MXPA99001950A - Catalytic nucleic acid and its medical use - Google Patents

Abstract

The present invention concerns nucleic acid molecules, or complex of molecules, which have no catalytic activity in the absence of a specific co-factor, and feature catalytic activity only in the presence of a specific co-factor. The present invention further concerns methods for the production of said molecules, or complex of molecules, particularly utilizing in vitro evolution, as well as their usage in diagnostics and therapeutics.

Description

CATALYTIC NUCLEIC ACID AND ITS MEDICAL USE DESCRIPTION OF THE INVENTION The present invention relates to molecules and methods for detecting specific agents in a medium. Ribozymes are typically RNA molecules which have catalytic activities similar to enzymes usually associated with cleavage, splicing or ligation of nucleic acid sequences. The typical substrates for the catalytic activities of ribozymes are RNA molecules, although ribozymes can catalyze the reaction in which DNA molecules (or can even be proteins) serve as substrates. The ribozymes which are active intracellularly work in cis, catalyzing only a renewal, and are usually self-modified during the reaction. However, ribozymes can be machined to act in trans, in a truly catalytic way, with a renewal greater than one and without being self-modifying. Two distinct regions can be identified in a ribozyme: the binding region which gives its specificity to the ribozyme through hybridization to a catalytic nucleic acid sequence (and possibly also to specific proteins), and a catalytic region which gives to the ribozyme the activity of cleavage, ligation or splicing. It has recently been proposed to use ribozymes for the purpose of treating genetic diseases or disorders by cleaving a target RNA, such as a viral RNA or messenger RNA transcribed from genes that must be blocked. This is proposed as an alternative to block RNA transcription by the use of antisense sequences. Due to the catalytic nature of the ribozyme, a single ribozyme molecule cleaves many target RNA molecules and therefore therapeutic activity is obtained in relatively lower concentrations than those required in an antisense treatment (WO 96/23569.) The use of ribozymes for diagnostic purposes it has seldom been mentioned WO 94/13833 describes a method for detecting nucleic acid molecules in a solution inspected by a specific ribozyme molecule having two regions, one complementary to the nucleic acid sequence to be detected, and The ribozyme is capable of binding specifically and reversibly both to a targeted target nucleic acid sequence and to the target co-target.When both target or co-target is linked, the ribozyme suffers. a conformational change which leads him to be active and be able to split the brand e co, and the free mark can be detected. After cleaving the target, the ribozyme is able to re-associate with an additional target, cleave more tags and produce more detectable signals. WO 94/13791 relates to an adjustable ribozyme molecule which, after binding to the ligand, alters its activity in a target RNA sequence. Again, as in WO 94/13833, the link to the target causes conformational change in the ribozymes which leads to being active. An example for such a change is the presence in the ribozyme of a redundant sequence which masks the core region of the ribozyme. Only after the link of the target sequence to the redundant sequence, the nucleus becomes masked, and in this way active. U.S. Patent 5,472,840 (Stqfano, JE) discloses a nucleic acid sequence comprising the MDV-1 motif (capable of autocatalytic replication in the presence of the Q-beta replicase enzyme) which becomes active only when it forms a specific structure, comprising the GAAA sequence, with a target nucleic acid molecule. The Stefano ribozyme is prepared by modifying an existing ribozyme and features only the cleavage activity in the presence of a very restricted range of target molecules. Lizardi, M.P. and Helena Porta (Biotechnology 13: 161-164 (1995)) describes an allosteric hammer head ribozyme which is inactive a priori, and is activated by specific interaction with allosteric DNA effector that is complementary to a one-stranded circuit in the RNA strand ribozyme. This publication describes a ribozyme which becomes active when a part of which becomes double-stranded. Previous publications describe ribozymes which become catalytically active, due either to a conformational change, or due to a hybridization reaction which leads to double chain. These types of reactions can also occur spontaneously, for example, if a ribozyme is inactivated due to the presence of a redundant sequence which masks its core region, this redundant sequence can be either broken or open, even in the absence of the target, and in this way the ribozyme will become catalytically active even without an objective. Spontaneous inversion to an active state, of course, leads to ribozymes to be impractical for diagnostic purposes. Patent applications Israel 112799 and 115772 (corresponding to PCT / US96 / 02380) describe methods for the detection of catalytically active ribozymes in a medium where the catalytically active ribozyme is typically used as a reporter for the presence of other biomolecules in a test sample. According to the methods described in these applications, the catalytically active ribozyme, if present in a test medium, produces a cascade reaction in which more catalytically active ribozymes are produced in a positive feedback form in one of several specified modes in these requests. The catalytically active ribozyme can be produced or activated only in the presence of a test molecule. In the following, the term "nucleozyme" will be used to denote an oligonucleotide or a complex formed between an oligonucleotide and a nucleic acid sequence or between an oligonucleotide and another molecule for example an oligonucleotide, a protein or a polypeptide, etc., the which has a catalytic activity. The term "protonucleozyme" will be used to denote a nucleic acid molecule or a complex of two or more such molecules, which has a priori no catalytic activity but which becomes catalytically active after the formation of a complex with a co factor. A proto nucleozyme is in fact a nucleozyme with a lost component, which is a missing component completed by the co-factor. The complex between the proto nucleozyme and the co-factor can also sometimes be referred to as a "catalytic complex" and is in fact a nucleozyme since it has catalytic activity. The protonucleonezyme may consist of deoxynucleotides (DNTP), ribonucleotides (rNTP) as well as other nucleotides such as 2"-O-methylnucleotides, or any combinations thereof.

The term "catalytic activity" is understood to encompass all possible catalytic activities, including cleavage, ligation, desempalme (cleave both ends of a short nucleic acid sequence to remove it from a larger sequence and ligate the ends of the cut), spliced (cleaving by opening a nucleic acid sequence, inserting another short nucleic acid sequence and ligating the ends of the cut), rearrangement, as well as additional catalytic activities such as phosphorylation, kinase-like activity, addition or deletion of other chemical portions, bio-staining , filling of lost nucleotide spaces, polymerization, etc. The term "co-factor" will be used to denote a molecule or a portion within a molecule (for example, a certain DNA sequence within a larger DNA molecule), which forms a complex with a proto nucleozyme to produce a catalytic complex , that is, an active nucleozyme. The co-factor completes an absent portion of the proto nucleozyme in such a way that it can become catalytically active, and changes to a nucleozyme. The present invention relates to novel proto nucleozymes and their use. The pro nucleozymes of the invention have substantially no catalytic activity, as they lose a critical component, which is essential for catalytic activity, the lost component is completed by the co-factor. In other words, in order to become catalytically active, the proto nucleozymes need to form a complex with the co-factor, in order to form a catalytic complex (the nucleozyme) which exerts catalytic activity. The term "that has substantially no catalytic activity" is meant to denote that the proto nucleozymes possesses either no catalytic activity or possesses catalytic activity which is much smaller (typically by several orders of magnitude) than that of the catalytic complex. As indicated above, there are known ribozymes which form a complex with a factor (also named objective), for example, a protein or other nucleic acid sequence which causes the ribozymes to undergo conformational change so that the catalytic activity of the ribozyme becomes more pronounced. However, in distinction from such ribozymes of the prior art, the proto nucleozyme of the invention, a priori loses an essential component and the co-factor provides the lost component. Examples of such lost components are sequences in the core region of the nucleozyme. The co-factor may be a protein which fills a space between two ends of nucleic acid strand of the nucleozyme or which fills such two ends; or it may be a nucleic acid sequence capable of joining between two free ends of the proto nucleozyme thus leading to a space.

Given the structural and functional characteristics of the proto nucleozyme, when it is in solution, it does not have the capacity to spontaneously become a catalytically active form, since its activation does not depend solely on a conformational change which can occur spontaneously but on a completion of a lost component. This feature is an additional distinction of inactive ribozymes of the prior art, which has the ability to spontaneously undergo conformational change, albeit at a low speed, and exert some catalytic activity even in the absence of a co-factor. Thus, unlike the inactive ribozymes of the prior art, such as those described above in the background section of the invention of this specification, when the ribozymes of the invention are used, there is substantially no background activity in the absence of the co-enzyme. factor. For example, when the proto nucleonzymes of the invention are used in the diagnosis, there is essentially no "noise", that is, there is a very high ratio of signal to noise and there are virtually no false positive results. For another example, when used in therapeutics, the proto nucleozymes of the invention, will exert a very high target specificity and only in the presence of an appropriate target, the catalytic complex (the nucleonezyme) will be formed and will exercise its activity. The proto nucleozymes of the invention can be prepared either by in vitro evolution, in a form to be described later, or by means of a rational design by synthesis of nucleic acid, for example, using a sequence of a known ribozyme and leaving a space of several nucleotides lost in their core region. The present invention provides a proto nucleozyme, which is a nucleic acid molecule or a complex of nucleic acid molecules, which essentially has no catalytic activity but can form a complex with a cofactor to form a nucleozyme which possesses catalytic activity, the proto nucleozyme lacks an essential component for the catalytic activity of the nucleozyme and the cofactor provides the component. The component that is lost in the proto nucleozyme and that is provided by the cofactor for the conversion of the proto nucleozyme into a catalytically active nucleozyme can be a segment of lost nucleic acid of one or more nucleotides (hereinafter referred to as "space") or a missing link between two nucleotides (hereinafter referred to as "notch"), etc. In this way the cofactor can be a segment of nucleic acid which can bridge the space or notch. For this purpose, such a cofactor nucleic acid segment has sequences which can hybridize with complementary sequences in the two strands on both sides of the space or notch and in the case of a space, it can also comprise the missing sequence. It should be noted that sometimes the cofactor segment can hybridize and provide a bridge between two nucleic acid lengths of the proto nucleozyme different from those that immediately pass through the space or notch. In addition, the cofactor can also sometimes be a macromolecule such as a protein, an oligosaccharide, etc., which can form a complex with the two terminal nucleic acids that pass through the space and notch and therefore lead to the junction of the two . The space or notch can be between two different oligonucleotide chains which are functionally linked together by the cofactor or can be between ends of the same oligonucleotide wherein the cofactor in such produces the function of a functional closed oligonucleotide circuit. Such proto nucleozymes can be obtained ("machined") by in vitro evolution or by means of rational design. The specific cofactor can be any molecule which can chemically interact with nucleotides in any form, for example by the formation of hydrogen bonds, by electrostatic bonds, by Van der Waals interactions, etc. Such molecules include, proteins, peptides, oligopeptides, antibiotics, phosphate nucleotides such as ATP, GTP, cyclic AMP, and others, carbohydrates, lipids, nucleic acid sequences (DNA or RNA sequences), etc. Sometimes it may be desirable to modify the proto nucleozyme in order to increase its resistance to the nuclease, which can be achieved by replacing some of the natural nucleotides, with non-natural nucleotides such as 2"-0-methylnucleotides (Usman et al., Nucleic Acids Symposium Series, 31: 163-164, 1994) A currently preferred, but not exclusive, use of the proto nucleozymes of the invention is as a diagnostic tool for detecting the presence of a certain agent in a medium. For this purpose, a proto nucleozyme will be designed in such a way that the agent will be a specific cofactor.When the agent is present in the tested medium, a catalytic complex will be formed and the catalytic activity, which will then be tested, will then serve as calibration. for the presence of the agent in the medium For this preferred embodiment, nucleozymes are preferred which have absolutely no catalytic activity or tasting activity lithic is not measurable, and then catalytic activity is essentially manifested only after the formation of the catalytic complex. In such a case, there will be an essentially zero background signal and a detection of the catalytic activity will then be an unambiguous indication ("all or nothing") of the presence of the agent (which is the specific cofactor) in the medium. Where a proto-nucleozyme having some small catalytic activity is used, the level of catalytic activity will have to be determined in order to test the presence of the agent in the medium. The present invention further provides a method for the detection of an agent in a medium, which comprises the following steps: (a) Contacting the medium with a proto nucleozyme, wherein the agent is the specific cofactor required for the formation of a catalytic complex; (b) Providing or maintaining proportions that allow the catalytic activity of the catalytic complex; and (c) Testing the presence of products of the catalytic activity in the medium, such presence indicating the presence of the agent in the medium. An example of the type of catalytic activity that can be determined in stage (c) above, is the excision of the nucleic acid sequence for example, the catalytic complex can excise, from an immobilized nucleic acid substrate, a small fragment carrying a detectable label. Then the detection of a free label in the reaction medium is indicative of the activity of the catalytic complex, which is in turn an indication of the presence of the agent tested in the medium. Other examples of catalytic activity that can be determined in step (c) are ligation, disassembly, assembly. etc. All these three previous catalytic activities result in a change in the distance between two nucleotide sequences which are the substrates for the reaction in the ligation and disassembly two sequences are joined and in the assembly two sequences are separated. One of these sequences can carry, for example, a portion containing a fluorescent compound (e.g. rhodamine) and the other sequence can carry a portion containing a fluorescence detection compound (e.g. fluorescein) or a portion that increases fluorescence; the change in the distance between the two sequences can then be determined by measuring the change in fluorescence emission, which stops (in the case of a fluorescence retainer) or increases (in the case of a fluorescence enhancer) when the two sequences are adjacent to each other, and is increased or stopped respectively when the two sequences are separated from each other. A preferred method for testing products with catalytic activity is by means of the cascade reaction of self-amplifying ribozyme described in the international application PCT / US96 / 02380 and corresponding to the patent applications of Israel 112799 and 115772, the contents of which they are incorporated herein by reference. Very briefly, the catalytic complex (or the nucleozyme) once formed catalyzes a reaction which leads to the activation of inactive nucleozymes a priori (identified in the applications referred to above as ribozymes), and those catalytically active nucleozymes then they act catalytically to activate additional ribozymes and successively, in a self-amplifying positive feedback cascade reaction. This amplified signal thus serves as a calibration for the presence of the catalytically active initial nucleozyme in the medium; the presence of such an initially catalytically active nucleozyme is in turn an indication of the presence of the specific cofactor which is the agent to be detected which forms a complex with the proto nucleozymes to produce the catalytic complex of the initial ribozyme in the medium . The unique ability of proto nucleozymes to be catalytically active in the presence of a specific cofactor also makes them useful as therapeutic agents in certain target therapies. In some diseases, cells carrying diseases differ from normal cells in the expression of certain expression products. This is the case, for example, in viral diseases in which diseased cells differ from other cells by the fact that they express viral proteins. The proto nucleozyme can be machined in such a way that after the formation of the complex with a specific viral protein (which will be the specific cofactor), it will have a certain cytotoxic catalytic activity or its catalytic activity will produce a cytotoxic reaction product. The catalytic activity can be objectified to a specific nucleic acid sequence or to a gene expression product in such a way that the expression of the unwanted gene (for example of a viral origin or of cellular origin) will be inhibited. For example, the catalytic activity can cleave the sequence of an mRNA thereby inhibiting the production of an undesired protein. Such proto nucleozymes can be provided in formulations that allow their entry into the cell, for example a liposome formulation, and while proto nucleozymes enter many cells, they will assume their catalytic activity and in this way will destroy only the desired cell population for example. , or inhibit the expression of unwanted genes only in cells that carry viruses. In addition to viral diseases, there are also other diseases where diseased cells express or contain products which are not found in normal cells or are found in late cells in only small amounts. Such is the case, for example, in cancer; in a variety of infectious diseases other than viral diseases; in various genetic diseases wherein diseased cells in diseased individuals contain a mutant gene and abnormal expression products; etc. Accordingly, the present invention provides a method for selectively destroying a population of specific cells, which contain or express a specific gene, the method comprising: (a) Providing a proto nucleozyme of a nucleozyme, in which the nucleozyme has catalytic activity which is cytotoxic to the cell or has cytotoxic reaction products, and where the agent is a specific cofactor for the proto nucleozyme; (b) Insert the proto nucleozyme into the cells that contain or express the agent or apply the proto nucleozymes to the tissue suspected to contain a population of cells that contain or express the agent, under conditions or using a vehicle, to insert the proto nucleozyme In the case where the proto nucleozyme is proposed to destroy cells containing viruses, the co-factor may be, for example, a virus-associated protein, for example in the case of HIV, the HIV-TAT protein. The cytotoxicity of the nucleozyme can be manifested in a variety of ways. For example, a nucleozyme, once it becomes active, can catalyze a reaction that produces the formation of cytotoxic reaction products. Such cytotoxic reaction products can be, for example, reaction products which competitively inhibit one or more metabolic or catabolic pathways in the cells. For another example, the catalytic activity of the cytotoxic ribozyme can itself produce the decomposition of substances which are produced inside cells or transported in the cell through cell membranes, which are essential for cell growth and survival. . Other examples can be nucleozymes which degrade mRNA, either in general or such that they have specific sequences, nucleozymes which decompose mRNA, nucleozymes which degrade a variety of proteins, and others. The present invention also provides a method for inhibiting the expression of undesired genes in cells, comprising: (a) providing a proto nucleozyme of a nucleozyme, in which the nucleozyme has catalytic activity which is objectified in a nucleic acid sequence specific or gene expression product for such that once within a cell, undesired gene expression will be inhibited; (b) insert the proto nucleozyme into cells that contain or express the gene, or apply the proto nucleozyme to tissues suspected of having a population of cells that contain or express the gene, under conditions or using a vehicle to insert the proto nucleozyme to the cells. For a modality of this last aspect, the cofactor of the proto nucleozyme is either a nucleic acid sequence of such a gene, or a product of transcription or expression of the gene. The inhibition of DNA expression by the latter method can be, for example, by decomposition of the specific mRNA, decomposition of a gene expression product, or decomposition of regulatory substances that regulate the expression of the gene. Prior methods where proto nucleozymes are inserted into cells may be useful in human therapy. The proto nucleozymes within the working structure of such therapies can be administered in vivo or they can be contacted with the ex vivo cells, into which the cells are then inserted back into the body. According to its therapeutic aspect, the present invention further provides a pharmaceutical composition for example for use in the destruction of a specific cell population, which comprises the proto nucleozyme and a pharmaceutically acceptable carrier. The carrier will preferably be of a type which allows the insertion of the proto nucleozyme into the cells, for example a liposome carrier or any other carrier known in the art. As explained above, the nucleozyme of the invention can be produced by rational design or by the in vitro evolution form. As will be further explained later, in vitro evolution does not require any advance decision on the exact mechanism of catalytic complex formation between the proto nucleozyme and the specific cofactor. The exact mechanism of activation develops during such evolution in vitro. The term "in vitro evolution" refers to a method for generating and selecting nucleic acid sequences (which may be DNA or RNA sequences, or sequences comprising both dNTP and rNTP comprising nucleotides found naturally or unnaturally) having desired characteristics, without an a priori knowledge of the exact construction of the sequence of nucleic acid selected. Typically, this involves production of a large number of random or partially random nucleic acid sequences, or complexes comprising one or more nucleic acid sequences, then providing the conditions required for the selection of those sequences which highlight a specific property ( for example, add a protein and select only those nucleic acid sequences which show a catalytic activity only in the presence of the protein). Then the selected nucleic acid sequences are amplified, for example, by polymerase chain reaction (PCR), and then the selection and amplification steps are repeated in many cycles, for example in the range of 10 to 100, resulting in an enrichment of the reaction mixture for those nucleic acid sequences or complexes which highlight the desired property. Sometimes it is useful to progressively increase the threshold criteria for selection in each turn of a selection and amplification. For example, as soon as the stages of in vitro development proceed, only species that have progressively greater affinity to the desired protein are selected. The in vitro selection methodologies for sounding RNA function and structures are summarized in a review by Conrad, R.C., Molecular Diversity 1: 69-78 (1995) and have been studied in models for autocatalytic replication of RNA by Giver et al. (G.R Fleischaker et al. (Editor), Self-production of Supramolecular Structures 137-146, (1994), Klewer Academic Publishers). In this way the present invention provides a method of in vitro evolution for the production of a proto nucleozyme of the invention which after the formation of a catalytic complex with a specific cofactor manifests a specific catalytic activity which is either absent or substantially minor in the proto nucleozyme. The method which has both positive and negative selection steps, comprises: (a) preparing a panel of different nucleic acid molecules, at least a part of the sequence in the honeycomb molecules is a random or partially random sequence of such that the part has a different sequence in different molecules of the panel; (b) add the cofactor to the panel and incubate under conditions that allow for catalytic activity; (c) separating between the nucleic acid molecule of the panel that characterizes the catalytic activity and which does not, to obtain a first selected panel of nucleic acid molecules that characterize the catalytic activity; (d) amplifying the nucleic acid molecules of the first selected panel; (e) incubating the first selected panel with a reaction mixture, which lacks the cofactor, under conditions that allow the catalytic activity; (f) eliminating the nucleic acid molecules that highlight the catalytic activity, whereby a second selected panel of nucleic acid molecules lacking the removed nucleic acid molecules is obtained; and (g) repeating steps (b) - (f) over a plurality of cycles, for example, about 10-100 cycles, to obtain the proto nucleozyme. For one embodiment, the molecules in the panel are comprised of an entirely random sequence with the exception of two short flanking sequences for binding of the PCR promoters. For another embodiment of the invention, the molecules in the panel are constructed based on a known nucleozyme sequence. For example, the molecule may be comprised of a constant sequence, derived from an enzyme linked to a semi-random or random sequence. Usually, a random sequence can be prepared, for example, using a nucleic acid synthesizer. In the above method, the positive section stages (steps (b) - (d)) precede the negative selection steps (steps (e) - (f)), which is a preferred embodiment of the invention. However, it is also possible to carry out the method in a reverse sequence, that is, first the negative selection stages and then the negative selection stages. Another example is a panel of molecules, all in base, to a sequence of known ribozymes and wherein the nucleotides in the total molecule or in a part thereof have a certain probability (for example 70-99%) of being identical to the known sequence. Such a semi-random sequence means that each nucleotide has a certain probability of being different than the corresponding nucleotide in the original ribozyme (for example this probability is 30-1%, respectively). The preparation of the panel begins with a known nucleozyme sequence and then the semi-random sequences are created based thereon by replacing some of the nucleotides with different, random nucleotides ("implant"). The level of implant is typically approximately 1-30%. The implant can also be performed in each cycle or once in several cycles so it is slowly brought to the development of a proto nucleozyme with a high specificity towards the cofactor. An example of how such a selection is made can be represented in the specific case where the catalytic activity is cleaved in cis. Initially, all panel members can be immobilized on a solid support. Those sequences which possess the cis-cleavage catalytic activity in the positive selection stages ie in the presence of the specific cofactor (steps (b) - (d)) are released into the reaction mixture and are collected and amplified, for example using RCP After amplification, the collected sequences are allowed to cleave in the absence of the specific cofactor, and those released in the negative selection stages (stages (e) - (f)), are eliminated. In the selection stages, it is sometimes desired to apply the exact conditions in which the proto nucleozyme will eventually be used. Where the proto nucleozyme is proposed for use within the working structure of a diagnostic assay, the selection conditions will typically be similar to those in the diagnostic assay. For example, where the cofactor is a blood protein, the proto nucleozyme is proposed for use in a diagnostic assay for detection of such a protein in the blood, selection (both positive and negative) in a reaction medium can be performed. it mimics the conditions (both chemical and temperature environmental) that will exist in the assay, for example composition similar to blood and room temperature. In the following, a method, of preferred in vitro evolution, useful in the preparation of protonucleozymes of the invention will be described. However, it should be noted that this method of in vitro evolution is an example, and the invention is not limited to the preparation of proto nucleozymes for this specifically described method, nor to in vitro evolution preparation methods in general. A preferred in vitro evolution method. A major problem in an in vitro evolution method for the preparation of a proto nucleozyme having the above defined characteristics, is the difficulty in separating proto nucleozyme candidates which highlight the catalytic activity only in the presence of the specific cofactor, and those which highlight the catalytic activity both in the presence of the cofactor as well as in its absence, or in the presence of other agents. In other words, there is a difficulty in eliminating unwanted candidates. Many times, especially in the initial cycles of in vitro evolution, there are molecules that highlight catalytic activity also in the absence of the specific cofactor (molecules which must be eliminated), but they do it in a very low efficiency; in this way the stage of negative selection in which such molecules are eliminated, may require extremely long incubation times, rendering the total process of in vitro evolution impractical (which requires multiple cycles of negative selection stages) . Under standard incubation times only a small percentage of the molecules which are acting but highlight a catalytic activity also in the cofactor's presence, but which must be eliminated in the negative selection stages, will actually characterize the catalytic activity in time. of incubation given. This may result in incomplete removal of unwanted molecules in the negative selection stage and may eventually lead to false positive results when the proto nucleozymes are used in the diagnosis. In principle, the molecules eliminated during the negative selection stage can be collected, and can be used to "take out", by hybridization, other identical molecules of the same species of proto nucleozyme candidates which must have been eliminated, but due to the Short incubation time does not have an opportunity to characterize the catalytic activity. However, the sequences of candidate proto nucleozymes which highlight the catalytic activity both in the presence of the specific cofactor and in the presence of other agents (to be eliminated), and the sequences of the protonucleozyme candidates which highlight the desired property in the presence of the cofactor (to be maintained), they can be very similar (the difference in the sequence of the two can be very small in relation to the large size of the proto nucleozyme candidate) so that it can be practically impossible to distinguish between the two by hybridization. The solution to this problem is the addition of a random tag sequence to each of the proto nucleozyme candidates in the initial oligonucleotide mixture comprising a panel of different proto nucleozyme candidates, such that after amplification, all the Oligonucleotides of the same species (ie, all the amplified molecules of an original progenitor molecule) have the same random tag sequence. This tag sequence is not part of the functional sequence of the nucleozyme (that is, the tag is a redundant sequence). The functional sequence is the part that when the proto nucleozyme reacts with the cofactor is the part that imparts the catalytic activity. The tag sequence is linked to a variable sequence (candidate to involve the functional sequence). While the variable sequence of different oligonucleotide species may be similar (eg since all variable sequences are "implanted" from an original known sequence of a ribozyme); the tag sequence is completely different from one species of oligonucleotide to the other. In this way, when, after a negative selection stage, the oligonucleotides are collected which highlight the catalytic activity in the absence of the cofactor in order to be eliminated, it is then possible to first excise the cleavable sequence, and thus collect separately only the tag sequences of the unwanted oligonucleotides. These tag sequences, which are random, have a very high probability of being different in each species of oligonucleotide molecules, and thus can be used to effectively "take out" complementary tag sequences from other members of the unwanted oligonucleotide species to be eliminated and in this way are able to discover "latent" oligonucleotides which, if the incubation time is sufficiently long, can highlight the catalytic activity even in the cofactor's presence. Since this "extruded" that is to say elimination process is based only on the hybridization of the single tag sequence (which is different from the tag sequences of the other species), the fact that the variable sequence of the oligonucleotides which are to be eliminated is very similar to the sequence variable of the oligonucleotides which must be maintained, does not interfere with the selection process. In this way, the in vitro evolution method, to obtain the proto nucleozyme of the invention, which together with a cofactor forms a catalytic complex having catalytic activity imparted by a functional sequence of the proto nucleoside, comprises: (a) Preparation a mixture of different candidate oligonucleotides to involve the proto nucleozymes each of which comprises a variable sequence that is a candidate to involve in the functional sequence and a tag sequence, each of the two sequences being different from the corresponding sequences of different oligonucleotides in the mixture, the variable sequence and the tag sequence that is linked to at least one cleavable sequence; (b) Process the mixture through negative and positive selection steps, each step optionally followed by an oligonucleotide amplification step, with at least one step of positive selection and at least one stage of negative selection, these steps that they comprise: (ba) a positive selection step comprising applying the specific cofactor and separating between the oligonucleotides they have and those that do not have catalytic activity to obtain a first selected mixture comprising a first group of oligonucleotides that highlight catalytic activity in the presence of cofactor; (bb) amplifying the first group of oligonucleotides in the first selected mixture to produce a plurality of copies of each of the first group of oligonucleotides to obtain a first amplified mixture; (bca) apply a reaction mixture lacking the specific cofactor and the separation between a second group of oligonucleotides which have no catalytic activity in the absence of the cofactor and a third group of oligonucleotides having the catalytic activity in the absence of the cofactor, to obtain a second selected mixture comprising the second group of oligonucleotides and a third selected mixture comprising the third oligonucleotide groups; (bcb) amplifying the third set of oligonucleotides in the third selected mixture to produce a plurality of copies of each of the third oligonucleotide group to obtain a second amplified mixture; (bcc) cleaving the cleavable sequence of the oligonucleotides in the second amplified mixture, separating between the variable and tag sequences and collecting the tag sequences; (bed) contacting the collected tag sequences with the second selected mixture under severe hybridization conditions and removing hybridized oligonucleotides and other oligonucleotides from the second mixture, whereby a fourth selected mixture of oligonucleotides lacking essentially oligonucleotides is obtained which they have catalytic activity in the absence of the cofactor; where the positive selection step precedes the negative selection step, the positive selection step is applied to the mixture prepared in step (a) and the negative selection step is applied in the first amplified mixture; and where the negative selection step precedes the positive selection step, the negative selection step is applied to the mixture obtained in step (a) and the positive selection step is applied in the fourth mixture. Preferably, the positive selection step must precede the negative selection stage. It is preferred that in the positive selection stage of each consecutive cycle the conditions become more and more severe, for example, shorter test times, harder sample preparation conditions etc .; while in the negative selection stage they become less and less severe, allowing even those oligonucleotides with a very small catalytic activity in the absence of the cofactor, to exert their catalytic activity (and thus be eliminated) for example using larger incubation, etc. Where the selection step is repeated a plurality of times, each stage or several steps may be performed in the presence of another agent which is not the cofactor. For example, where the cofactor is a certain protein, each stage in the negative selection can be incubated in the presence in the medium of another non-cofactor protein that is present in a different negative selection step. It should be noted that by the previous in vitro evolution method, the proto nucleozymes that are converted into nucleozymes by simple conformational change are eliminated. Such proto nucleozymes can become a catalytically active form (ie, in the nucleozyme) even spontaneously, without the cofactor, albeit in a very low probability. Due to the highly sensitive and unique negative selection stage used in the previous in vitro evolution method, all the proto nucleozymes are identified and eliminated, retaining only those proto nucleozymes that do not have essentially catalytic activity and need the cofactor, which completes the lost component, to become catalytically active. The present invention will now be illustrated in the following non-limiting examples with an occasional reference to the accompanying drawings: BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic representation of a method for preparing a pro nucleozyme of the invention, wherein the specific cofactor is a protein; Figure 2A, 2B, and 2C show nucleic acid constructs which is a proto nucleozyme where the cofactor is a nucleic acid sequence. Figure 3 shows an improvement in the negative selection stage of the method of Figure 1; Figure 4A shows a three-dimensional structure of the ribozyme Group intron I; Figure 4B shows the conserved nucleotide structure and conserved structure of the ribozyme intron Group I; Figure 4C and 4D show several modifications of the Sun Y ribozyme; Figure 4E shows several regions of the Sun Y ribozyme; Figure 5 shows a comparison in the production of ligation product (%) between different Sun Y constructions; Figures 6 and 7 show electrophoresis gels of the ribozyme ligation products in different experiments obtained using various proportions of ribozymes to substrates taken at different times; Figure 8 shows a graph of the concentration of ligation product (nM) created by a Sun Y P910 ribozyme at different ribozyme to substrate ratios as a function of time. Figure 9 shows a double reciprocal plot (or Lineweaver Burt) based on the parameters in Figure 8; Figure 10 shows the effects of various concentrations of SDS, GITC, urea and phenol (reextracted chloroform) or ribozyme reaction; Figure 11 shows the effect of various concentrations of the RCP inhibitors isopropanol, EtOH, urea, GITC and SDS in the ribozyme reaction; Figure 12 shows the effect of phenol (either not extracted or reextracted with chloroform) and a commercial specimen kit, in the ribozyme reaction; Figure 13 shows the effect of varying the concentrations of GITC in the ribozyme reaction (expansion of Figure 10); Figure 14 shows the effect of varying concentrations of ATA, Spdermidine, DMF, DMSO, Triton, and Tween 40 in the ribozyme reaction; Figure 15 shows the effect of varying concentrations of Spermidine, Triton X-100 and Tween 40 in the ribozyme reaction (expansion of Figure 14); Figure 16 shows the construction of the native Sun Y ribozyme; Figure 17 shows the construction of Sun Y in which fragment B (indicated in bold in the text) is replaced by 16 clamidai rRNA sequences; Figure 18 shows an electrophoresis gel indicating the ligation activity of several intron group Chlamydia group I ribozymes; Figure 19 shows fluorescently labeled RNA constructs used as ribozyme substrates; Figure 20 shows the effect of PAGE de-normalizing after monitoring FRET; Figure 21 shows relative fluorescence as a function of time indicating the binding activity of the ribozyme in the presence of 5% SDS mM urea or without additions; Figure 22 shows a schematic representation of a reserve which suffers selection by in vitro evolution; and Figure 23 shows the binding activity of proto nucleotides which is dependent on cofactor (also named "effector"). In the description the following terms and definitions will be used: Group I intron - An intron having a three-dimensional structure as represented in Figure 3A and a conserved nucleotide structure and secondary structure represented in Figure 3B. 6.2 - a substrate for Sun Y ligation that has the sequence: 5'CCCUCU 3"- Pl - shown on the right side of Figure 4E.P = when N is G P1RL = Pl as in Figure 4E when N = 5 ' GGAGAAU 3 'P2 - shown in the ribozyme of Figure 4E P2A = a modification of P2 where the sequence 5' GGC UUA GAG AAG AAA UUC UUU AA 3"is inserted between # (-23) and 0 P2H = a modification of P2 where the sequence S "GGU AA 3" is inserted between # (5) and 0 P5SA = a modification of P5 where the sequence 5 'AGC UAU A 3' is inserted between bases # 28 and # 32 P5LA = a modification of P5 where the sequence 5"AGC UAU AGA CAA GGC AAU CC 3" is inserted between bases # 28 and # 32 P9. O - shown in the ribozyme of Figure 4E P9L = a modification of P9.0 with the sequence 5 'AUU CUC 3"P910 = a modification of P9.0 with the sequence 5" AUU CUC CAC C 3' P9A = a modification of P9 with the sequence 5 'AAA GCC AAU AGG CAG UAG CGA AAG CUG CGG 3"[substitution of P9 after P9.0] P9JD = a modification of P9 with the sequence 5' GGG GUG ACC CGA UC 3 '[substitution of P9 after P9.0] F910 = a sequence of 10 base pairs derived from F9 having a sequence: 5 'AUU CUC CAC C 3' The following is a list of chimeras of various Sun U constructions used herein : Table 1 EXAMPLES In the following examples, the numbers in parentheses indicate the number of the relevant stage in the respective figure. Example 1. In vitro evolution where the cofactor is a protein i. Prepare a random panel of nucleic acid sequences (1) A random panel of DNA sequences is prepared in a standard nucleic acid syntheses using a program to generate random sequences. A typical sequence is shown in step (1) of Figure (1) and comprises a (P) promoter of approximately 20 bp, a sequence which is cleavable in cis by a catalytically active ribozyme named in the "substrate" figure ( S) of approximately 10 bp (both sequences are constant and not random), a sequence generated in a random (R) of 50-8000 p.b. which averages at 100, and two sequences, one upstream of the random sequence and one current below it, which serves as a promoter for the PCR (M). ii. Preparation of immobilized nucleic acid sequences (2 and 3) The DNA sequences of i are transcribed. above, using a promoter with biotin (B) at the 5 'end (Fig. 1, step 2). Then, the biotin is allowed to react with avidin which is present on a solid support, such as Stepravidin beads (S), such that each molecule of the randomized arrangement becomes immobilized on a solid support (Fig. 1). stage 3). iii. Negative selection stage (4 and 5) A reaction mixture is added to the nucleic acid sequences prepared above which lacks the cofactor protein tested but contains magnesium ions which are necessary for ribozyme catalytic activity. Alternatively, it is possible to add a reaction mixture which contains another protein (Pro ') which is different from the specific cofactor proposed (Fig. 1, step 4a). The molecules that may be required for catalytic activity such as Mg ++ or GTP are then added to the reaction medium (Fig. 1, step 4b). Those nucleic acid sequences that are either spontaneously excised (step 4b (ii)) or possess a normal ribozyme cleavage activity with the unprobated cofactor (Pro ') (step 4b (i)) are released into the medium and removed . After this negative selection, only nucleic acid sequences that show no activity are maintained (step 4b (iii)). iv. Positive selection stage (5) Then the specific cofactor that is protein (Pro) is added to the reaction mixture (Figure 1, step 5a).

Magnesium is then added to the reaction mixture in order to allow the catalytic activity of cis cleavage of the random nucleic acid sequences (Fig. 1, step 5b). The nucleic acid sequences in the medium can be divided into three groups, as shown in step 5b; Group i. Proto nucleozymes of the invention that are activated only after the formation of a catalytic complex with the specific cofactor protein; Group ii. Nucleic acid sequences, which form a complex with the protein cofactor, but do not highlight any catalytic activity; Group iii. (comprising most of the nucleic acid sequences) nucleic acid sequences that do not complex with the protein cofactor. The last stage of positive selection comprises elimination of those nucleic acid sequences that are released from immobilized beads (group i). v. Amplification of the separated sequence (6) The sequences selected in iv are amplified. previous. The amplified products are transcribed, bound to biotin and bound to a solid support as specified in (ii) above).

Then steps (2) - (6) are repeated for approximately 10-100 cycles, wherein the reaction mixture is gradually enriched with the proto nucleozymes having the novel activity of the invention, ie they are catalytically active only in the presence of the specific cofactor protein. It should be noted that the positive selection stage may precede the negative selection stage. Example 2. Prepare haptens that link cofactors As a preliminary step prior to the construction of the DNA sequence of step 1 (in Figure 1), it is possible to find first short sequences (haptens) that are able to bind the desired cofactor (protein). The procedure for the preparation of such haptens is as follows: 1. Short random sequences (approximately 80 base pairs) are prepared, and reacting with the protein cofactor is allowed. 2. Those sequences that link the protein cofactor are identified, for example, by the use of antibodies against the protein, by absorption of a protein to a membrane capable of absorbing proteins, etc. and they are separated from other sequences that do not bind to the protein. 3. Repeat steps 2 and 3 above for 10-30 cycles so that the reaction mixture is enriched with haptens capable of binding the specific protein cofactor. These short haptens then serve as part of the random sequence (R) shown in step 1 of Figure 1. Example 3. Preparation of semi-random nucleic acid sequences Sometimes, for purposes of in vitro evolution, it is desired that the panel Recently prepared sequences resemble to some degree a known sequence, for example to that of the ribozyme. If, for example, it is desired that the semi-random sequence have an average 70% similarity to the specific nucleic acid sequence, it is possible to provide the nucleic acid synthesizer instead of four pure solutions containing each of the nucleotides (A, T, G and C), with four bottles each containing a solution composed of 70% of a nucleotide (for example A), and 10% each of three other nucleotides (10% G, 10% T and 10% C). By instructing the synthesizer to construct a sequence that uses these four mixtures, it is possible to obtain a sequence which has a 70% similarity with a known sequence. Of course other percentages of similarity can be used. Example 4 In vitro evolution where the cofactor is a nucleic acid sequence I. A proto nucleozyme for use in the detection of a short nucleic acid sequence cofactor (Fig. 2A): A known ribozyme sequence having a region is chosen of specific nucleus (C). The core region is evaluated for sequences that mostly resemble the nucleic acid cofactor. The similar sequences are removed all together (Fig. 2, step 2a), or replaced by a completely random sequence (R) (Fig. 2, step 2b). Sequences are set upstream and downstream of the absent core region or the random region with two flanking sequences (F) which are complementary to the sequences flanking the proposed cofactor (stage 3 (a) and (b)) . The original ribozyme sequences of the ribozyme obtained by step 3 (b) are then implanted, i.e. become semi-random (R ') as explained in Example 3 (step 4 (b)), for example having a 70% similarity to the original ribozyme sequence. With respect to the two parts of the precursor obtained in step 3 (a), only the original ribozyme sequence of one part becomes semi-random (R ') while the other part remains constant (step 4 (a)). The sequences obtained in 4 (a) or 4 (b) can then undergo the same steps of in vitro evolution of Example I (ii) - (v).

Alternatively, it is possible to construct a sequence comprising 5'-3 '; a completely random sequence ®, a flanking sequence (F) and a semi-random sequence (R ') having a 70% similarity to a known ribozyme (Fig. 2B) or the same sequence in the 3'-5' orientation. (Fig. 2) . II. A proto nucleozyme for use with the detection of large nucleic acid sequence cofactors Essentially the procedure of Example is repeated 4 (1), however, without inserting the flanking sequences (F) into the sequence. Since the cofactor to be detected is itself a very large nucleic acid sequence (eg, ribosomal RNA), it is assumed that even without manipulation, some of the large cofactor sequences will be complementary (to some degree) to core sequences. that have not been eliminated or replaced by random sequences. III. A proto nucleozyme for use in the detection of a nucleic acid sequence cofactor that is known Sometimes, it is not known a priori exactly which is the sequence of the target cofactor, for example, it can be one of many sequences of a virus or a bacteria the presence of which will be detected. In such a case, it is better to randomly remove some sequences from the nucleus of a ribozyme, and then take it to the remaining semi-random parts. It is desired that the deleted sequences be completed by the sequences of unknown bacteria. When steps (ii) - (v) of Example 1 are repeated with the construction prepared above, it is necessary that in the negative selection, step (iii), the sequences of viruses or bacteria that are added to the reaction medium are similar to one to be detected, in order to eliminate the nucleic acid sequences that are activated by other viruses or bacteria - In the repeat cycles, other types of viruses or bacteria can be added to the negative selection stage. Example 5. Improvement in the selection stage using a tag i sequence. Prepare random panel of nucleic acid sequences (1) A panel of DNA sequences is prepared on a standard nucleic acid synthesizer using a program to generate desired sequences. A typical sequence is shown in Figure 3 step (1) and comprises a promoter (P) of about 20 bases, a substrate S that can be cleaved in cis by a catalytically active oligonucleotide, a first promoter for PCR amplification (PCR 1 ), a first Ci restriction site of approximately 4-8 bases, a random tag sequence (TAG) of approximately 15 bases, a second C2 restriction site of approximately 4-8 bases (both sequences are constant and nonrandom), a variable sequence (V9 of 50-8000 bp that average in 100, the variable sequence that is a candidate capable of involving a functional sequence.) The variable sequence may be similar to variable sequences of other sligonucleotide species, for example since all the sequences are implanted, that is to say that it has a certain percentage of identical nucleic acid with those of a known ribozyme and a certain percentage of randomness, and a second sequence of CPR promoter (RCP 2). The variable sequence is a candidate to involve a sequence which after forming the complex with a specific cofactor, which completes an absent component becomes catalytically active. ii. Preparation of immobilized nucleic acid sequence (2 and 3) The DNA sequences of i are transcribed. above, using a promoter with biotin (B) at the 5 'end (Fig., step 2). Then, the biotin is allowed to react with avidin which is present in a solid support, such as Stepravidin beads (S), such that each molecule of the randomized arrangement becomes immobilized on a solid support (Fig. stage 3). iii. Positive selection stage (4) Then add the specific assayed agent which is protein (which serves as the selected group of conditions) (Pro) to the reaction mixture (Fig. 3), stage 4a). Magnesium and / or other factors required for catalytic activity are then added to the reaction mixture in order to allow the catalytic activity of cis cleavage of the variable oligonucleotide sequences in the substrate sequence S (Fig. 3, step 4b). The oligonucleotide sequences in the medium can be divided into three classes, as shown in step 4b; Group i. Oligonucleotide activated only after the formation of a catalytic complex with the specific cofactor Pro and substrate S cleaved and are released in this way to the medium. Oligonucleotides, which highlight catalytic activity even without protein; Group iii. (comprising most of the nucleic acid sequences) oligonucleotides that do not emphasize the catalytic activity in the presence of the cofactor. The last stage of the positive selection comprises collecting those oligonucleotides which are released from the immobilized beads. (Class i and Class ii). iv. Amplification of the separate sequence (5) If desired, the protein bound to the oligonucleotides can be eliminated by denaturation through heating and phenol chloroform extraction. The sequences collected in the previous step 4 are reverse transcribed and amplified by PCR. Since the sequence of the substrate S is cleaved (step 4b) this sequence must be reconstructed using either reverse transcription or by PCR proposes promoters containing the sequence of the cleaved substrate and in this way the amplified and reconstructed product is again identical to l. The amplified products are transcribed, bound to biotin and bound to a solid support as specified above (step 6). v. Negative selection step (7) The amplified and immobilized oligonucleotides of step 6 are subjected to a negative selection step. In this stage the catalytic activity is determined either in the absence of any protein, or in the presence of non-cofactor agents, similar to the specific cofactor which is added to the reaction mixture, the agents that are a protein similar to the specific cofactor (Pro) (Step 7a); Magnesium and / or other agents required for catalytic activity are added (step 7b). Oligonucleotides released into the medium by cleavage of the substrate sequence belong to three classes: Class i. In which the non-cofactor protein is carried around the cleavage of the substrate sequence; Class ii. Which includes cleaved oligonucleotides without need for any other external cofactor. Class iii. Which are not split at all. Both classes i are collected. AND ii. (7c) and the protein is removed (step 7d). The sequences are reverse transcribed and amplified by PCR in order to produce a double-stranded construct (step 8). Suitable restriction enzymes are added in order to cleave the Cx and C2 sequences, a cleavage site forming a blunt end and a strut end (step 9). The tip end is completed with the aid of biotinylated nucleotides and then the separated TAG sequence is immobilized on a solid support (avidin) and denatured in such a way that a denatured immobilized tag sequence of a chain remains. The immobilized tag sequences of step 10 are contacted with oligonucleotides collected from the step 4 (b) (Class i and Class ii) (stage 11). The molecules of the group which hybridize with the immobilized TAG sequence (stage 12) are eliminated in such a way that only molecules remain which highlight cleavage in the presence of the specific cofactor (Pro) and do not highlight cleavage activity without any protein or in the presence of a cofactor agent (Pro '). The oligonucleotides obtained in step 11 are again subjected to all preceding steps for 2-1000 cycles, preferably 10-100 cycles, more preferably 20-30 cycles. Example 6 Ligation of PIRL and 6.2 substrates: Comparison between 15 different Sun and Y ribozymes A comparison of the ability to bind PIRL and 6.2 substrates among the following 15 ribozymes derived from Sun and SNYP9L is performed; SNYP2AP9JD; SNYP2P9L; SNYP5SAP9L; SNYP910; SNYP2P5SAP9; SNYP2P910; SNYP2HP5SAP9; SNYP2HP9L; SNYP5LAP9L; SNYP2AP9A (ABOVE); SNYP2P5LAP9L; SNYP2AP9A (BELOW); SNYP9L; SNYP9JD; The reaction conditions are as follows: Molar proportion of ribozyme: PIRL: 6.2 = 1: 5: 5 Final concentration of ribozyme: 0.5 μμM. Pre-incubate the reaction mixture containing the ribozyme, PIRL, and substrate 6.2 at 58 ° C for 2 minutes in a buffer containing 30 mM tris HCl, pH 7.4 (at 25 ° C) / 10 mM NH4C1 / 0.4 M KCl / 20% ethanol. The solution is cooled to 45 ° C and the reaction is initiated by the addition of 90 mM final MgCl 2 in a reaction volume of 5 ul. The reaction is stopped after 30 minutes by the 2-fold addition of 10 ul of charge buffer containing 7 M urea. The samples are heated for -3 minutes at 80 ° C and charged on a 20% Paa gel which contains 7 M urea. Radioactive gels are analyzed with the help of a Biorad Phosphoimager system. The results are shown in Figure 5. As can be seen, several ribozymes which are derived from the Sun ribozyme Y maintain the ability to bind substrates. Example 7 Kinetic studies on Sun and P910 ribozymes using the PIRL and 6.2 substrates The Sun Y P910 ribozyme is used for ligation of the PIRL and 6.2 substrates. The reaction conditions are as follows: Molar proportions between the ribozyme: PIRL: 6.2 = 1: 2.5: 2.5 to 1:20:20 Final end of ribozyme: 0.25 μM. Pre-incubate the reaction mixture containing the ribozyme and PIRL substrates and 6.2 at 58 ° C for 2 minutes in a buffer containing 30 mM tri-HCl pH 7.4 (at 25 ° C) / 10 mM NH4C1 / 0.4 M KCl / 20% ethanol. The solution is cooled to 45 ° C and the reaction is initiated by the addition of final MgCl 2 concentration of 90 mM in a final reaction volume of 5 μl. The reactions are stopped at the times designated by the 2x addition of 10 μl of a charge buffer containing 7 M urea. The samples are heated for 2-3 minutes at 80 ° C and loaded onto a 20% PAA gel which contains urea 7 Radioactive gels are analyzed in a Biorad Phosphoimager system. The results are shown in Figures 6 and 7. These results are used to form a kinetic graph represented in Figure 8, which is used to create a Lineweaver-Burt graph of Figure 9. The calculated Km is 4.7 μm and the Kcat calculates is 6 min. "1 These results indicate a vast improvement in Km and an 1800 improvement in Kcat as compared to previously obtained results (data not shown) with the native Sun Y ribozyme Example 8 effect of lysis conditions on kinetics of ribozyme Conventional amplification systems are based on protein enzymes., are necessary either tedious purification stages or have to be worked very special and smooth lysis conditions. Briefly, ribozymes reactions are very robust and insensitive to harsh conditions, such as high concentrations of detergent strong ionic SDA (up to the solubility limit of 5% at 45 ° C in the presence of 400 mM KCl). high concentrations of Triton X-100 non-ionic detergent (10% at 45 ° C are even stimulants). Denaturing agents such as urea (100 mM has no effect). EDTA nuclease inhibitor. ATA ribonuclease inhibitor. organic solvents such as isopropanol, ethanol The effects of all additives on ribozyme reactions are analyzed by polyacrylamide gel electrophoresis and are shown hereinafter. I. Effect of denaturing agents (SDA, GITC, urea and phenol) in the reaction rate ribozyme Reaction conditions: 0.5 .mu.M Incubate 5 uM and 5 uM PI BS and substrate 6.2 32P brand 58 ° C in a reaction buffer containing SDS, GITC, urea or chloroform in concentrations indicated in Figure 9 or 25% (v / v) of the above aqueous supernatant, the phenol or the phenol / chloroform phase. The arrow at the top of the figure indicates a re-extraction of the aqueous phase with chloroform; again 25% of the aqueous supernatant is added to the ribozyme reaction. After cooling slowly to 45 ° C the reaction is initiated by the addition of MgCl 2 to a final concentration of 150 mM. After 60 minutes the reaction is stopped by stopping with 8 M urea. The results are shown in Figure 10. As can be seen, the addition of denaturing agents or phenol after re-extraction with chloroform does not significantly change the ribozyme as compared to a control. II. Effect of RCP inhibitors (isopropanol, EtOH, urea, GITC and SDS) on the ribozyme reaction rate 0.5 μM of Sun Y and 5 μM of the substrate are preincubated 6. 2 of 32P mark and 5 μM of Pl BS at 58 ° C in reaction buffer containing the indicated concentrations of several PCR inhibitors (EtOH as indicated, all others contain 10% EtOH) and cooled slowly. After the reaction reaches 45 ° C, the reaction is initiated by the addition of MgCl 2 to a final concentration of 50 mM. After 60 minutes, the reaction is stopped by stopping with 8 M urea. The results are shown in Figure 11. As can be seen, several agents that inhibit the PCR reactors do not significantly affect reactions based on ribozymes, thus making the more attractive ribozyme-based reaction agents for detection purposes. III. Effect of phenol and commercial specimen preparation equipment on the ribozyme reaction rate. Pre-incubate 0.5 μM of Sun Y and 5 μM Pl BS and 5 μM of 32P-PIP at 58 ° C in reaction buffer containing the following additives: 25% (v / v) of the aqueous layer on the phenol or phenol / chloroform. The arrow indicates re-extraction with chloroform; again 25% of the aqueous layer is added. The following solutions are added from the commercial Amplicor CT / NG (Chlamydia, trachomatia / Neisseria gonorrhoeae) Specimen Prep (Roche) equipment: 25% or 10% (v / v) of the urine wash buffer; 25% or 10% CT / diluent NG Specimen; 25%, 10% or 8.3% CT / NG lysis buffer containing a non-ionic, non-specific detergent. After the temperature reaches 45 ° C, the reaction is initiated by the addition of MgCl 2 to a final concentration of 150 mM. The reaction is stopped after 60 minutes stopping with 8 M urea. The results are shown in Figure 12. As can be seen, both phenol, which is used extensively in several laboratories for specimen preparation, (after re-extraction by chloroform) and a commercially available specimen kit does not affect the ribozyme reaction. The laboratory procedure performed in the presence of phenol must include a re-extraction step by chloroform before subjecting the specimen for ribozyme reaction. IV Effect of various concentrations of GITC on the ribozyme reaction rate 0.25 μM of Sun Y and 5 μM of substrates 6.2 labeled with 32P and 5 μM of Pl BS at 58 ° C in reaction buffer containing GITc in 100 mM are preincubated. which increases from 0 to 1 M and cools slowly. After the reaction reaches 45 ° C, the reaction is initiated by the addition of MgCl 2 to a final concentration of 150 mM. After 30 minutes the reaction is stopped by stopping with 8 M urea. The results are shown in Figure 13. As can be observed up to 300 mM GITC does not affect the reaction rate. V. Effect of several additives (ATA, Spermidine, DMF, DMSO, Triton, Nonidet and Tween 40) in the ribozyme reaction. 0.5 μM of Sun Y and 5 μM of Pl BS and 5 μM of substrates 6.2 marked with 32 P are incubated. 58 ° C in reaction buffer containing the indicated additives. After cooling slowly to 45 ° C, the reaction is initiated by the addition of MgCl 2 to a final concentration of 150 mM. After 60 minutes the reaction is stopped by stopping with 8 M urea. Relative product yields are indicated: the control value is set without additive to 100. The significant stimulation is printed in bold with Triton X-100. The results are shown in Figure 14. As can be seen, most additives do not significantly reduce the ribozyme reaction while Triton X-100 increases ribozyme activity by 80%. SAW. Effect of additives (Spermidine, Triton x-100, and Tween 40) 0.5 μM of Sun Y and 5 μM of Pl BS and 5 μM of substrates 6.2 marked with 32P at 58 ° C are incubated in reaction buffer containing the indicated additives in Figure 14. After cooling slowly to 45 ° C, the reaction is initiated by the addition of MgCl 2 to a final concentration of 150 mM. After 60 minutes the reaction is stopped by stopping with 8 M urea. Relative product yields are indicated: the control value is set without additive to 100. The significant stimulation is printed in bold with Triton X-100. The results are shown in Figure 15. As can be seen, most of the additives used only Tween 40 at a concentration of 205 significantly inhibits the reaction. The Triton X-100 in all tested concentrations allows to increase the activity by EXAMPLE 9 Proto Chlamydia Nucleozymes A portion of the catalytic core of a Sun Y ribozyme is substituted with 16 Chlamydia trachomatis rRNA sequences. Corresponding changes in the ribozyme are introduced to accommodate the replacement of Chlamydia. Figure 16 shows the wild-type ribozyme with the sequence being substituted in bold. 17 different ribozyme sequences are tested for splicing activity. Figure 17 shows the actual sequence of the ribozyme (option 29) which is active when part of its core sequence is replaced by the chlamydia sequence (the last one shown in bold). The experiments are carried out as follows: In Figure 18, 2 μM each of Chlamydia-dependent ribozymes are reacted with 4 μM of substrate for 18 hours at 45 ° C. The wild-type Sun Y ribozyme is used as a positive control. Reactions are stopped with 5 mM EDTA and 3.5 M urea, final concentration, and then run on a 20% PAA-7M urea gel. The results are shown in Figure 18. These results show that a ribozyme (option 29) where part of its nucleotides are replaced by 16 rRNA sequences of Chlamydia trachomatis can be catalytically active. This ribozyme without the Chlamydia sequence can serve as a proto nucleozyme of the invention and is used in an assay to detect the presence of chlamydia, since the Chlamydia sequences (which serve as cofactors) can complete their missing sequences and thus a catalytically active nucleozyme. Example 10 Detection of the ribozyme ligation reaction using fluorescent energy transfer (FRET) A convenient model for detecting ribozyme activity, which may be cleaving, splicing or binding, is to use a fluorescent dye such as fluorescein (F) and a retaining dye CY-5. When the two dyes come into contact (for example, due to ligation or splicing), the reproter dye (F) can absorb light, but the fluorescence is stopped by FRET: the energy is transmitted to the retaining dye CY-5 which emits its fluorescent light distinctly different at 670 nm. As a consequence of the ligation or splicing reaction, the nucleotide labeled with F is released. Now, the reporter dye can emit its own fluroescence, characteristic at 522 nm. Two small RNAs are selected which are well characterized substrates by binding of Group I ribozymes. Is each RA? with only the dye: the AR? large carries the fluroescein dye "reporter" (F) at its 5 'end; - the AR? small carries the "stop" dye CY-5 at its 5 'end. The ribozyme constructions are shown in Figure 19. Prior to the ribozyme reaction, both substrates of AR? they hybridize to form a notch yoke. The incoming light (hv, represented in the Figure as a solid arrow) is absorbed by fluorescence (F) and transferred to CY-5 (dotted arrow) which subsequently emits its characteristic fluorescence at 670 nM (hv; zigzag arrow). After the reaction, the fluorescein-labeled nucleotide (F-UGG) is released and the absorbed light is emitted directly from the fluorescein with the characteristic wavelength of 522 nm. Diagram 11: An example of real-time monitoring The FRET can be measured by calculating the proportion of reporter dye F (at 522 nm) to an inert signal at 500 nm. This ratio is plotted against the time axis: After incubation intervals of 5 minutes, the fluorescence is measured for 30 seconds. Each measurement is evident by a small peak: due to photobleaching, the signal falls during the measurement period and recovers during the dark interval. The results are shown in Figure 21. Reaction conditions: 1 μM Sun Y and 1 μM of Pl BS marked with 5 'fluorescein and 5 μM of PIP labeled with 5'-CY-5 are incubated in the Abi 7700 cousin for monitoring of real time. Incubation is performed at 45 ° C for 180 minutes, with data collection at 5 minute intervals. The relative fluorescence change of the fluorescein dye reporter is shown. For additional analysis, aliquots of 10 μl of the denaturing samples are analyzed in polyacrylamide gel electrophoresis (shown in Figure 19). The comparison shows that GITC stops the fluorescent signal, but does not inhibit the ribozyme reaction. The results are shown in Figure 20 which shows the effect of denaturing on the reaction rate and detection. In the absence of ribozyme, there is no change of FREI signal. In the presence of the ribozyme, without additional additives, the signal changes from the initial value "4.2" to the final value "6.2". Similar changes are observed if 200 mM urea or 5% SDS have been added, indicating that these additives do not significantly change the reaction rate or the detectable signal. Although 100 mM guanidinium isothiocyanate (GITC) does not inhibit the ribozyme reaction (Fig. 12), it is impossible to include GITC if fluorescein is used as a reporter dye since GITC is a very strong stopper for fluorescein. However, the effect of GITC is specific to the dye since no arrest is observed with other fluorochromes such as CY-5 (data not shown). Example 12 I. Production of objective dependent ribozyme proto nucleosomes In vitro evolution is used to prepare proto nucleozymes that are catalytically active only in the presence of a cofactor. The selection scheme is similar to that used by Barte and Szotsta (Science, 261: 1411-1418 (1993)), A reserve of ARB random sequence (N90) starts with a 5 'triphosphate. The reserve is captured in an affinity matrix complementary to the 3 'end of the pool and mixed with an RNA substrate complementary to the 3' end of the pool and mixed with an RNA tag substrate with a DNA sequence. After ligation, the RNA can be eluted from the column, reverse transcribed via a cDNA promoter, and the PCR is amplified via a promoter that is complementary to the newly ligated DNA tag. A hosted PCR reaction using an internal promoter generates an annealing that can be transcribed again to regenerate selected ligases. This cycle can be used for the selection of the fastest and most active ligases. A schematic reservation of RNA sequences is shown in which the selection is made in Figure 22, together with the 3 'end sequence and its complementary cDNA promoter. After several rounds of selection the reserve is cloned and the individual clones are assayed, for activity and dependence on the cofactor sequence. The major class of selected ligases can efficiently catalyze the addition of the labeled RNA-DNA substrate thereto. Once of course it is performed under standard assay conditions, and ligation products of unbound ribozymes are separated by denaturing PAA gel gel. In the absence of either Mg + 2 or the cofactor, which is a specific nucleic acid sequence, no reaction is observed. The relative amount of the radioactive label in bound and unbound ribozymes is determined using a Phosphoimager, and the degree of reaction as a function of time. The initial velocities and the total degree of the reactions are determined using the fixation procedure in standard curves, and are shown in Figure 23. As can be observed in the absence of the cofactor or Mg + 2, no bound products are produced. The clones are examined additionally for specificity. A series of cofactors of DNA and RNA nucleotide sequence are synthesized and their ability to activate the proto nucleozymes is determined. The values are calculated as described above. '-' indicates that the activity can not be measured in relation to the baseline. Experiments carried out in duplicate or triplicate indicate that the relative activities of "active" cofactors (for example lines (1) and (9)) and inactive (for example lines (6) and (10)) are consistent among the experiments. The results are shown in Table III: A variety of potential cofactors are synthesized and tested for their ability to increase their ligase activity; The results of these experiments are shown in Table III. Oligonucleotide cofactors extending in complementarity towards the 5 'end of the RNA fade towards the 3' end show little or no activation relative to no effector control (Table III, lines (2) and (3)). These results seem to indicate that the position of the cofactor is essential for the activity, and that the 3 'end of the cofactor may play a role in the inactivation. A cofactor is synthesized with a substitution of G to A at the 3 'terminus. The decoupling C: A is much less active than the totally complementary effector (line (4)). However, the loss of activity observed with the change from G to A in the effector can be suppressed by a compensatory change or C to U in the ribozyme (line (5)). These results seem to indicate that the 3 'end of the ribozyme is acting as a "terminal guide sequence" (TGS). The cofactor that ends in a dideoxy nucleotide may not activate catalysis (line (6)). In contrast, a cofactor all active catalysis RNA also close (relative activity = 0.72; line (7)) as the original all-DNA effector. Taken together, these results emphasize that the ribozyme is able to specifically recognize its specific oligonucleotide cofactor. II. Activation in the presence of random sequences The experiment described in I above is repeated with a 12 mer cofactor in the presence of a random sequence of 12 nucleotides, or in the presence of both random sequence and the cofactor, either when both are in the same concentration (1.3 μM) or where the random sequence is in an excess (10: 1). The results are shown in Figure 23. As can be observed a 12 mer cofactor is capable of activating the proto nucleozyme at various concentrations while the random sequence reserve does not show such activation. On the other hand, the random sequence reservation, even in excess concentration does not interfere with the ability to correct the cofactor to activate the proto nucleozyme. These results show the specificity of the proto nucleozyme of the invention to its specific cofactor and demonstrate that the presence of other sequences does not interfere with this specificity.

Claims (20)

1. CLAIMS 1. A proto nucleozyme, characterized in that it is a nucleic acid molecule or a complex of nucleic acid molecules, with essentially no activity but which can form a complex with a specific non-nucleic acid cofactor to form a nucleozyme possessing catalytic activity; the cofactor which is a molecule or portion within the molecule; the protonucleozyme lacking an essential component for the catalytic activity of the nucleozyme, and the cofactor provides the component so it converts the proto nucleozyme into the nucleozyme.
2. 2. A proto nucleozyme, characterized in that it is a nucleic acid molecule or a complex of nucleic acid molecules, with essentially no activity but which can form a complex with a specific nucleic acid cofactor to form a nucleozyme possessing catalytic activity; the cofactor which is a nucleic acid molecule or a nucleic acid sequence within a nucleic acid molecule; the protonucleozyme lacking a segment of one or more nucleotides or lacking a link between the two nucleotides the completion of which converts the proto nucleozyme into the catalytically active nucleozyme, and the cofactor provides an absent segment or junction.
3. 3. A proto nucleozyme according to claim 1 or 2, characterized in that the nucleozyme is a ribozyme.
4. 4. A proto nucleozyme according to claim 1 or 2, characterized in that it is a product of in vitro evolution.
5. 5. A proto nucleozyme according to claim 1, characterized in that the non-nucleic acid cofactor is selected from the group consisting of: proteins, peptides, oligopeptides, antibiotics, phosphate nucleotides, carbohydrates and lipids
6. 6. A method for the detection of a agent in a medium which comprises the following steps: (a) Contacting the medium with a proto nucleozyme according to any of claims 1-4, wherein the agent is the specific cofactor required for the formation of the catalytic complex; (b) Provide or maintain conditions that allow the catalytic activity of the catalytic complex; and (c) Testing the presence of products of the catalytic activity in the medium, such presence indicating the presence of the agent in the medium.
7. 7. A method according to claim 6, characterized in that the catalytic activity is cleavage.
8. 8. A method according to claim 6, characterized in that the catalytic activity is ligation.
9. 9. A method of in vitro evolution for the production of a pro nucleozyme according to claim 1, the method characterized in that it comprises: (h) preparing a panel of different nucleic acid molecules, at least a part of the sequence in the molecules of the honeycomb is a random or partially random sequence, in such a way that the part has a different sequence in different molecules of the panel; (i) add the cofactor to the panel and incubate under conditions that allow the catalytic activity; (j) separating between the nucleic acid molecule of the panel that characterizes the catalytic activity and which does not, to obtain a first selected panel of nucleic acid molecules that characterize the catalytic activity; (k) amplifying the nucleic acid molecules of the first selected panel; (1) incubating the first selected panel with a reaction mixture, which lacks the cofactor, under conditions that allow the catalytic activity; (m) removing the nucleic acid molecules that highlight the catalytic activity, whereby a second selected panel of nucleic acid molecules lacking the removed nucleic acid molecules is obtained; and (n) repeating steps (b) - (f) over a plurality of cycles, for example, about 10-100 cycles, to obtain the proto nucleozyme.
10. 10. A method according to claim 9, characterized in that the cycles are repeated 10-100 times.
11. A method according to any of claims 9 or 10, characterized in that the panel molecules of nucleic acid molecules in step (a) have a sequence that is derived from a known nucleozyme sequence with some proportion of the nucleotides which are replaced by other nucleotides.
12. 12. A method according to claim 10, characterized in that some proportion is approximately 1-30%.
13. 13. A method in accordance with the claim 11 or 12, characterized in that they comprise at least one amended cycle, wherein the step of amplifying the selected panel molecules also comprises replacing a small proportion of the nucleotides in the molecules of the selected panel.
14. 14. A method according to claim 13, characterized in that it comprises a plurality of such amended cycles.
15. 15. A method of in vitro evolution for the production of the proto nucleozyme according to claim 1, wherein the nucleozyme together with a cofactor forms a catalytic complex having catalytic activity imparted by a functional sequence of the proto nucleozyme, the method characterized in that it comprises: (c) Preparing a mixture of different candidate oligonucleotides to involve the proto nucleozymes each of which comprises a variable sequence that is a candidate to involve in the functional sequence and a tag sequence, each of the two sequences being different from the corresponding sequences of different oligonucleotides in the mixture, the variable sequence and the tag sequence which is linked to at least one cleavable sequence; (d) Process the mixture through negative and positive selection steps, each step optionally followed by an oligonucleotide amplification step, with at least one step of positive selection and at least one step of negative selection, these steps that they comprise: (ba) a positive selection step comprising applying the specific cofactor and separating between the oligonucleotides they have and those that do not have catalytic activity to obtain a first selected mixture comprising a first group of oligonucleotides that highlight catalytic activity in the presence of cofactor; (bb) amplifying the first group of oligonucleotides in the first selected mixture to produce a plurality of copies of each of the first group of oligonucleotides to obtain a first amplified mixture; (bca) apply a reaction mixture lacking the specific cofactor and the separation between a second group of oligonucleotides which have no catalytic activity in the absence of the cofactor and a third group of oligonucleotides having the catalytic activity in the absence of the cofactor, to obtain a second selected mixture comprising the second group of oligonucleotides and a third selected mixture comprising the third oligonucleotide groups; (bcb) amplifying the third set of oligonucleotides in the third selected mixture to produce a plurality of copies of each of the third oligonucleotide group to obtain a second amplified mixture; (bcc) cleaving the cleavable sequence of the oligonucleotides in the second amplified mixture, separating between the variable and tag sequences and collecting the tag sequences; (bed) contacting the collected tag sequences with the second selected mixture under severe hybridization conditions and removing hybridized oligonucleotides and other oligonucleotides from the second mixture, whereby a fourth selected mixture of oligonucleotides lacking essentially oligonucleotides is obtained which they have catalytic activity in the absence of the cofactor; where the positive selection step precedes the negative selection step, the positive selection step is applied to the mixture prepared in step (a) and the negative selection step is applied in the first amplified mixture; and where the negative selection step precedes the positive selection step, the negative selection step is applied to the mixture obtained in step (a) and the positive selection step is applied in the fourth mixture.
16. 16. A method for selectively destroying a specific cell population, which contains or expresses a specific agent, the method characterized in that it comprises: (a) Providing a proto nucleozyme according to claim 1, which when present in the complex Catalytic has catalytic activity which is cytotoxic to the cell or has cytotoxic reaction products for the cells, and wherein the agent is a specific cofactor for the proto nucleozyme; (b) Insert the proto nucleozyme into the cells that contain or express the agent or apply the proto nucleozymes to the tissue suspected to contain a population of cells that contain or express the agent, under conditions or using a vehicle, to insert the proto nucleozyme in the cells.
17. 17. A method for inhibiting the expression of undesired genes in cells comprising: (a) Providing a proto nucleozyme according to claim 1, wherein the nucleozyme has catalytic activity which is targeted to a nucleic acid sequence or product of specific gene expression in such a way that once activated within a cell, the expression of the gene will be inhibited; (b) Insert the proto nucleozyme into the cells that contain or express the gene, or apply the proto nucleozymes to the tissue suspected to contain a population of cells that contain or express the gene, under conditions or using a vehicle, in order to insert the proto nucleozyme in cells.
18. 18. A method in accordance with the claim 17, characterized in that the cofactor of the proto nucleozyme is either a nucleic acid sequence of the gene or a transcription product of the gene.
19. 19. A pharmaceutical composition characterized in that it comprises a proto nucleozyme according to any of claims 1-5 and a pharmaceutically acceptable carrier.
20. 20. A pharmaceutical composition according to claim 19, characterized in that the carrier comprises liposomes.