell. Endogenous substances, however, may nevertheless be produced as a result of manipulation of cellular metabolism to, for example, make the cell express the gene encoding the substance.

[0166] As used herein, the term "exogenous" refers to a substance the source of which is external to a cell, cell extract, or reaction system. An exogenous substance may nevertheless be internalized by a cell by any one of a variety of metabolic or induced means known to those skilled in the art.

[0167] As used herein the term "modified base" refers to a non-natural nucleotide of any sort, in which a chemical modification may be found on the nucleobase, the sugar, or the polynucleotide backbone or phosphodiester linkage.

[0168] As used herein, the term "gene" means the coding region of a deoxyribonucleotide sequence encoding the amino acid sequence of a protein. The term includes sequences located adjacent to the coding region on both the 5'and 3' ends such that the deoxyribonucleotide sequence corresponds to the length of the full-length mRNA for the protein. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed, excised or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0169] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

[0170] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides referred to as the "3' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.

[0171] The term "gene of interest" as used herein refers to a gene, the function and/or expression of which is desired to be investigated, or the expression of which is desired to be regulated, by the present invention. In the present disclosure, the td gene of the T4 bacteriophage is an example of a gene of interest and is described herein to illustrate the invention. The present invention may be useful in regard to any gene of any organism, whether of a prokaryotic or eukaryotic organism.

[0172] The term "hybridize" as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid strands) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the melting temperature of the formed hybrid, and the G:C (or U:C for RNA) ratio within the nucleic acids.

[0173] The terms "complementary" or "complementarity" as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, for the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be partial, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

[0174] The term "homology," as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe which can hybridize (i.e., it is the complement to the single-stranded nucleic acid sequence under conditions of low stringency as described).

[0175] As known in the art, numerous equivalent conditions may be employed to comprise either low or high stringency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e.g, the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

[0176] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid selections are conducted. With "high stringency" conditions a relatively small number of nucleic acid catalysts will be selected from a random sequence pool, while under "low stringency conditions" a larger number of nucleic acid catalysts will be selected from a random sequence pool.

[0177] Numerous equivalent conditions may be employed to comprise low or high stringency conditions; factors such as the length of incubation of the reaction, the presence of competitive inhibitors of the reaction, the buffer conditions under which the reaction is carried out, the temperature at which the reaction is carried out are considered and the hybridization solution may be varied to generate conditions of low stringency selection different from, but equivalent to, the above listed conditions.

[0178] The term "antisense," as used herein, refers to nucleotide sequences that are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to tile "sense" strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a complementary strand. Once introduced into a cell, the transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may also be generated. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand. The term is also used in reference to RNA sequences that are complementary to a specific RNA sequence (e g., mRNA). Included within this definition are antisense RNA ("asRNA") molecules involved in genetic regulation by bacteria.

[0179] Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. Once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term "antisense strand" is used. in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation. (i.e., "negative") is sometimes used in reference to the antisense strand with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.

[0180] A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.

[0181] "Transformation," as defined herein, describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. The term "transfection" as used herein refers to the introduction of foreign DNA into eukaryotic cells.

[0182] Transfection may be accomplished by a variety of methods known to the art including, e.g., calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Thus, the term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into the genomic DNA. The term also encompasses cells that transiently express the inserted DNA or RNA for limited periods of time. Thus, the term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term "transient transfectant" refers to cells that have taken up foreign DNA but have failed to integrate this DNA.

[0183] As used herein, the term "selectable marker" refers to the use of a gene that encodes an enzymatic activity and which confers the ability to grow in medium lacking what would otherwise be an essential nutrient (e.g., the HIS3 gene in yeast cells); in addition, a selectable marker may confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989),pp. 16.9-16.15.

[0184] As used herein, the term "reporter gene" refers to a gene that is expressed in a cell upon satisfaction of one or more contingencies and which, upon expression, confers a detectable phenotype to the cell to indicate that the contingencies for expression have been satisfied. For example, the gene for Luciferase confers a luminescent phenotype to a cell when the gene is expressed inside the cell. In the present invention, the gene for Luciferase may be used as a reporter gene such that the gene is only expressed upon the splicing out of an intron in response to an effector. Those cells in which the effector activates splicing of the intron will express Luciferase and will glow.

[0185] As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector." The term "vector" as used herein also includes expression vectors in reference to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0186] As used herein, the term "amplify", when used in reference to nucleic acids refers to the production of a large number of copies of a nucleic acid sequence by any method known in the art. Amplification is a special case of nucleic acid replication involving template specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

[0187] As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer may be single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact length of the primers will depend on many factors, including temperature, source of primer and the use of the method.

[0188] As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g. ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0189] As used herein, the term "target" when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted oat from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence. As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence.

[0190] To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be PCR amplified".

[0191] With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies, e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of .sup.32 P-labeled deoxynucleotide triphosphates, such as DCTP or DATP, into the amplified segment. In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

[0192] As used herein, the term "metabolic pathway" means a physical or chemical process found in a living organism by which a substance is produced and maintained, e.g., anabolism, and also the transformation by which energy is made available for the use of a living organism, e.g., catabolism.

[0193] As used herein, the term "bioproduct" means a substance produced by a physical or chemical process, the components of which are found in a living organism(s).

[0194] As used herein, the term "biosynthetic process" means a process by which a substance is produced using the physiological process found in a living organism.

EXAMPLE 1: GPITH1P6

Engineering of an RCANA for In vivo Detection Applications

[0195] The first example illustrates how to make an RCANA construct and demonstrates self-splicing of the RCANA out of a gene in response to an effector molecule.

1 Construction of a RCANA. Oligos GplWt3.129: 5-TAA TCT TAC CCC GGA ATT (SEQ ID NO:1) ATA TCC AGC TGC ATG TCA CCA TGC AGA GCA GAC TAT ATC TCC AAC TTG TTA AAG CAA GTT GTC TAT CGT TTC GAG TCA CTT GAC CCT ACT CCC CAA AGG GAT AGT CGT TAG-3' and GpITh1P6.131: 5-GCC TGA GTA TAA GGT GAC TTA TAC TTG TAA TCT (SEQ ID NO:2) ATC TAA ACG GGG AAC CTC TCT AGT AGA CAA TCC CGT GCT AAA TTA TAC CAG CAT CGT CTT GAT GCC CTT GGC AGA TAA ATG CCT AAC GAC TAT CCC TT-3'

[0196] were annealed and extended in a 30 .mu.l reaction containing 100 pmoles of each oligo, 250 mM Tris-HCl (pH 8.3), 40 mM MgCl.sub.2, 250 mM NaCl, 5 mM DTT, 0.2 mM each dNTP, 45 units of AMV reverse transcriptase (RT: Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) at 37.degree. C. for 30 min. The extension reaction was diluted 1 to 50 in H.sub.2O.

[0197] A PCR reaction containing 1 .mu.l of the extension dilution, 500 mM KCl, 100 mM Tris-HCl, (pH 9.0), 1% Triton.RTM. X-100, 15 mM MgCl.sub.2, 0.4 .mu.M of GpIWt1.75: 5'-GAT AAT ACG ACT CAC TAT AGG GAT CAA CGC TCA GTA GAT GTT TTC TTG GGT TAA

[0198] TTG AGG CCT GAG TAT AAG GTG-3' (SEQ ID NO:3)),0.4 .mu.M of Gp1Wt4.89:5'-CTT AGC TAC AAT ATG AAC TAA CGT AGC ATA TGA CGC AAT ATT AAA CGG TAG CAT TAT GTT CAG ATA AGG TCG TTA ATC TTA CCC CGG AA-3' (SEQ ID NO:4), 0.2 mM each dNTP and 1.5 units of Taq polymerase (Promega, Madison, Wis.) was thermocycled 20 times under the following regime: 94.degree. C. for 30 sec, 45.degree. C. for 30 sec, 72.degree. C. for 1 min. The PCR reaction was precipitated in the presence of 0.2 M NaCl and 2.5 volumes of ethanol and then quantitated by comparison with a molecular weight standard using agarose gel electrophoresis.

[0199] The RCANA construct was transcribed in a 10 .mu.l high yield transcription reaction (AmpliScribe from Epicentre, Madison, Wis. The reaction contained 500 ng PCR product, 3.3 pmoles of .sup.32P [.sup.32P]UTP, 1.times.AmpliScribe transcription buffer, 10 mM DTT, 7.5 mM each NTP, and 1 .mu.l AmpliScribe T7 polymerase mix. The transcription reaction was incubated at 37.degree. C. for 2 h. One unit of RNase free-DNase was added and the reaction returned to 37.degree. C. for 30 min. The transcription was then purified on a 6% denaturing polyacrylamide gel to separate the full length RNA from incomplete transcripts and spliced products, eluted and quantitated spectrophotometrically.

[0200] In vitro Assay. The RNA (4 pmoles/12 .mu.l H.sub.2O) was heated to 94.degree. C. for 1 min then cooled to 37.degree. C. over 2 min in a thermocycler. The RNA was divided into 2 splicing reactions (9 .mu.l each) containing 100 mM Tris-HCl (pH 7.45), 500 mM KCl and 15 mM MgCl.sub.2, Plus or minus theophylline (2 mM). The reactions were immediately placed on ice for 30 min. GTP (1 mM) was added to the reactions (final volume of 10 .mu.l) and the reactions were incubated at 37.degree. C. for 2 h.

[0201] The reactions were terminated by the addition of stop dye (10 .mu.l) (95% formamide, 20 mM EDTA, 0.5% xylene cyanol, and 0.5% bromophenol blue). The reactions were heated to 70.degree. C. for 3 min and 10 .mu.l was electrophoresed on a 6% denaturing polyacrylamide gel. The gel was dried, exposed to a phosphor screen and analyzed using a Molecular Dynamics Phosphorimager (Sunnyvale, Calif.).

[0202] Activation was determined from the amount of circular intron in each reaction. Circularized introns migrate slower than linear RNA and can be seen as the bands above the dark bands of linear RNA in the +Theo lanes of the gels of FIGS. 2a and 2b.

[0203] In vivo Screening of Group I Aptazymes. The RCANA constructs as well as the wild type and a negative control were ligated into a vector that contains the T4 td intron with Eco R I and Spe I flanking the P6 region, transformed and mi