Nucleic acid sensor molecules

Nucleic acid sensor molecules and methods are disclosed for the detection and amplification of signaling agents using enzymatic nucleic acid constructs, including hammerhead enzymatic nucleic acid molecules, inozymes, G-cleaver enzymatic nucleic acid molecules, zinzymes, amberzymes and DNAzymes; kits for detection and amplification; use in diagnostics, nucleic acid circuits, nucleic acid computers, therapeutics, target validation, target discovery, drug optimization, SNP detection, SNP scoring, proteome scoring and other uses are disclosed.

DETECTION OF TARGET SIGNALING MOLECULES In one embodiment, the invention features several approaches to detecting signaling agents, ligands and/or target signaling molecules in a system using nucleic acid molecules. In all cases, activity of the nucleic acid is modulated via interaction of the nucleic acid with the target signaling agent, ligand and/or target signaling molecule. In one embodiment, the present invention utilizes at least three oligonucleotide sequences for proper function: nucleic acid sensor molecule, reporter molecule, and target signaling molecule. The nucleic acid sensor molecule is comprised of a sensor component, enzymatic nucleic acid component, and a linker between them which can be present or absent. The nucleic acid sensor molecule ( FIG. 6 ), is in its inactive state when the sensor component binds to the nucleic acid sensor molecule in the enzymatic nucleic acid component. The sensor component can bind to the substrate binding regions or nucleotides that contribute to the secondary or tertiary structure of the enzymatic nucleic acid component. For example, the sensor component can bind to nucleotides located within the nucleic acid sensor molecule, which can disrupt catalytic activity. The reporter molecule can be able to bind to the nucleic acid sensor molecule, but a catalytic activity would be inhibited since the molecule is structurally inactive. Alternatively, the sensor component can bind to the substrate binding region(s) of the enzymatic nucleic acid component, which can prevent the reporter molecule from binding to the nucleic acid sensor molecule. The sensor component cannot be cleaved because the cleavage site would contain either a chemical modification which prevents cleavage or an inappropriate sequence. For example, hammerhead ribozymes need to have a NUH motif in the molecule to be cleaved (H is adenosine, cytidine, or uridine) for proper cleavage. By adding a guanosine at the H position in the RNA to be cleaved, cleavage can be inhibited. In the presence of the target signaling molecule, the sensor component can disassociate from the enzymatic nucleic acid component and bind to the target signaling molecule preferentially. The sensor component can preferentially bind to the target signaling molecule which results in the formation of a more stable complex. For example, the sensor component can bind to more nucleotides on the target signaling molecule than on the nucleic acid sensor molecule. Binding to a larger number of nucleotides can have increased chemical stability and therefore is preferred over binding to a smaller number of nucleotides. When the sensor component is bound to the target signaling molecule and the reporter molecule binds to the nucleic acid sensor molecule, a reaction can be catalyzed on the reporter molecule by the enzymatic nucleic acid component. For example, the reporter molecule can be cleaved. The cleavage event can then be detected by using a number of assays. For example, electrophoresis on a polyacrylamide gel would detect not only the full length reporter oligonucleotide but also any cleavage products that were created by the functional nucleic acid sensor molecule. The detection of these cleavage products indicate the presence of the target signaling molecule. In addition, the reporter molecule can contain a fluorescent molecule at one end which fluorescence signal is quenched by another molecule attached at the other end of the reporter molecule. Cleavage of the reporter molecule in this case results in the disassociation of the florescent molecule and the quench molecule, resulting in a signal. This signal can be detected and/or quantified by methods known in the art (for example see Nathan et al., U.S. Pat. No. 5,871,914, Birkenmeyer, U.S. Pat. No. 5,427,930, and Lizardi et al., U.S. Pat. No. 5,652,107, George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, and Shih et al., U.S. Pat. No. 5,589,332). Alternatively, the sensor of the signaling molecule can comprise a separate oligonucleotide sequence, as shown for example in FIG. 11 , system M. 
 Target Sites Targets for useful nucleic acid sensor molecules can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat. No. 5,525,468 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Nucleic acid sensor molecules to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such nucleic acid sensor molecules can also be optimized and delivered as described therein. Hammerhead, hairpin, Inozyme, Zinzyme, Amberzyme and DNAzyme-based nucleic acid sensor molecules are designed that can bind and are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706; Denman, 1993, Biotechniques, 15, 1090) to assess whether the nucleic acid sensor molecule sequences fold into the appropriate secondary structure. Those nucleic acid sensor molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA. Nucleic acid molecules of the differing motifs are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. Hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver-based nucleic acid sensor molecule cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684; and Caruthers et al., 1992, Methods in Enzymology 211,3-19. 
 Nucleic Acid Molecule Synthesis The nucleic acid molecules of the invention, including certain nucleic acid sensor molecules, can be synthesized using the methods described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59. Such methods make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 &mgr;mol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 &mgr;mol scale can be done on a 96-well plate synthesizer, such as the PG2100 instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 &mgr;L of 0.11 M &equals;6.6 &mgr;mol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 &mgr;L of 0.25 M&equals;15 &mgr;mol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 &mgr;L of 0.11 M &equals;13.2 &mgr;mol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 &mgr;L of 0.25 M &equals;30 &mgr;mol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by calorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used. Cleavage from the solid support and deprotection of the oligonucleotide is typically performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 ° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 &mgr;L of a solution of 1.5 mL N-methylpyrrolidinone, 750 &mgr;L TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65 ° C. After 1.5 h, the oligomer is quenched with 1.5 M NH 4 HCO 3 . Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is heated at 65 ° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH 4 HCO 3 . An alternative deprotection cocktail for use in the one pot protocol comprises the use of aqueous methylamine (0.5 ml) at 65° C. for 15 min followed by DMSO (0.8 ml) and TEA.3HF (0.3 ml) at 65° C. for 15 min. A similar methodology can be employed with 96-well plate synthesis formats by using a Robbins Scientific Flex Chem block, in which the reagents are added for cleavage and deprotection of the oligonucleotide. For anion exchange desalting of the deprotected oligomer, the TEAB solution is loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that is prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the RNA is eluted with 2 M TEAB (10 mL) and dried down to a white powder. For purification of the trityl-on oligomers, the quenched NH 4 HCO 3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile. Alternatively, for oligonucleotides synthesized in a 96-well format, the crude trityl-on oligonucleotide is purified using a 96-well solid phase extraction block packed with C18 material, on a Bahdan Automation workstation. The average stepwise coupling yields are typically >98% (Wincott et al. 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted as larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction. To ensure the quality of synthesis of nucleic acid molecules of the invention, quality control measures are utilized for the analysis of nucleic acid material. Capillary Gel Electrophoresis, for example using a Beckman MDQ CGE instrument, can be ulitized for rapid analysis of nucleic acid molecules, by introducing sample on the short end of the capillary. In addition, mass spectrometry, for example using a PE Biosystems Voyager-DE MALDI instrument, in combination with the Bohdan workstation, can be utilized in the analysis of oligonucleotides, including oligonucleotides synthesized in the 96-well format. The nucleic acids of the invention can also be synthesized in two parts and annealed to reconstruct the nucleic acid sensor molecules (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). The nucleic acids are also synthesized enzymatically using a variety of methods known in the art, for example as described in Havlina, International PCT publication No. WO 9967413, or from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Other methods of enzymatic synthesis of the nucleic acid molecules of the invention are generally described in Kim et al., 1995, Biotechniques, 18, 992; Hoffman et al., 1994, Biotechniques, 17, 372; Cazenare et al., 1994, PNAS USA, 91, 6972; Hyman, U.S. Pat. No. 5,436,143; and Karpeisky et al., International PCT publication No. WO 98/28317) Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204). The nucleic acid molecules of the present invention are preferably modified to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Nucleic acid sensor molecules are purified by gel electrophoresis using known methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water. The sequences of the nucleic acids that are chemically synthesized, useful in this study, are shown in Table III. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the nucleic acid (all but the binding arms) is altered to affect activity. The nucleic acid construct sequences listed in Table III can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such nucleic acids with enzymatic activity are equivalent to the nucleic acids described specifically in the Table. 
 Optimizing Nucleic Acid Molecule Activity Synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. W092/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Bichem. Sci. 17, 334; Usman et al., International Publication No. WO93/15187; Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. All these references are incorporated by reference herein. Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are preferably desired. There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35, 14090). Sugar modifications of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. , 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al, 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers ( Nucleic acid Sciences ), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated by reference herein in their totalities). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid sensor molecule molecules without inhibiting catalysis. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention. While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, many of these modifications can cause some toxicity. Therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules. Nucleic acid molecules having chemical modifications which maintain or enhance activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in the presence of biological fluids, or in cells, the activity can not be significantly lowered. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective diagnostic agents, whether utilized in vitro and/or in vivo. Improvements in the synthesis of RNA and DNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19; Karpeisky et al., International PCT publication No. WO 98/28317) (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above. In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure. In one embodiment, the invention features modified nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39. These references are hereby incorporated by reference herein. In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′—Nh 2 or 2′-O—NH 2 , which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Karpeisky et al., WO 98/28317, respectively, which are both incorporated by reference herein in their entireties. Various modifications to nucleic acid (e.g., nucleic acid sensor molecule) structure can be made to enhance the utility of these molecules. Such modifications enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells. 
 Administration of Nucleic Acid Molecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). Other approaches include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J. Neuro Virol., 3, 387-400. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al, PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819, all of which are incorporated by reference herein. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a patient. The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art. The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells. By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, including CNS delivery of the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058. All these references are hereby incorporated herein by reference. The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al. J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392; all of which are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incorporated by reference herein. The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present. Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water. The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; Skillern et al., International PCT Publication No. WO 00/22113; Conrad, International PCT Publication No. WO 00/22114; and Conrad, U.S. Pat. No. 6,054,299; all of these references are hereby incorporated in their totalities by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856; all of these references are hereby incorporated in their totalities by reference herein). Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269-280; Peterson et al., 2000, Cent. Nerv. Syst. Dis., 485-508; Peel and Klein, 2000, J. Neurosci. Methods, 98, 95-104; Hagihara et al., 2000, Gene Ther., 7, 759-763; and Herrlinger et al., 2000, Methods Mol. Med., 35, 287-312. AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat. No. 6,180,613. In another aspect of the invention, nucleic acid molecules of the present invention are preferably expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510, Skillern et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299) inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510). In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner which allows expression of that nucleic acid molecule. In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences). Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). All of these references are incorporated by reference herein. Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J, 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566; all of these references are incorporated by reference herein). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein. The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra). In another aspect the invention features an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. 
 EXAMPLE The following are non-limiting examples showing techniques useful in isolating nucleic acid molecules of the instant invention. 
 Example 1 
 Diagnostic Screen A series of sensor molecules with trans-acting sensor component sequences were designed. Table III shows the sequences that were used in this test. Sequences with names beginning with R- were the reporter sequences used in this experiment, and those were beginning with SM- were nucleic acid sensor molecules. Sequences beginning with S- were sensor component sequences that were designed to bind to portions of the sensor molecule sequences (to varying degrees) and to prevent the sensor molecule from binding and cleaving reporter molecules; these sequences are shown in lower case because they were synthesized using 2′-O-methyl nucleotides in order to increase binding affinity. The one sequence labeled T-2a represents the target signaling molecule sequence which was designed to bind to the sensor component sequences so as to prevent them from inhibiting the sensor molecule activity. The system construct is shown in FIG. 15 . FIG. 16 shows the results of testing some of these sensor molecule/sensor component combinations in a cleavage assay. The reporter molecules were 5′-end labeled with 32 P-phosphate and incubated for 12 or 60 minutes in either: (1) buffer alone (50 mM Tris, pH 7.5, 10 mM MgCl 2 ), or in the presence of (2) 10 nM sensor molecule, (3) 10 nM sensor molecule plus 20 nM sensor component, (4) 10 nM sensor molecule plus 200 nM sensor component, or (5) 10 nM sensor molecule plus 20 nM sensor component and 500 nM target signaling molecule. At the end of the incubation the reactions were loaded onto a PAGE gel to separate cleaved reporter from uncleaved reporter. The gel was imaged on a Molecular Dynamics phosphorimager and quantitated to determine the percent of reporter molecule cleaved under each set of conditions. Control reactions were carried out to ensure that addition of sensor component or target signaling sequence, without sensor molecule, did not result in reporter cleavage; only 0.2-0.4% of reporter was cleaved under these conditions. FIG. 16 shows that sensor molecule alone results in 40-60% cleavage of the reporter molecule after 1 minute, and 85% cleavage after 60 minutes for three sensor molecules. When 20 nM sensor component is added to the reaction, the cleavage activity is reduced by 30-70%. When 200 nM sensor component is added, the cleavage activity is reduced by 50-99%. Finally, addition of 500 nM target signaling molecule to a reaction containing 10 nM sensor molecule and 20 nM target signaling molecule results in almost complete recovery of the cleavage activity up to the level observed with sensor molecule alone. 
 Example 2 Auto-ligating Nucleic Acid Molecules FIG. 17 is a schematic representation of the method of the invention used to isolate nucleic acid molecules capable of auto-ligation reactions useful, for example, in diagnostic applications. FIG. 17 a shows the general selection scheme used for isolating active sequences. A random pool of nucleic acid, such as RNA is combined with a substrate molecule comprising the structure R1—O—R2-Biotin, wherein R1 is selected from the group consisting of methyl, hydrogen, phosphate, nucleoside, nucleotide, oligonucleotide, R2 is selected from the group comprising molecules capable of generating a detectable signal, such as molecular beacons, small molecules, fluorophores, chemophores, ionophores, radio-isotopes, photophores, peptides, proteins, enzymes, antibodies, nucleic acids, and enzymatic nucleic acids, L represents a linker which can be present or absent, and “—” represents a covalent bond. Catalytically active sequences are biotinylated. The reaction mixture is passed over a solid support derivatized with Avidin, resulting in the capture of the biotinylated, catalytically active sequence pool. The support bound sequences are amplified by methods known in the art. FIG. 17 b shows the selection of the initial pool of sequences that provide ligation activity, and subsequent selection of molecules that are active in the presence of a ligand. Initially, selection of catalytic sequences takes place in the absence of the ligand. The active molecules isolated from the first round of selection that initially bind to the Avidin derivatized support are eliminated. Molecules that pass through the support are re-selected in the presence of the ligand. The re-selected pool that binds to the support after reaction in the presence of the ligand is amplified by methods known in the art and transcribed for subsequent rounds of selection. FIG. 17 c shows another selection strategy for isolating nucleic acid molecules capable of autoligation in the presence of a ligand. In this case, an initial selection takes place in the absence of the ligand to select sequences with autoligation activity. This pool is mutagenized by methods known in the art. The resulting mutagenized pool is selected for ligand binding activity by methods known in the art, for example, by using ligand affinity chromatography or gel shift assays. The resulting pool is mutagenized by methods known in the art. The original selection (for activity) is repeated in the presence of the ligand of diagnostic interest, with counterselection for molecules that react in the absence of the ligand. 
 Example 3 
 Isomerase Nucleic Acid Molecules FIG. 18 is a schematic representation of the method of the invention used to isolate nucleic acid sensor molecules capable of catalyzing isomerization reactions useful, for example in diagnostic applications. R1 and R2 represent compounds, which can be the same or different, capable of generating a detectable signal or quenching a detectable signal when an isomerization event takes place, comprising molecular beacons, small molecules, fluorophores, chemophores, ionophores, radio-isotopes, photophores, peptides, proteins, enzymes, antibodies, nucleic acids, and enzymatic nucleic acids, L represents a linker which can be present or absent, and “—” represents a covalent bond. FIG. 18 a shows the general selection scheme used for isolating active sequences. A random pool of nucleic acid sequences are passed over the complex of interest, derivatized to a solid support. The representative example of the complex shown in the figure consists of two fluorescent molecules joined together via a cis-carbon double bond linkage. Alternatively, a trans-carbon double bond linkage can be used. The selection pool is enriched and mutagenized throughout multiple generations to generate a diverse pool of “cis” binding sequences. Cis-binding nucleic acid molecules are then loaded onto the resin and the corresponding trans isomer of the complex is used to elute sequences that bind the trans-isomer tighter than the cis-isomer. FIG. 18 b shows how the concentration of cis-isomer on the resin and the concentration of trans-isomer eluant can be manipulated in order to select sequences that prefer binding to one isomer over the other, and can therefor drive the reaction in the desired direction. FIG. 18 c shows a selection scheme for isolating ligand dependent nucleic acid isomerase molecules from the initial selection pool from FIG. 18 a . A counter-selection takes place in which sequences that are bound to the cis-isomer complex are eluted with the ligand of diagnostic interest. An additional counter-selection takes place in which sequences that are bound to the cis-isomer complex are eluted with the ligand of diagnostic interest. A selection then takes place in which sequences remaining from the counter-selection rounds that are bound to the cis-isomer complex are eluted with a mixture of the ligand of diagnostic interest and the trans-isomer complex, the eluted ligand dependent nucleic acid catalyst sequences are amplified and transcribed by methods known in the art. 
 Example 4 
 Detection of HCV RNA A nucleic acid sensor molecule of the instant invention can be utilized to detect the presence of hepatitis C virus (HCV) in a sample of human blood. A system comprising a human blood sample, a reporter molecule such as a high turnover enzyme, and a nucleic acid sensor molecule attached to a solid support surface is used. The nucleic acid sensor molecule comprises an enzymatic nucleic acid component including an HCV specific sensor component, wherein in response to an interaction of HCV RNA or HCV core proteins with the nucleic acid sensor molecule, the enzymatic nucleic acid component catalyzes a chemical reaction. The reaction can comprise cleavage and release of a reporter molecule when HCV RNA is used as a target signaling agent (see for example FIG. 19 ), or when HCV core proteins are used as a target signaling agent (see for example FIG. 20 ). Alternatively, the reaction can comprise the attachment of the reporter molecule to the nucleic acid sensor molecule in the presence of the HCV target (see for example FIG. 23 or FIG. 24 ). In the case of a sensor molecule that ligates a reporter molecule, the system is subjected to conditions under which free reporter molecules are removed from the system, for example, by washing the surface of the solid support. The reporter molecule in the system can comprise a conjugated enzyme, such as luciferase, alkaline phosphatase, or horseradish peroxidase. Covalent attachment of the reporter molecule to the nucleic acid sensor molecule takes place in the presence of HCV RNA or core protein. The system is subjected to conditions that cause free reporter molecule to be removed from the system, for example, washing the surface of a solid phase system. A substrate for the conjugated enzyme is contacted with the system under conditions where conversion of the substrate by the immobilized enzyme generates an amplified signal, for example a precipitate, that is detected on the surface of the system (see FIG. 23 or FIG. 24 ). A system in which cleavage of a reporter molecule rather than ligation is used to detect the presence of a target signaling molecule is shown in FIG. 22 . An example for the attachment of a reporter enzyme to a nucleic acid sequence is shown in FIG. 21 . A system comprising a solution phase and a solid phase is used, wherein a biotin conjugated Zinzyme sensor molecule is used to detect the presence of a target signaling molecule (for example HCV RNA). In the presence of the HCV RNA target signaling molecule (“target” in the figure), the reporter molecule component of the sensor molecule is released from the sensor molecule when the sensor molecule interacts with the target signaling molecule in solution. The solution phase components are passed through a solid phase derivatized with avidin, streptavidin, or neutravidin. The eluent is assayed to indicate the presence of the high turnover enzyme by providing substrate for the enzyme. Enzyme activity is indicative of the presence of the HCV RNA in the system. Alternatively, the sensor molecule is attached to a solid support, for example covalently, wherein a sample is passed through or is passed over the support bound sensor molecule. The eluent is assayed to indicate the presence of the high turnover enzyme by providing substrate for the enzyme. Enzyme activity is indicative of the presence of the HCV RNA in the system. The use of nucleic acid sensor molecules as described herein is amenable to point of care applications, enabling the simple and efficient detection of analytes in a clinical setting. 
 Example 5 
 Nucleic Acid Sensor Circuit FIG. 25 describes a process whereby a nucleic acid signaling molecule is used in a nucleic acid circuit. The nucleic acid sensor molecule can be used to open or close an electronic circuit. In response to a target signaling agent, for example current, the nucleic acid sensor molecule catalyzes a chemical reaction comprising ligation in response to a predetermined current or cleavage in response to a predetermined current. The nucleic acid circuit is thereby modulated between an open and a closed state based on the predetermined input current that is applied to the circuit. A plurality of such circuits that comprise nucleic acid sensor modulation can be used in a variety of electronic devices, and can substitute solid state or silicon-based circuits in such devices. For example, computer processors comprising a plurality of nucleic acid sensor molecule based-circuits can be used in a computer device. Open and closed nucleic acid sensor molecule based-circuits can be used to generate or respond to binary code, creating a readable output. Processing of nucleic acids by nucleic acid sensor molecules can be used to generate more complex code, for example where particular nucleic acid sequences represent different code variables. 
 Example 6 
 Target Inhibition of Nucleic Acid Sensor Molecule FIG. 26 shows a non-limiting example of target signaling molecule inactivation of a zinzyme sensor molecule. In the absence of the target (SEQ ID NO. 31), the zinzyme sensor molecule (SEQ ID NO. 32) catalyzes the cleavage of a reporter molecule (SEQ ID NO. 33). Reaction conditions: 140 mM KCl10 mM NaCl, 20 mM HEPES pH 7.4, 1mM MgCl2, 1 mM CaCl2, 400 nM Nucleic acid sensor, 400 nM Target, Trace of labeled reporter (˜10 nM), 25 &mgr;l reaction volume, Nucleic acid sensor, target and reporter were heated at 75° C. for 3 min, cooled to 37° C. and cleavage initiated by the addition of MgCl2 and CaCl2. 
 Example 7 
 Target Activation of Nucleic Acid Sensor Molecule FIG. 27 shows a non-limiting example of target signaling molecule activation of a zinzyme sensor molecule. In the presence of the target (SEQ ID NO. 34), the zinzyme sensor molecule (SEQ ID NO. 35) catalyzes the cleavage of a reporter molecule (SEQ ID NO. 36). Reaction conditions: 140 mM KCl, 10 mM NaCl, 20 mM HEPES pH 7.4, 1 mM MgCl2, 1 mM CaCl2, 400 nM Nucleic acid sensor, 400 nM Target, Trace of labeled reporter (˜10 nM), 25 &mgr;l reaction volume, Nucleic acid sensor, target and reporter were heated at 75° C. for 3 min, cooled to 37° C. and cleavage initiated by the addition of MgCl2 and CaCl2. 
 Example 8 
 Protein (Erk) target Activation of nucleic Acid Sensor Molecule One method for protein detection contemplated by the invention utilizes a catalytically attenuated enzymatic nucleic acid molecule that is fused to a high affinity RNA ligand for a target protein in such a way that target association induces catalytic activity. A variation of combinatorial selection methods can be easily and quickly used to create high affinity RNA ligands (RNA sensor domains) for specific proteins. Combinatorial selection of RNA aptamers has been automated and multiplexed, providing a high throughput method for their production. Very much like antibodies, RNA aptamers display picomolar affinities for their targets and can discriminate between protein homologs, isoforms, and even different activation states of the same protein. Alternately, RNA sensor domains can be obtained from natural sources, such as the RNA binding domains of a virus (e.g. rev response elements and TAR elements of HIV) or eukaryotic RNA binding proteins (e.g. protein kinase PKR, promoters, RNA polymerase, ribosomal RNA binding domains etc.). In addition, a random sequence can be attached to an attenuated enzymatic nucleic acid molecule and through the use of combinatorial selection, allosteric nucleic acid molecules can be isolated that are modulated in the presence of a target signaling agent or molecule. This approach relies upon binding of a protein target to an RNA aptamer domain in the nucleic acid sensor molecule to induce catalytic activity. To accomplish this activation, the sensor and enzymatic nucleic acid molecule domains are fused via a third element, a communication module, that is responsible promoting enzymatic nucleic acid molecule catalysis upon target binding. The communication module is a nucleic acid sequence or sequences that promote a conformational rearrangement of the enzymatic nucleic acid molecule domain into its active structure upon target binding. Two routes exist for the production of communication modules: rational design or combinatorial selection. One approach utilizes rational design where pre-made communication module or modules are fused to preexisting enzymatic nucleic acid molecule and aptamer domains in a modular strategy. An RNA sensor domain that binds to protein ERK2 (Erk) was appended to a variant of the hammerhead enzymatic nucleic acid molecule through a communication module developed through rational design. The salient feature of this design strategy is that substrate-binding elements in the enzymatic nucleic acid molecule domain are sequestered by complementary allosteric effector sequences present in the communication module in the absence of target. Target association with the sensor domain forces an alternative RNA conformation in which the substrate binding elements become available for interaction with cleavage substrate, thus promoting catalysis. FIG. 28 shows a non-limiting example of a nucleic acid sensor molecule that is modulated by a protein target signaling molecule, Erk. In the presence of the target protein (Erk), the nucleic acid sensor molecule (SEQ ID NO. 39) catalyzes the cleavage of a reporter molecule. Reaction conditions: 100 mM KCl, 1 mM MgCl2, 10 mM Tris 7.5, 10 &mgr;M ERK protein, 1 &mgr;M HH enzymatic nucleic acid molecule, Vf&equals;19 &mgr;l, 34° C. for 30 minutes, trace 5′ labeled substrate (1 &mgr;l). This nucleic acid sensor displays little catalytic activity in the absence of the ERK2 protein but is activated approximately one hundred fold in the presence of recombinant ERK2 ( FIG. 31 al ). No nucleic acid sensor activation is observed if bovine serum albumin (BSA) replaces ERK 2 in the reaction, indicating that activation specifically requires ERK2. An enzymatic nucleic acid molecule that does not contain the ERK2 sensor component displays nearly identical activity in the presence or absence of the protein target ( FIG. 31 a ). To examine the dependence of activation on the concentration of ERK2, various amounts of ERK2 were added to different reactions ( FIG. 31 b ). One half-maximal nucleic acid sensor molecule activation is promoted by ˜800 pg/&mgr;l ERK2. Because the parental RNA sensor component displays an affinity of 8 pg/&mgr;l for ERK2, the sensitivity of this sensor molecule activation by ERK2 can likely be increased a further hundred fold by combinatorial optimization of the sensor molecule. Thus, this technology has a sensitivity comparable to that displayed by standard antibody based ELISA assays. The specificity of allosteric activation also compares favorably with antibody based approaches. ERK2 is a member of the mitogen activated protein kinase (MAPK) family, different members of which are implicated in a wide range of cellular processes including cancer (ERK2) and inflammation and apoptosis (P38 and JNK). These kinases are highly homologous, displaying up to 45% amino acid sequence identity. To examine the specificity of ERK2 responsive nucleic acid sensor molecule (allozyme), applicant attempted to activate the nucleic acid sensor molecule with P38 and JNK. These proteins did not activate the allozyme ( FIG. 32 a ), nor did bovine serum albumin ( FIG. 31 a ). ERK function is up regulated by a specific phosphorylation event that alters its structure. The RNA sensors used for the allozyme described here preferentially associate with the unactivated form of ERK2. Phosphorylated ERK2 was substituted for unactivated ERK2 in an allozyme reaction to assess its ability to activate enzymatic nucleic acid catalysis. Phosphorylated protein fails to activate the allozyme ( FIG. 32 b ). Thus, protein responsive nucleic acid sensor molecules (allozymes) can not only distinguish between different protein homologs, but also between different activation states of the same protein. Another approach used in the design of nucleic acid sensor molecules involves combinatorial selection of nucleic acid sensor molecules that are capable of catalysis in the presence of a predetermined target. For example, the evolution of protein binding nucleic acid sensor molecules to a protein, such as ERK2, can take place with modification of a known enzymatic nucleic acid motif. A variable region is introduced into the sequence and selective pressure is applied in iterative rounds of isolation and amplification, for example the isolation and amplification of sequences that cleave a substrate in cis in the presence of the target molecule. In a non-limiting example, such a random region is introduced into the Zinzyme stem-loop region (5′-CCGAAAGG-3′ ) shown in FIG. 3 . 
 Example 9 
 Half-zinzyme Nucleic Acid sensor Molecule (Halfzyme) Applicant has developed a generalizable methodology for the production of nucleic acid sensor molecules that are activated by target nucleic acids. This technology is based on enzymatic nucleic acids that, in the absence of a target nucleic acid, are catalytically inactive because they lack portions of the catalytic core and substrate recognition elements. In this ‘half-ribozyme’ or ‘halfzyme’ system, catalysis can occur if a specific target nucleic acid supplies the sequences required for catalysis in trans. Although many enzymatic nucleic acid motifs can be used for the halfzyme strategy, one system uses the Zinzyme motif ( FIG. 3 ) in which the substrate nucleic acid is attached to the enzymatic nucleic acid. This motif is small (about 32 nucleotides), carries modifications that confer a half-life in serum of greater than 100 hours, and has minimal target sequence requirements (5′-N3-RG-N3-3′, where N&equals;any nucleotide and R&equals;A or G). Thus, this motif is readily synthesized, has the ability to detect different sequences, and can be used directly in serum or other biological fluids. Applicant has tested the feasibility of the halfzyme approach using the Zinzyme motif and the Hepatitis C Virus genome as a model target. A synthetic oligoribonucleotide representing loop IIIB of the 5′ untranslated region (UTR), a universally conserved region of the HCV genome, activates catalysis of a rationally designed, sequence matched halfzyme. In the absence of oligoribonucleotide target no nucleic acid sensor molecule activity is detected. Other regions of the HCV 5′-UTR (see FIG. 34 ) can be similarly used in the design of other halfzymes contemplated by the invention. In this example, the halfzyme is activated by a target sequence derived from intact HCV genome. The 5′-UTR of HCV folds into a compact three-dimensional structure independent of the remaining portion of the HCV genome. To disrupt this structure so that UTR-derived loop IIIB sequences are accessible for activation of the halfzyme, a simple 20 minute pre-treatment step was inserted into the assay. Pre-treatment of the HCV 5′-UTR with a DNA oligonucleotide complementary to stem III and RNase H ( FIG. 30 a ) is sufficient to activate halfzyme catalysis to the same extent as that observed with a short synthetic oligoribonucleotide ( FIG. 30 b ). Thus, the halfzyme used in these studies can efficiently detect the presence of a conserved sequence element derived from the HCV genome. Target capture by a halfzyme is determined by the affinity of the halfzyme for its target and can be described in molarity by a dissociation constant. The value of this dissociation constant can be rationally engineered into the halfzyme, allowing 100% target capture when halfzyme used in the assay is in excess of this concentration. A primary concern of any technology aimed at detecting low concentrations of nucleic acids is its sensitivity. The halfzyme approach is unique because catalysis is only promoted in the presence of a sequence-matched target and because 100% target capture can be achieved by manipulating halfzyme concentration. Therefore, single molecule detection is theoretically possible by this approach provided that an adequate signal amplification system is in place. Given the enormous flexibility of possible signal amplification and detection systems accommodated by the technology, signal detection should not define the limit of sensitivity of this technology. In practice, the limit of sensitivity of this approach is dictated by the uncatalyzed rate of substrate cleavage promoted under the assay conditions used. Therefore, the salient issue in terms of sensitivity becomes the relative rate of catalyzed versus uncatalyzed substrate cleavage. A virtue of the system is that both the assay conditions and halfzyme activity can be manipulated to maximize this rate differential. FIG. 29 shows a non-limiting example of a “half-zinzyme” nucleic acid sensor molecule with a PEG linker that is modulated by the 5′-UTR of the Hepatitis C virus (HCV 5′-UTR). The figure shows both inactive and active forms of the zinzyme sensor molecule (SEQ ID NO. 42). In the presence of the target signaling oligonucleotide (SEQ ID NO. 43) which represents the stem loop IIIB of the HCV 5′-UTR, the zinzyme sensor demonstrates an activity increase of three logs in cleaving the reporter molecule component of the sensor molecule as shown in the graph (&plus;oligo target) as compared to the sensor molecule in the absence of the target. In the presence of the full length 350 nt. HCV 5′-UTR, the zinzyme sensor molecule demonstrates an almost one log increase in activity in cleaving the reporter molecule component of the sensor molecule. Reaction conditions: 140 mM KCl, 10 mM NaCl, 20 mM HEPES pH 7.4, 1 mM MgCl2, 1 mM CaCl2, 400 nM Nucleic acid sensor, 400 nM Target, Trace of labeled reporter (˜10 nM), 25 &mgr;l reaction volume, Nucleic acid sensor, target and reporter were heated at 75° C. for 3 min, cooled to 37° C. and cleavage initiated by the addition of MgCl2 and CaCl2. 
 Example 10 
 Nucleic Acid Sensor Ligase A ligase derived from the Bartel class I ligase (Ekland et al., 1995, Science, 269, 364-370) was prepared. Three different constructs carried various 3′ truncations. These segments were supplied in trans as oligonucleotide HCV sequence. One ligase, termed HZBART-2 showed ligation rate 107 fold above background ligation ( FIG. 33 ). Ligation reactions were performed at room temperature in 30 mM Tris, pH 7.5, 200 mM KCl, 60 mM MgCl2 and 0.6 mM EDTA. Halfzyme ligases (1 &mgr;M) with corresponding effector oligonucleotide (1 &mgr;M) were heated in water at 90° C. for 2 min and cooled at room temperature for 10 min followed by the addition of salt, buffer and 32P-labeled substrate oligonucleotide (0.1 mM final concentration). Reactions were carried out for 60 min at room temperature and stopped by the addition of 1 volume of gel loading buffer (7M urea, 100 mM EDTA) and snap cooling on ice. Products were separated on 20% denaturing polyacrylamide gel electrophoresis. 
 Other Uses The nucleic acid sensor molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of a specific RNA in a cell. The close relationship between nucleic acid sensor molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple nucleic acid sensor molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with nucleic acid sensor molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple nucleic acid sensor molecules targeted to different genes, nucleic acid target molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of nucleic acid sensor molecules and/or other chemical or biological molecules). Other in vitro uses of nucleic acid sensor molecules of this invention comprise detection of the presence of mRNAs associated with a disease-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology. In a specific example, nucleic acid sensor molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first nucleic acid sensor molecule is used to identify wild-type RNA present in the sample and the second nucleic acid sensor molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both nucleic acid sensor molecules to demonstrate the relative nucleic acid sensor molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis can require two nucleic acid sensor molecules, two substrates and one unknown sample, which are combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. 
 Additional Uses Potential usefulness of sequence-specific nucleic acid sensor molecules of the instant invention have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant describes the use of nucleic acid molecules to detect gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells. The nucleic acid sensor molecules of the invention represent a new class of therapeutic agents capable of modulating the expression of target genes, peptides, proteins, and other biologically active molecules in vivo as described herein. The therapeutic activity of nucleic acid sensor molecules of the invention can respond to both internal and external stimuli in a patient, for example the presence of a gene, pathogen, SNP, peptide, protein, RNA, metabolite, neurotransmitter, co-factor, drug, toxin, or physical stimuli such as light, gravity, temperature, and pressure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention, are defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. Other embodiments are within the following claims. 1 TABLE I Characteristics of naturally occurring ribozymes Group I Introns Size: ˜150 to >1000 nucleotides. Requires a U in the target sequence immediately 5′ of the cleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH of guanosine to generate cleavage products with 3′-OH and 5′-guanosine. Additional protein cofactors required in some cases to help folding and maintenance of the active structure. Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others. Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies &lsqb; i, ii &rsqb;. Complete kinetic framework established for one ribozyme &lsqb; iii, iv, v, vi &rsqb;. Studies of ribozyme folding and substrate docking underway &lsqb; vii, viii, ix &rsqb;. Chemical modification investigation of important residues well established &lsqb; x, xi &rsqb;. The small (4-6 nt) binding site can make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a “defective” beta-galactosidase message by the ligation of new beta-galactosidase sequences onto the defective message &lsqb; xii &rsqb;. RNAse P RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portion of a ubiquitous ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA &lsqb; xiii &rsqb;. Reaction mechanism: possible attack by M 2&plus; -OH to generate cleavage products with 3′-OH and 5′-phosphate. RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates. Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA &lsqb; xiv, xv &rsqb;. Important phosphate and 2′ OH contacts recently identified &lsqb; xvi, xvii &rsqb; Group II Introns Size: >1000 nucleotides. Trans cleavage of target RNAs recently demonstrated &lsqb; xviii, xix &rsqb;. Sequence requirements not fully determined. Reaction mechanism: 2′-OH of an internal adenosine generates cleavage products with 3′-OH and a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Only natural ribozyme with demonstrated participation in DNA cleavage &lsqb; xx, xxi &rsqb; in addition to RNA cleavage and ligation. Major structural features largely established through phylogenetic comparisons &lsqb; xxii &rsqb;. Important 2′ OH contacts beginning to be identified &lsqb; xxiii &rsqb; Kinetic framework under development &lsqb; xxiv &rsqb; Neurospora VS RNA Size: ˜144 nucleotides. Trans cleavage of hairpin target RNAs recently demonstrated &lsqb; xxv &rsqb;. Sequence requirements not fully determined. Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OH ends. Binding sites and structural requirements not fully determined. Only 1 known member of this class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see text for references) Size: ˜13 to 40 nucleotides. Requires the target sequence UH immediately 5′ of the cleavage site. Binds a variable number nucleotides on both sides of the cleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OH ends. 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent. Essential structural features largely defined, including 2 crystal structures &lsqb; xxvi, xxvii &rsqb; Minimal ligation activity demonstrated (for engineering through in vitro selection) &lsqb; xxviii &rsqb; Complete kinetic framework established for two or more ribozymes &lsqb; xxix &rsqb;. Chemical modification investigation of important residues well established &lsqb; xxx &rsqb;. Hairpin Ribozyme Size: ˜50 nucleotides. Requires the target sequence GUC immediately 3′ of the cleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage site and a variable number to the 3′-side of the cleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OH ends. 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent. Essential structural features largely defined &lsqb; xxxi, xxxii, xxxiii, xxxiv &rsqb; Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection &lsqb; xxxv &rsqb; Complete kinetic framework established for one ribozyme &lsqb; xxxvi &rsqb;. Chemical modification investigation of important residues begun &lsqb; xxxvii, xxxviii &rsqb;. Hepatitis Delta Virus (HDV) Ribozyme Size: ˜60 nucleotides. Trans cleavage of target RNAs demonstrated &lsqb; xxxix &rsqb;. Binding sites and structural requirements not fully determined, although no sequences 5′ of cleavage site are required. Folded ribozyme contains a pseudoknot structure &lsqb; xl &rsqb;. Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OH ends. Only 2 known members of this class. Found in human HDV. Circular form of HDV is active and shows increased nuclease stability &lsqb; xli &rsqb; i Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5-7. ii Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17. iii Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71. iv Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80. v Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70. vi Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58. vii Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9. viii Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12. ix Zarrinkar, Patrick P.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8. x Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D.C.) (1995), 267(5198), 675-9. xi Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5′-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11. xii Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994), 371 (6498), 619-22. xiii Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol. Chem., 247, 5243-5251 (1972). xiv Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA enzyme. Science (Washington, D.C., 1883-) (1990), 249(4970), 783-6. xv Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci, USA (1992) 89, 8006-10. xvi Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210-18. xvii Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2′- hydroxyl-base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A. (1995), 92(26), 12510-14. xviii Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25. xix Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77. xx Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38. xxi Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2′-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70. xxii Michel, Francois; Ferat, Jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61. xxiii Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of 2′-hydroxyl groups within a group II intron active site. Science (Washington, D.C.) (1996), 271(5254), 1410-13 xxiv Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1), 31-49. xxv Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76. xxvi Scott, W. G., Finch, J. T., Aaron, K. The crystal structure of an all RNA hammerhead ribozyme: A proposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-1002. xxvii McKay, Structure and function of the hammerhead ribozyme: an unfinished story. RNA, (1996), 2, 395-403. xxviii Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. U.S Pat. No. 5,633,133. xxix Hertel, K. J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic framework for the hammerhead ribozyine reaction. Biochemistry, (1994) 33, 3374-3385. Beigelman, L., et al., Chemical modifications of hammerhead ribozymes. J. Biol. 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(1993), 21(18), 4253-8. 2 TABLE II Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNA A. 2.5 &mgr;mol Synthesis Cycle ABI 394 Instrument Phosphoramidites 6.5 163 &mgr;L 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole 23.8 238 &mgr;L 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 &mgr;L 5 sec 5 sec 5 sec N-Methyl 186 233 &mgr;L 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 &mgr;L 100 sec 300 sec 300 sec Acetonitrile NA 6.67 &mgr;L NA NA NA B. 0.2 &mgr;mol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 &mgr;L 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 &mgr;L 45 sec 233 min 465 sec Acetic Anhydride 655 124 &mgr;L 5 sec 5 sec 5 sec N-Methyl 1245 124 &mgr;L 5 sec 5 sec 5 sec Imidazole TCA 700 732 &mgr;L 10 sec 10 sec 10 sec Iodine 20.6 244 &mgr;L 15 sec 15 sec 15 sec Beaucage 7.7 232 &mgr;L 100 sec 300 sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 &mgr;mol Synthesis Cycle 96 well Instrument Equivalents:DNA/ Amount: DNA/2′-O- Wait Time* 2′-O- Reagent 2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66 40/60/120 &mgr;L 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210 40/60/120 &mgr;L 60 sec 180 min 360 sec Acetic Anhydride 265/265/265 50/50/50 &mgr;L 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50 &mgr;L 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500 &mgr;L 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 &mgr;L 30 sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec Acetonitrile NA 1150/1150/1150 &mgr;L NA NA NA *Wait time does not include contact time during delivery. 3 TABLE III Nucleic acid sequences Seq.ID RPI&num; Name Sequence No. 15404 R-2.1 & 2.7 AAGCACUAAUGGAGA 1 17161 R-3.1 AAGCACUAACAGUAA 2 15400 SM-2.1 UCUCCAU CUGAUGAGGCCGUUAGGCCGAA AGUGCUUG 3 17159 SM-2.7 UCUCCAU CUGAUGAGGCCGUUAGGCCGAA AGUGCUUG CGAGUG 4 17160 SM-3.1 UUACUGU CUGAUGAGGCCGUUAGGCCGAA AGUGCUUG CGAGUG 5 17162 s-2.1 caagcacuuucucaucagauggaga 6 17163 s-2.2 cacucgcaagcacuuucucaucagauggaga 7 17164 s-2.3 cacucgcaagcacccuaucaggcagua 8 17165 s-2.4 cacucgcaagcacccuaucagguggaga 9 15405 T-2a UACUGCCUGAUAGGGUGCUUGCGAGUG 10 UPPER CASE &equals; RIBO lower case &equals; 2′-O-methyl