MXPA97004726A - Ribozimas varkud satell - Google Patents

Abstract

A ribozyme capable of dividing a separate substrate RNA molecule, said ribozyme having three base pair regions, generally, but not limited to, in a proposed "I" configuration, wherein the base pair regions " upper "e" lower "comprise between about 4 and 80 bases, inclusive, of which at least about 50% are paired with each other, and where the" connecting "region between the upper and lower base pairs regions comprises between 4 and 20 bases, approximately, inclusive, of which at least about 50% are paired

Description

RIBOZItlflS VflRKUD SflTELLITE BACKGROUND OF THE INVENTION This invention relates to rLbozunas. The following is a brief description of the publications that relate to the pbozímas, in par-t ícular, with the nbozímas Varkud Satellite (VS). None of them is admitted to be prior art with respect to the claims appended hereto, and all of them are incorporated herein by reference. There are currently six basic varieties of enzyme nucleic acids that occur in nature. Each one of them can catalyze the hydrolysis of RNA phosphodiester ligatures in t ans (and, in this way, can divide other ORN molecules), ba or physiological conditions. Table I summarizes some of the characteristics of these pbozímas. In general, the enzymatic nucleic acids act by first binding to a target ORN. Such binding occurs by means of the target binding portion of an enzymatic nucleic acid, which is maintained in close proximity with an enzymatic portion of the molecule acting to divide the target flRN. In that way, the nucleic acid enzyme only recognizes first and then binds to a target ORN, through the formation of complementary base pairs; and once it binds to the correct site, it acts enzymatically to cut the target RNA. The strategic division of said target RNA destroys its ability to digest the synthesis of an encoded protein. After an enzymatic nucleic acid +? Co has joined and divided its destination ORN, it is released from that ORN to search for another destination and can repeatedly join new destinations and divide them. The enzymatic nature of a phocyan is advantageous over other technologies, both in terms of the technology opposed to transfer (where a nucleic acid molecule usually simply binds to a nucleic acid target to block its translation) , since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an oligonucleotide opposed to transfer. This advantage reflects the capacity of the pbozirna to act enphatically. Thus, a single ribozuna molecule is capable of dividing many target ORN molecules. Traditionally, ribozyme is a highly specific inhibitor, the specificity of the division depending not only on the mechanism of base stop formation of the target ORN binding, but also on the mechanism of cleavage of the target RNA. Individual inequalities or base substitutions near the division site can completely eliminate the catalytic activity of a ribozyme. Similar inequalities in the molecules of opposition to the transfer do not prevent their action (Uoolf and coauthors, 1992, Proc. Nati Acad.c. USO 89, 7305-7309). Therefore, the specificity of the action of a ribozyme is greater than that of an oligonucleotide opposed to the transfer, which binds to the same ORN site. It has been found that a small number of ORNs isolated from a variety of natural sources possesses a self-monitoring activity that is involved in multimeric transcripts from processing to monomers, apparently as part of the reproduction cycle. Several different ORN sequences and secondary structures appear to be capable of such activity. These include the hammerhead, which is found in several viral satellite ORNs of plant,? N ORN viroí and in the transcription of? ODN nuclear satellite of a ne t (reviewed by Syrnons, 1992, nn ?, Rev. Biochern. 61, 641), - the hair pin (or paper fastener) in the filament minus the satellite of the tobacco blight virus and related viruses (Buzayan and coauthors, 1986 Nature, 323, 349; Feldstein and co-authors, 1990, Proc. Nati, Acad. Sci. USA, 87, 2623); in genomic ORNs and atigenonuuses of the hepatitis delta virus (VHD, Sharrneen and coauthors, 1988, 3. Vi rol., 62, 2674, Kuo and coauthors, 1988, J. Virol., 62 4439, Perrotta and Been, 1991, Nature 350, 434); and the ORN of Varkud Satell? te_ (VS) in the itocondpos of certain isolates of Neuros ora (Saville and Collins, 1990 Cell 61, 685). In their natural contexts, the ribozymes mentioned above, as well as others, such as the introns of group I (Cech, 1990 Onnu.

Rev. Biochem. 59, 543) and those from the IT group (Ni che! And coauthors, 3989 Gene 82, 5), perform amolecular self-dissemination and, in some cases, ligation reactions. The structure-function studies of the group T introns (Zaug and Cech, 1986, Science 231, 470, Szostak, 1986, Nature 322, 83) and posteriorly of the hammerhead ribozymes (Uhlenbecl. ', 1987 Nature 328, 596), hair pin ribozimies (Feldstein and coauthors, 1990, supra, Harnpel and co-authors, 1990, Nucleic Ocids Res. 18, 299) and ribozymes of VHD (Perrotta and Been, 1992, Biochemi try 31, 16; Branch and Robertson, 1991, Proc. Nat. Ocad. Sci. USO, 88, 10163), have been facilitated by altering these RNAs to effect intrarenal interdivision reactions. In a transcription reaction, an RNA, the substrate, contains the site to be divided. A separate ORN, the ribozirna, provides the sequences required to catalyze the division. A ribozirna of trans-action, occurring in nature, has been discovered: the ORN component of RNase P, which divides the pre-tORN precursors into tras (Guerper-Ta ada and co-authors, 1983 Cell 35, 849). The trans-division reactions of most ribozymes have been designed in such a way that substrate binding occurs through the formation of multiple tatson-Cpck base pairs in the ribozyna. Interactions that are not Wat-C-riel and tertiary interactions at the junction to the substrate are also involved and may be essential for the proper binding (Pyle and co-authors, 1992 Natu e 358, 123; D? ~ Ha? Jy coauthors, 1993 Nucí. cids Res. 21, 1797; S ith and coauthors, 1992, 3. Biol. Chem. 267, 2429; Guerper-Takada and Oltman, 1993, Bioche i try 32, 7152). With hammerhead, hairpin and group T ribozimies, it has been found that very few specific nucleo-tides in the substrate are necessary for trans division, provided that the region or adjacent regions are complementary. to the binding site in the ribozirna. This property has allowed the design of ribozymes that can divide different sequences to those that are recognized by the ribozyme that occurs in nature. Some engineered ribozirins also function in vivo in unnatural host cells, which has led to the possibility of their use as therapeutic agents in dominant inherited alterations and against retroviruses and ORN viruses (reviewed by Castanotto and coauthors, 1992, Critical Revie s ín Eul - aryot? C Gene Expression 2, 331).

BRIEF DESCRIPTION OF THE INVENTION This invention relates to novel catalytic nucleic acid which performs the same type of division of ORN as the hammerhead, hairpin and HDV ribozones, leaving products with cyclic phosphate 2 ', 3' and OH ends 5 '(Saville and Collins, 1990, supra); but it is different in the sequence, the secondary structure, the selection of the division site and the functional properties with respect to the ribozymes that divide in t-rans known in the art (Collins and Olive, 1993 Bioche i try 32, 2795; and < coauthors, 1993, rlol. Biol., 232, 351). This invention incorporates the construction and use of enzymatic nucleic acid molecules, for example, those derived from ORN of the Neurospora Varkud Satellite (VS), which can catalyze a trans-vission reaction, wherein an ORN of Separate substrate is divided into its specific descent site. The minimum substrate can form a stalk-stem base structure, of the hairpin type, stable (Figure 6). The recognition of the substrate by means of catalytic nucleic acid involves multiple interactions, including tertiary interactions. The catalytic nucleic acid includes a binding domain to the ORN target, which interacts with the nucleotides of the target ORN (preferably with the 3 'bases of the cleavage / ligation site), and an enzymatic portion (which may include a part or the entire RNA binding portion of the substep), which has the enzymatic activity. The nucleic acid binds to the target RNA, preferable, with bases 3 'of the cleavage / ligation site and causes the cleavage of the RNA substrate at that cleavage site. Thus, in one aspect, the invention incorporates a nucleic acid molecule that catalyzes the splitting of a separate, double-stranded ORN target molecule in a manner specific to the sequence. By "trans-division" is meant that the ribozirna is capable of acting in the trans position to divide another ORN molecule that is not covalently bound to the ribozyna proper. In such a way, ribozyme is not able to act-on itself in a reaction of intramolecular division. By "base pairs" is meant a nucleic acid that can form hydrogen bonds and /? other ligatures with another sequence of ORN, either by means of traditional interactions of the Uatson-Crick type or other non-traditional interactions (for example, of the Hoogsteen type). The enzymatic ORN molecules of this invention can be designed to divide the ORN (minimum length of between 8 and 20 nt), which have only one preference for at least one nucleotide or immediately 5 'with respect to the division site, and the availability of an adjacent 2 'hydroxyl group, for division to occur. The 2'-hydroxyl group is generally provided by the substrate ORN molecule. Therefore, these molecules of enzymatic RNA provide significant in vitro and in vivo activities that can be used for diagnostics and for therapeutic procedures. Thus, in a third aspect, the invention incorporates a ribozirna capable of dividing a separate substrate ORN molecule. The ribozyme has three regions of base pairs, generally in an "I" configuration. The upper and lower base pair regions of the proposed "I" include in-t 10 and 80 bases, inclusive, of which about 50% monkeys form pairs with each other. The connecting region of the "T" proposed between said regions of upper and lower base pairs includes between about 8 and 20 bases, inclusive, of which at least about 50% are formed in pairs. By "ribozyme" is meant any enzyme-nucleic acid molecule which usually contains at least some pbonucleotides, which is active to divide an ORN molecule without forming a covalent bond with that substrate. In such a manner, the molecule generally lacks a nucleophilic attack group that is capable of causing it to divide the substrate and form a covalent bond with that substrate (at least temporarily). A "separate ORN molecule" is one that is not covalently linked to the ribozirna and that may contain no pbonucleotides within its length. Preferably it is an ORN molecule that occurs in nature, such as viral rnORN or a pathogenic ORN molecule. The proposed "I" configuration is generally shown in Figures 5B to 8. That structure may contain other nucleic acid strands joined to different portions of the "I", but those skilled in the art will recognize that it is advantageous to have the least amount of these extra chains that are possible, so that the interactions of secondary structure are reduced and when the size of the molecule is kept as small as possible. The "1" proposal has an "upper" region and a "lower" region, which are described above, and are connected by an intermediate region ("connector"). Together, these regions provide enzymatic activity for ribozyme. While the formation of base pairs in these regions is important, those skilled in the art will recognize that other types of base pair formation activities are also useful in this invention, for example, base pair formation. Hoogsteen These regions, as noted, may include regions that are not formed in pairs at the ends of the pairs regions., or even within, or between these regions of pairs, as long as the enzymatic activity is not eliminated. By means of a formation of 50% of base pairs it is meant that throughout the region at least half of the bases in that region interact with other bases to maintain the ribozíma in the general configuration of " I ". In the preferred embodiments there are at least 70% or even 80% base pair formation, as illustrated in the accompanying figures. The proposed "T" configuration means that it is a non-limiting structure. Those who are ordinary experts in the field will recognize modifications (insertions, omissions, base substitutions and / or chemical modifications) to the structure of "I" proposal, which can be easily generated using known techniques in this field. Additionally, structures other than the proposed "I" configuration can easily be generated by those skilled in the art, and are also within the scope of this invention. In other preferred embodiments, the "connector" region additionally includes a single filament region of between about 3 and 7 bases, inclusive; for example, the single-filament region is adjacent to the "upper" base pair region as shown in Figures 6-8; the "upper" region includes a "left" portion and a "right" portion, each of which has at least about 6 to 30 bases, and the "lower" region also includes a "left" portion and a "portion" right ", each of which has between about 6 and about 30 bases inclusive. Said regions are delineated by means of the "connector" region noted above, and as shown in the figures. In other preferred embodiments, the "lower" region and / or the "connecting" regions at least include a bulky nucleotide (e.g., A), which is a base not formed in pairs, which may be available for interaction with the proteins; the "higher" base pair region includes bases not formed in pairs with other bases in the "higher" base pair region that are available to form base pairs with a substrate ORN, for example, as shown in figures 8 and 9; where bases that are not formed in pairs include at least 3 bases. In addition, the substrate for the riozyme has a region of base pairs of at least 2 base pairs, for example, the substrate has the sequence 3 'GONN 5', where the division of the ribozi a is between each N ( each N, independently, is any basis, throughout this document, the term N or N ', independently is any base or any base equivalent). In other preferred embodiments, the "lower" base pair region has non-base-formed bases at its 5 'end, available for base pair formation with? N substrate ORN; the ribozyme makes contact with the ORN substrate only 3 'with respect to the division site; the substrate of ORN s double-stranded RNA and the nucleic acid molecule is able to make contact with the double-stranded RNA substrate only in the 3 'direction of the dividing site and causes the splitting of the RNA substrate at the site of division; the RNA substrate is single-filament ORN and the ribozirna is able to make contact with the single-strand ORN substrate only 3 'with respect to the division site and causes division of the ORN substrate at the division site . In a more preferred embodiment, the ribozyme is derived from Ne? Rospora VSN ORN. That is, the ribozyme has the essential bases of the ORN VS molecule, held together in a suitable configuration as described above, so that the RNA substrates can be divided into the cleavage site. Said essential bases and said configuration are determined as described below; Those who are experts in the field will recognize that it is now routine to determine such parameters. An example of said ribozirna is that which has approximately 80 to 90% of the sequence shown in Figures 5 to 8. In other embodiments, the ribozirna is an enzymatic entity active to cut an ORN duplex having at least two Base pairs; the ribozyme is enzymatically active to cut 5 'with respect to the 5' NAGNnGUCN 3 'sequence (see Figure 6B), wherein each N is independently any nucleotide base; n and m are independently an integer between 3 and 20 inclusive; and the sequence forms at least two intramolecular base pairs; the ORN substrate binds to the ribozyme at a site distant from the division site; the ribozyme is a circular molecule; wherein the circular molecule makes contact with a separate ORN substrate and causes the division of the ORN substrate at a cleavage site; and the ribozyme includes RNA. In other aspects, the invention incorporates a cell that includes nucleic acid which encodes the previous one; an expression vector having nucleic acid encoding that ribozirna in a manner that allows the expression of the ribozyme within a cell; and a cell q? e includes said expression vector. Other aspects also include an expression vector in which the ribozyme encoded by the vector is capable of dividing a separate ORN substrate molecule, selected from a group consisting of viral ORN, messenger ORN, pathogen ORN and cellular ORN. . In other related aspects, the invention incorporates a method for dividing an ORN substrate from a single strand into a cleavage site, by causing the base pair formation of the ORN substrate with a nucleic acid molecule only 3 'with respect to to the division site (figure 7). Said method includes contacting the ORN substrate with a nucleic acid molecule having an enzymatic activity dividing the ORN substrate, which divides a separate ORN substrate, into a dividing site. This nucleic acid molecule includes a substrate binding portion of ORN, which forms base pairs with the ORN substrate only 3 'with respect to the cleavage site; and an enzymatic portion (which may include part or all of the substrate binding portion of ORN), which has the enzymatic activity. The nucleic acid molecule is capable of forming base pairs with the ORN substratum only 3 'with respect to the cleavage site and causes cleavage of the ORN substrate at the di isLon site. The contact is carried out under conditions in which the nucleic acid molecule causes the division of the ORN substrate at the division site.

In the preferred embodiments of the above aspects, the ORN nucleic acid molecule of Neurospora VS is derived; the nucleic acid molecule is active to pair 5 'with respect to the RNA duplex substrate (Figure 6) of the sequence 5' -OOGGGCGUCGUCGCCCCGfl, or 5'-NNNNNNNNNNNNNNNNNNN, where N independently can be any specified nucleotide base, where the sequence forms a duplex structure > of at least two base pairs; the nucleic acid molecule is RNA; the nucleic acid is a mixture of nbonucleotides and deoxypho- nol-cleotides; the nucleic acid contains at least one nucleotide containing modifications of sugar, phosphate and / or base or combinations thereof; the nucleic acid molecule may contain abasic substitutions and / or q? e are not nucleotide; the nucleic acid molecule makes contact with the target RNA sequence; the nucleic acid molecule is circular; and the nucleic acid molecule is active to cut a single-filament ORN (FIG. 7) 5 'with respect to the sequence OOGGGCG or NNNNNNN or OOGGGCGUCGUC or NNNNNNNNNNNNN, wherein each N independently can be any specified nucleotide base; wherein the sequence forms at least 2 base pairs with a complementary sequence in the 5 'region of the enzyme nucleic acid molecule; wherein the substrate ORN has at least one nucleotide 5 'with respect to the division site. By "derivative" it is meant that the enzymatic portion of the ribozirna "I" proposed is essentially the sequence shown in Figures 50 and 60. In another additional preferred embodiment, the nucleic acid molecule derived from the ORN of N urospora VS makes contact with a separate ORN duplex substrate molecule, by means of base pair interactions (Figures 8 and 9) and causes division of ORN of duplex substrate at the division site. This interaction improves the speci ty of the ORN split reaction. In another aspect, the invention incorporates the synthesis and assembly of the nucleic acid enzyme in one or more pieces, where the nucleic acid makes contact with a separate substrate ORN molecule and divides the substrate ORN at the site. of division. In another aspect, the invention incorporates a circular nucleic acid molecule that has an enzymatic activity that cleaves a separate RNA substrate into a "j" t-1 or a split-off nucleic acid. methods described in the art (eg, Been and coinventores, UO 93/14218; P? ttaraj? and co-authors, 1993 N? cleic Ocids Res. 21, 4253; Blumenfeld and co-inventors, UO 93/05157) Other aspects and advantages of the invention will be apparent from the following description of the preferred embodiments of the ism, and from the claims.

DESCRIPTION OF THE PREFERRED NUMBERS First, the drawings will be briefly described.

THE DRAWINGS Figure 1 shows a diagramatic representation of a hammerhead ribozimus donunium known in the art. The TT stem can be 2 base pairs long, or may even lack base pairs and consist of a loop region. Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2b is a diagrammatic representation of the hammerhead ribozirna as divided by Uhlenbeck (1987, Nature, 327, 596) on a substrate and an enzymatic portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Na ure, 334, 585) in two portions; and Figure 2d is a similar diagram showing the hammer head divided by Jef Fries and Syrnons (1989, Nucleic Ocids Res., 17, 1371) in two portions. Figure 3 is a diagrammatic representation of the general structure of the hair pin ribozim. ? f This provi es spiro 2 (H ") with less than 4 base pairs (ie, it is l, 2, 3 or 4) and the esp -a 5 can be provided 1 optionally with a length of 2 or more bases (preferably from 3 to 20 bases, that is, rn is from L to 20 or more). The turn 2 and the turn 5 can be connected covalently by means of one or more bases (ie, r is> 1 base). Eepiras 1, 4 or 5 can also be extended in 2 or more base pairs (e.g., 4 to 20 base pairs) to stabilize the ribozyme structure and preferably is a protein binding site. In each case, each N and N 'independently is any normal or modified base, and each dash represents a potential base pair formation interaction. These nucleotides can be modified in sugar, base or phosphate. The complete formation of base pairs is not necessary in the turns, but is preferred. The turn 1 and the turn 4 can be of any size (ie, o and p is in each case independently from 0 to any number, for example, 20) as long as the formation of base pair is maintained. The essential bases are shown as specific bases in the structure; But those who are experts in the field will recognize that one or more can be modified1. chemically (basic, basic, sugar and / or phosphate modifications) or replaced by other bases without significant effects. The turn 4 can be formed to pair L r of two separate molecules, that is, without a connecting ripple. The connector-curl, when present, can be a cleotid ribon with or without modifications in its base, its sugar or its phosphate. The symbol "q" is > 2 bases The connector curl can also be IB replaced with a linkage molecule that is not a nucleotide. The symbol H refers to the bases 0, U or C. The symbol Y refers to the bases of pipmidi a. The symbol "" refers to a chemical union. "Figure 4 is a representation of the general structure of the ribozyme domain of the hepatitis delta virus, known in the art (Perrotta and Been, 1991, supra). Figure 5 is a representation of the general structure of the ORN domain of Neurospora VS autodivisor. B is a linear diagram representing the ribozirna motion "I". The figure shows the "upper" and "lower" base pair regions linked by the "connector" region. IV (left) and V (right) show the left and right regions within the "upper" region respectively. II (left) and VI (right) show the left and right regions within the "lower" region, respectively. Figure 6 is a diagrammatic representation of a division catalyzed by the VSN ORN enzyme, den trans division, of a double stranded duplex ORN. (fl) Stem I is an intramolecular loop formed within the substrate ORN. Stems II to VI are intramolecular loops formed within the ribozyme. (B) is the schematic representation of the minimum requirement of the substrate sequence for division by the ribozimm "I". N refers to any base. N 'refers to any base that is complementary to N. Y 39 It refers to a pipmidma. Figure 7 is a diagrammatic representation of a trans-division catalyzed by the VS RNA enzyme, of a single-stranded ORN. (0) Stem I is an inter-molecular loop formed between the substrate ORN and ribozirna. Stems II to VI are intramolecular loops formed within the ribozyme. (B) is an alternative strategy to facilitate the division of a single-filament ORN by ribozirna T. Figure 8 is a diagrammatic representation of the VSN autodivisor ORN The base pair formation interactions between the nucleotides in loop 1 (6630, U631 and C632) with complementary nucleotides in loop 5 (C699, 0698 and G697) are shown as bold lines. Figure 9 is an enlarged view of the interaction between loop 1 and loop V. (0) shows base pair formation of G630 with C699; U631 with O698 and CB32 with G697. (B) shows the interaction of base pair formation between the nucleotides of loop 1 with the nucleotides of loop V, where N can be any base (for example, A, U, G, C) and N 'can be any base that is complementary to N. By "complementary" is meant a sequence of nucleotides that can form one or more hydrogen bonds with another nucleotide sequence, through the parental formation interactions of traditional Uatson-Cpck bases or other non-traditional types (for example, the Hoogsteen type).

Figure 10 shows the time course of the division of double-stranded ORN (df), by VS RNA. A diagram of the fraction of the substrate ORN divided as a function of time is shown, Figure 11 shows the velocity of the division of RNA by the ribozyme of VS as a function of the concentration of the ribozyme. Figure 12 shows the effect of temperature variation on the reaction of RNA cleavage catalyzed by ribozin VS. Figure 13 shows the effect of pH on the ORN cleavage reaction catalyzed by the ribozyme VS. Figure 14 shows the effect of the idyne concentration on the RNA cleavage reaction catalyzed by the ribozyme VS. Figure 15 shows the effect of the concentration of Mg2 + on the division reaction of ORN, catalyzed by the ribozyme VS. Figure 16 shows the kinetics of the division reaction of ORN catalyzed by the ribozyme VS. (0) shows the effect of ribozyme concentration on the trans division reaction under the optimal reaction conditions. (B) shows the effect of substr-ato ORN concentration on the division reaction in rans under the optimal reaction conditions. Figure 17 shows the increase in the division reaction of ORN catalyzed by the ribozyme VS. The numbers 0, 5 and 30 minutes refer to the duration of the premubation of ORN VS with 100 rnM of viomycin, before the start of the lysis with ORN. - viomicma refers to the catalysis of ORN in the absence of vioinicma. Figure 18 shows the reduction that depends on the vionicma in the concentration of the magnesium chloride required for the catalysis.

THE DESTINATION SITES The destinations for useful ribozymes can be determined as described in Draper and co-inventors, UO 93/23569, Sullivan and co-inventors, UO 94/02595, as well as Draper and co-inventors, "Method and reagent for treatment or arthritic conditions". US Patent No. 08 / 152,487, filed on November 12, 1993, which are hereby incorporated by reference in their entirety. Instead of repeating the guides provided in those documents, in the present, specific examples are given below, not limited to what is known in the art. The ribozirnas pair * said destinations are generally designed as described in those applications and are synthesized to be tested m v tro and in vivo as well as described. Said ribozirna ?, can also be optimized and supplied as described there.

? '? The ribozyme activity can be optimized by chemically synthesizing the ribozymes with modifications that prevent their degradation by serum ribonucleases, modifications that increase their efficiency in the cells and the elimination of the bases containing loops, to shorten the ORN synthesis times and reduce the needs of chemical substances. See, for example, Eckstein and co-inventors, international publication No. UO 92/07065; Perrau.lt and co-authors, 1990 Nature 344: 565; Pieken and coauthors, 1991 Science 253: 314; Usrnan and Cedergren, 1992, Trends in Biochem. Sci. 17: 334; Usman and co-inventors, international publication No. UO 93/15187; and Roesi and co-inventors, international publication UO 91/03/162, as well as Usman, N. and co-inventors, US patent application No. 07 / 829,729, and Sproat, B., European patent application No. 92110298.4; Chowrira and Burke, 1992, supra; Chowrira and coauthors, 1993, .1. Biol. Chem. 268, 19458, which describe various chemical modifications that can be made in the sugar portions of the enzymatic ORN molecules. All these publications are incorporated here for reference. Ribozymes are added directly or can be complexed with cationic lipids, packaged in liposomes or otherwise delivered to target cells. The ORN or complexes with ORN can be administered locally to relevant tissues ex vivo or in vivo, by means of injection, aerosol inhalation, infusion pump or tent, with or without their incorporation into the hiopolymers. S? Llivan and coauthors, his ra, describe the general methods for supplying the ORN molecules? Enzyme? Ca <; _. The ribozymes can be adrift to cells by a variety of methods known to experts who are familiar with the art, including, but not limited to: encapsulation in liposomes by iontophoresis or by incoporation in other vehicles, such as or hydrogels, cyclodext squabbles, biodegradable nanocapsules and bioadhesive microspheres. For some indications, ribozymes ex vivo can be administered directly to cells or tissues with or without the carriers mentioned above. Alternatively, the combination of ORN / vehicle is delivered locally by direct injection or by the use of a catheter, an infusion pump or a store. Other delivery routes include, but are not limited to: intravascular, intramuscular, subcutaneous or articular injection; aerosol inhalation, oral route (tablet or pill form, topical, system, ocular, intrapen toneal and / or intrathecal.) More detailed descriptions of pouch supply and administration are provided in Sullivan and coauthors, supra and in Draper and co-authors , dele, which have been reported here as reference.Another means of accumulating high concentrations of one or more ribozymes within the cells is to incorporate the ribozyme coding sequences into an ODN expression vector. The ribozyme sequences are excited from a promoter for eukaryotic ORN polymerase I (pol I), ORN polymerase II (pol II) or ORN poly-erasa III (pol III) Transcripts from the pol II or pol III promoters will be expressed at high levels in all cells, the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (increments, silence managers, etc.), who are present in the vicinity. Prokaryotic ORN polymerase promoters are also used as long as the prokaryotic ORN polymerase enzyme is expressed in the expressed cells (Elroy-Stein, O. and Moss, B., 1990, Proc. Nati. Ocad. Sci, USO, 87 , 6743-7; Gao, X. and Huang, L., 1993, Nucleic Ocids Res., 21, 2867-72; Lieber, fl., And co-authors, 1993, Methods Enzyrnol., 217, 47-66; Zhou, Y., and co-authors, 1990, Mol. Cell, Biol., 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from said promoters can function in mammalian cells (eg, Kashani-Sabet, M. and coauthors, 1992, Flntisense Res. Dev., 2, 3-15; Ojwang, 3.0. and co-authors, 1992, Proc. Nati, Ocad, Sci. USO, 89, 10802-6, Chen, C.3., and co-authors, .1992, Nucleic Ocids Res., 20, 4581-9; Yu, M., and coauthors, 1993, Proc. Nati, Ocad. Sci. USO, 90, 6340-4, L'H? illier, P.3 and coauthors, 1992, EMBO 3. 11, 4411-8, Liesziewicz, 3. and co-authors , 1993, Proc. Nati, Ocad. Sci. USO, 90, 8000-4)). The activity of said ribozymes can be increased by their release from the primary transcript, by a second ribozyme (Draper and co-inventors, PCT U093 / 23569 and S? Llivan and co-vendors, PCT U094 / 02595, both incorporated herein in their entirety as a reference; Ohka a coauthors, 1992, N? Cleic Acids Syrnp. Ser., 27, 15-6; Taira and coauthors, 1991, Nucleic Ocids Res., 19, 5125-30; Ventura and co-authors, 1993, Nucleic Acids Res., 21, 3249-55; Chowrira and coauthors, 1994, 3. Biol.

Chem. 269, 25856). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including, but not limited to, plasmid DNA vectors, viral ODN vectors (such as adenoviruses or vectors associated with adeno), or viral ORN vectors (such as retroviruses and alpha-virus vectors). In a preferred embodiment of the invention, a transcription unit inserting a ribozin "I" that divides the target ORN is inserted into a plasmid ODN vector or an adenovirus or a viral DNA vector associated with adeno or an iral retro vector. Viral vectors have been used to transfer genes to the lung and these vectors lead to transient gene expression (Zabner and coauthors, 1993, Cell 75, 207; Carter, 1992, Curr. Biology, 3, 533) and both vectors lead to transient gene expression. The adenovirus vector is supplied as recombinant adenoviral particles. The ODN can be supplied alone or complexed with vehicles (as described for the previous ORN). The ODN, the ODN / vehicle complexes or the recombinant adenovirus particles are administered locally to the treatment site, for example, by the use of an injection catheter, an infusion pump, or are added directly to the cells or ex vivo tissues. In another aspect of the invention, the ibozirins are expressed which divide the target molecules from transcription units inserted in ODN or ORN vectors. Recombinant vectors are preferably ODN plasmids or viral vectors. Viral vectors expressing the ribozyme could be constructed based on, but not limited to, adeno-associated viruses, retroviruses, adenoviruses or alpha-viruses. Preferably, recombinant vectors capable of expressing lae ribozrins are delivered locally, as described above, and persist in the target cells. Alternatively, viral vectors that provide the transient expression of ribozymes can be used. Said;, vector-is could be administered repeatedly, as necessary. Once expressed, ribozírnas divide the destination rnORN. The delivery of vectors expressing the ribozirna could be systemic, for example, by intravenous or intramuscular administration, mediant €? administration to target cells explanted from the patient, followed by the reintroduction into the patient of the same, or by means of other files that would allow reintroduction into the desired target cell.

Thus, the ribozymes of the present invention that divide into destination rnOPN and thereby inhibit and / or reduce target activity have many potential therapeutic uses, and there are reasonable ways of supplying the ribozymes in various possible indications. The development of an effective ribozyme that inhibits specific function is described in the art. By "inhibit" is meant that the activity or level of the target ORN is reduced below that observed in the absence of the ribozirna; and preferably it is below that level which is observed in the presence of an inactive ORN molecule, capable of being linked to the same site in the ORN, but incapable of dividing that ORN. By "vectors" is meant any technique that is based on nucleic acid and / or viral based, used to deliver a desired nucleic acid.

EXAMPLES The following materials and methods are used in the following examples: CLONES AND MUTflGENESIS DIRECTED TO THE SITE The Gil clone has been previously described (Guo and coauthors, 1993, 3. Mol, Biol, 232, 351) and contains bases 617 to 881 of the ORN of VS in the vector TZ19R. Mutations were made in clone Gil or DG11 (from which the Scal, Oval, flcyl sites in the vector had been destroyed to facilitate future subcloning, stopping only a single site for each enzyme within the VS sequence). Substitutions on the 5 'or 3' side of a loop were made by oligonucleotide-directed mutagenesis (Kunkel and coauthors, J 87, in Methods Enzyrnol, editors Uu and Gross an, lathe 154, page 367, Ocade c Press, San Diego , CO).; Compensatory molecules were also prepared in this manner, except that a single restriction site separated the 5 'and 3' mutations, in which case recombinant ODN techniques were used to assemble the two mutations into a single clone. Two separate isolates of each mutant were identified and the sequence was established from the T7 promoter to the SspI site, which was the 3 'end of the obtained transcripts used to measure the division rates.

Lfl MEASUREMENT OF AUTODIVISION SPEED The ORN were synthesized by transcription of T7 from plasmid templates lysed with SspI (ORN of VS nt 183). The precursor ORNs were obtained from wild-type and active mutants using reduced magnesium concentrations during transcription (Collins and Olive, 1993, Biochemistry 32, 2795). The transcription reactions were extracted once with each phenol / chloroform. isoamyl alcohol (CIO) and once with CIO and ee precipitated with ethanol. The ORN (approximately 50 nM) was dissolved in water, premubated at 37 ° C and mixed with one fifth volume of 5X buffer (final concentrations: 50 rnM Tps-HCl pH 8, 50 inM KCl, 2 mM spermidine, 10 mM and MgCl2). Aliquots were removed at various times, the pr-ecursor- and ORNs of the product were separated by electrophoresis and quantified using a Phosphorilizer, as previously described (Collins and Olive, supra). The first-order autodivision velocities were determined from the slope of graphs of fractions of non-divided ORN against time.

MUTAGENESIS DIRECTED TO THE SITE The inventors constructed base substitution mutants directed to the site, which could be predicted to break the turns by changing one or more bases on the 5 'or 3' side of the predicted turns. The compensatory mutations that would restore a loop, but using a different base pair, were also constructed. The autodivision rates were measured for the wild type, for the 5 'and 3' mutants and for the compensatory mutant, denoted 5". The data for the representative mutants are shown in table 2.

DNA TEMPLATES AND THE SYNTHESIS OF RNA The fragments of ODN VS were cloned in pTZITR or 19R vectors (Pharmacia). Clone Gil (see Guo and coauthors, 1993, supra) contains the nts of VS 617 to 881, numbered as in Saville and Collins (1990, Cell 61, 685); the division site is between nucleotides G620 and O621. The ORN of substrates were transcribed (see below) from Gil or its derivatives that had been linearly raised at the Oval site (nucleotide 639) or at the SspE site (nucleotide 783) to form the ORNs designated Gll / Ova and Gll / Ssp, respectively. These ORN begin with nine vector nts (5'gggaaagcu, see Figure 5), followed by the VS sequence. A mutant directed to the site, Gil, clone 621U, which contains a single O to U substitution, which immediately follows the site of a? Todivision, was also used. Clone 0-3 that contains the Vs sequences downstream of the Oval site (numbers 640-881), in a derivative of pTZ19R that runs from the Xbal and SphT sites in the multiple cloning site (constructed for- not related to the project described here). Transcripts of clone 0-3 digested with Sspl (VS 783 nucleotide) begin with 9 vector nucleotides (5 'GGGOOOGCU) followed by 144 nucleotides of VS ORN; this ORN is designated the ribozírna Ova or Rz. The ORNs were prepared by transcription of 3J ORN polynerase of bacteriophage T7 i v 1 ro, from the linearized plasmid ODN. The transcription reactions (usually 300 μl) contained 10 to 20 μg of the appropriately linearized template; 1 mM each of NTP (Pharmacia), 5 M of dithiothreitol, IX of polymerase regulator T7 (Bethesda Research Laboratories: 40 rnM Tris-HCJ, pH 8.0, 8 rnM MgCl2, 25 rnM NaCl, 2 M of spermidine-HCl3); 300 U of ORNguard (Pharmacia), 150 to 200 units of ORN T7 poly erasa (Bethesda Research Laboratories) for 2 hours at 37 ° C. The radioactive transcripts were prepared as above, except that an additional 30 Ci of L "a-32p] GJP was added (or, for specific experiments, ATP or UTP).

Subsequently the samples were treated with DNase I (Pharmacia; 5 U / μg ODN template) for 15 minutes; then EDT was added at 10 rnM, the ORN was extracted with phenol: chloroform: isoamyl alcohol, chloroform: isoamyl alcohol (CIO) and ethanol, was precipitated in the presence of 0.3 M sodium acetate, pH 5.2. The precipitated ORN and two volumes of sec-chalker dye (95% forrnarnide, 0.5X TBE, 0.1% xylene-cyanol, 0.1% bromophenol blue) were dissolved in water and heated to 75 ° C for 3 hours. minutes and fractionated by electrophoresis on denaturing polyacrylamide genes (40: 1 acrylamide: bis-acrylamide), of appropriate concentration, containing 8.3 M of urea and IX of TBE (135 M of Tris, 45 mM of boric acid, 2.5 M of EDTO). The ORN either by autoradiography or by UV shading. The bands of interest were cut, eluted overnight at 4 ° C in water and filtered to remove residual polyacrylamide. The ORN was precipitated with ethanol in the presence of 0.3 M sodium acetate and dissolved in water. The concentrations were determined spectrophotometrically, assuming that 1 O26O corresponds to an ORN concentration of 40 mg / ml.

FINAL MARKING OF ARN The ORNs were marked at the 5 'ends using the polynucleotide qasease T4 and L ~ g-32P] OTP, or at the 3' ends using T4 ORN-ligase and 5 'I ^^ Pl pCp. The labeled ORN at the end was fractionated on denaturing polyacrylamide gels and detected by autoradiography. In order to eliminate the 5 'triphosphates before the 5' end was labeled, some ORN were treated with 1 U of calf intestinal alkaline phosphatase (Boehringer-Mannhein) in a 10 μl reaction containing 50 rnM of Trie. -HCl pH 8.0, 0.1 rnM EDTO at 55 ° C, for 30 minutes. The reactions were determined by extraction with phenol: CIO and with CIO.

TRANS-DIVISION REACTIONS The transection of the substrate NOS (S) was carried out by means of the ribozyme Ova (Rz), after pre-incubation of gel-purified S and Rz of the appropriate reaction solution IX, for 2 minutes. The reactions were initiated by adding ribozirna to the substrate in a final volume of 20 μl. In a typical reaction, < ..e removed 10 aliquots of 1.5 μl at specified times, was terminated by the addition of 13.5 μl of the stop mix (70% forrnarnide, 7 inM EDTO, 0. 4x TBE, 0.07% xylene-cyanol and 0.07% bromine blue phenol) and stored on ice. The mixtures were fractionated by means of electrophoresis on 20% polyapyl lick gels, denaturing them. The effects of temperature, pH, MgCl2 and spernidium na- (HCl) 3 on the trans-division reaction were analyzed by incubating equimolar concentrations of Rz and S (0.05 μM each) in solutions described in the legends of the figure. A final study of the effects of MgCl2 under conditions that were otherwise "optimized" was carried out at 30 ° C, 50 rnM Tris-HCl pH 8.0, 2 M spermidine, 25 mM KCl. Experiments were carried out to establish conditions of a single change (Figure 10) at 30 ° C in 50 mM Tps-HCl pH 7.1, 25 M MgCl2, 25 mM KCl, 2 mM esperrnidma. The analysis of the effect of pH on the conditions of a single change (Figure 13) were made as above, except that the concentrations of Rz and S were 5 μM and 0.13 μM, respectively. 50 rnM of Tris-HCl was used for pH 7.1 and 8.9; J6.5 rnM of PIPES / 44 rnM of Tris for pH 6. The quantities of substrate and products were quantified using the programs Phosphorlmager "and IrnageOuant" version 3.0 (Molecular Dymanics, Sunnyvale, CO, EUO). The estimates of the initial division rates were derived from the graphs of the fraction of the substrate divided against time, using the Grafit program (Erithacus Software Ltd, Staines, United Kingdom). It was possible to divide up to 90% of the substrate in 60 minutes, at an approximately equimolar concentration of ribozyme, the curve indicating the presence of approximately 10% unreacted starting material. The curves were not adjusted to complete 100%, and the nature of the non-reactive substrate was not further characterized.

EXAMPLE 1 ANALYSIS OF MUTATIONAL RNA FROM VS AUTODIVISOR As a starting point for the prediction of the structure, the inventors used the MFOLD program of Zuker et al. (Zuker, 1989, Science 244, 48) to obtain five main families of thermodynamic models that are reasonable for the minimum autodivisor ORN. The models differed in the number or length of the loops and / or the participants in the predicted pair formation for a given region of the sequence, and varied from the structure predicted to be very stable to the bipolar doublings, 10% less stable than the structure of lower free energy.Lae structures within this scale of free energy have been found to predict most turns in other ORN (Jaegar and coauthors, 1989 Proc. Natj. O. Sci. USA 86, 7706). These various structural models were tested to take advantage of site-directed mtotagenesis. Of the various models evaluated, the one shown in Figure 5O was the most consistent with the data obtained from the division activity of all the mutants. In general, mutations on the 5 'or 3' side of the predicted loops II to VI, corrected the ribozyme or decreased the activity well below that obtained with the wild-type sequence. The compensatory substitutions that restored a loop but with a different base sequence restored the activity usually to that of the wild type or higher, but always at a level at least greater than that of the 5 'or 3' mutants. These data showed that the regions of each of the turns play roles that are not specific to the sequence, but that are presumably involved in the proper folding of the ORN. In some cases, mutations on the 5 'and 3' side did not reduce activity to the same extent. For example, the mutant Va5 'does not show essentially activity, but Va3' retains more than half the activity of the wild type. It may be that the particular substitutions selected do not alter the loop equally well, or that one of the bases makes a specific contribution to the local or tertiary structure (Cech, 1988, Gene 73, 259).

In some positions the activity could not be restored by the attempted compensatory substitutions; even when restoration was possible in other positions of the same loop. This was especially common in predicted base pair pairs adjacent to natural breaks in a loop, such as adenosines not formed in pairs at positions 652 and 718 (Table 2, eg, positions ITc and Tile). The G653C mutant showed no activity, as did each of the three substitutions in the predicted complementary position C771; the double G653C: C771G showed some restoration of activity, but was still ten times slower than the wild type (mutant lie). Sirnily, the pair 0661: U717 immediately above the unformed O718 could not be replaced by U: 0 (lile mutant), even when the s? Gui € > This pair, C662: G716, could be suctitized by a G: C (mutant Tllb). The omission of unpaired adenosm to torque also decreased activity, as severely as in the case of 0652. These observations suggest that specific local structures may be especially important in these areas, or that some of these bases may be involved. in interactive and / or additional interactions. The structure and sequence requirements of coil I appear to be more complex than what is implied by the model in figure 5. Although several base substitutions severely decreased activity (e.g., rnu-tantes Ia5 ', Ic3' ) other mutations that could be expected to have an equally disturbing effect on the loop (rn? tantes Ia3 ', Ib3", Ic5') decreased the activity only slightly. No position has been found in which the compensatory substitutions that have rehearsed the activity well above the level of the individual mutants.This may be due, in part, to the stem of 5 GC pairs, possibly prolonged by non-Uatson-Cp c interactions, which could be predicted to be rn The stability and stability of loop I is supported by the sounding of the chemical structure and the difficulties in determining the sequence in that region. Taken together, these observations suggest They suggest that certain bases in turn I could be involved in alternative secondary structures or in tertiary interactions that are crucial to the activity. Based on the above data, the inventors have constructed a model for the secondary structure of the ORN a? T-odivisor of VS, which contains the minimum contiguous region of VS ORN required for autodivision. In five of the six turns proposed in the model, site-directed substitution mutations directed to the site that alter the loop diminish or eliminate activity. Compensatory substitutions restore activity, usually at the wild type level or even higher. These data provide strong support for a presumably structural role, independent of the sequence, for the portions of those five turns. Several observations suggest that the formation of the active structure is more complicated than what is implied above. While the mutants directed to the site of turns II to VI indicate that portions of those turns have a structural role independent of the sequence, the mutants in the spiral I show a more complex pattern. Mutations at certain positions in turn I inactivated the ribozyme, but compensatory substitutions did not restore activity. Traditionally, there is evidence of site-directed mutagenesis and compensatory substitutions for a tertiary interaction, which requires the unwinding of at least the upper base pair in turn T (G628: C632) to allow an interaction with the curl V (see figures 8 and 9). Taken together, these observations suggest that a substantial conformational change in the T-turn may occur under natural conditions. The model predicts that the VSN ORN contains certain structural aspects found or predicted in other ORNs. Spiral VI crowned with the GUOO tetrahedron is an example of a GNRO curl that is common in rflRN (Uoese and coauthors, 1990 Proc. Nati. Ocad. Sci. USE 87, 8467) and contains internal hydrogen bonding and interactions. that are stacked, that stabilize the curl structure (He? s and Pardi, 1991 Science 253, 191; Santa-Lucia and co-authors, 1992 Science 256, 217). The secondary structure of the VSN ORN is different from the hammerhead and hairpin ribozirnas because, although a short loop could be formed upstream of the division site in the VSN ORN, it is not required for activity. (Guo and coauthors, 1993, supra), co o in these two ribozymes (Foster and Syrnone, 1987 Cel 1 50, 9, Berzal-Herranz and coautoree, 1993 EMBO 3. 12, 2567). In addition, the ORN of VS does not contain the series of bases that are known to be important for the activity of hammerhead ribozirnas (Synon, 1992 Onn, Rev. Biochem. 61, 641) or of hair pin (Berzal-Herranz et al. coauthors, s? pra). Like the VSN ORN, the VHD ribozyme (Been, 1994 TIBS 19, 251) requires only a single nucleotide upstream of the cleavage site, and a GC-rich loop is downstream of the cleavage site in both ribozymes. Beyond these similarities, however, secondary structures have nothing in common.

EXAMPLE 2 REACTION OF TRANS-DIVISION CATALYZED BY VS RNA The trans reaction described below was constructed using various restriction fragments of VS ODN cloned in T7 promoter vector to construct pairs of non-overlapping regions of VSN ORN. One member of each pair, the substrate (S), contained the expected cleavage site, which followed nucleotide G620 (numbered as in Saville and Collins, 1990 supra); the other, the enzyme or ribozi a (Rz) contained the remainder of the VS sequence, terminating at the SspI site at nucleotide 783. In preliminary experiments, these transcripts were mixed at approximately a 1: 1 ratio and incubated under known conditions for support self-division (Collins and Olive, 1993, supra). Most combinations showed little or no division; however, the almost complete division of an ORN substrate of 32 nucleotides, ending in the Oval site (nucleotide 639), was observed during a one hour incubation with a ribozyme that starts at the Oval site and q? e ends at the SspI site (nucleotide 783); no division was observed in the absence of the ribozyme. The electrophoretic mobility of the two cleavage products was approximately that expected for division after nucleotide 620, which is the intramolecular autodivision site of VSN ORN. The inventors selected to examine this trans-division reaction in greater detail.

EXAMPLE 3 TRANS-DIVISION OCCURS ON THE SAME SITE AS SELF-DIVISION To determine the precise cleavage site, the Gll / Ova substrate was labeled, with Pl and P2 at its 5 'ends and the sequence was determined by partial enzymatic digestion using the RNase TI or U2. The products of dividing a substrate that contains a substitution 3 'of a single base of the division site (O621U) have also been characterized to resolve possible ambiguities due to the anomalous migration (ie some bands. the substrate and Pl are identical in sequence from the 5 'site to the cleavage site, all the RNase sequence-determining bands migrated together, as expected. The entire Pl igro stretch together with the thirteenth RNase TI fragment Gll / Ova nucleotides, which ends in G620, which is the intramolecular a? todivision site at the ORN of VS. Odernás, the 3 'end of Pl was found to be guanosim 2'3' cyclic phosphate, This indicates that both the site and the chemical path of the division in trans are the same as in the autodivision reaction, as expected from the discovery of a cyclic phosphate at the 3 'end of Pl, a group 5 was found. '-h? droxyl in the extr emo 5 'of P2, which was evidenced by its extreme labeling by Cg-32p] or? p and? a q? mass of the T4 polynucleotide, without the previous treatment with phosphatase. The alkaline hydrolysis ladles of P2 labeled at the 5 'end only contained 18 of the 19 expected bands. This is the result of a compression artifact that involves the formation of a structure of such lo-rizo very stable in the longer ORN; this is described later in detail. However, the nucleotides of the 5 'terminal of P2 derived from the division of Gll / Ova S and the mutant O621U were O and U, respectively, which confirms that the division occurred between nucleotides 620 and 621, as in autodivision reaction.

EXAMPLE 4 MINIMUM LENGTH OF SUBSTRATE RNA In order to determine the minimum required sequence downstream of the cleavage site, the inventors essentially used the approach that was described by Foster and Syrnons (1987 supra). The Gll / Ssp ORN, marked at the 5 'end, was partially hydrolyzed by treatment at high pH, then incubated with or without the ribozyme. Incubation in the absence of ribozyme confirmed the inventors 'prior discovery that the total ORN length of Gll / Ssp and the default derivatives lacked ten or less n? Cleidee at the 3' end and that they can self-divide (Guo). and coauthors, 1993, supra). Incubation with the ribozyme resulted in the disappearance, or at least the decrease in intensity, of the bands q? E corresponding to the ORNs q? E ending at nucleotide 639 or greater. A few ORNs were not completely divided under these conditions, which indicates that there are relatively lesser substr ats. The substrate of length rninirna ends in residue 639, which coincidentally corresponds precisely to the ORN used in figure 6, which was synthesized by carrying out the transcription of a template linearized in the Oval site. Therefore, only 19 nucleotides are required downstream from the division site for trans-division by the Ova ribozyme. A parallel experiment using labeled ORN at the 3 'end showed that only a single nucleotide upstream of the division site is necessary for trans-division. Taken together with the results of the ORN tagged at the 5 'end, these data show that the minimum contiguous region of the natural ORN required for trans division consists of a nucleotide going upstream of the division site and 19 nucleo- t gone :; downstream.

EXAMPLE 5 THE MINIMUM SUBSTRATE RNA CONSISTS PRIMARILY OF A RIZO OF PIN FOR THE HAIR The prediction of ORN structure using the MFOLD program of Zuker et al. (Zuker, 1989, supra) suggests that the terrnodynamically more reasonable structure of substrate ORN would be a structure containing the hair pin shown in Figure 6. During In the characterization of the trans division products, the inventors noted several observations that were consistent with that structure. P2 migrated faster than expected with respect to size markers for an ORN of 19 nucleotides, suggesting that it contained a structure that was not completely denatured, even in a gel containing 8.3 M urea Some guanosine residues (623 -625, 527 and 633) and adenosine (621 and 622) in S and P2 were weakly divided or not at all by the TI and / or U2 RNases, not even when sequencing reactions were carried out under denaturing conditions putatively of 50 ° C, 1 rnM of EDTO and 7 M of urea. Only 18 of the 19 expected bands were observed in the partial alkaline hydrolysis products of P2, labeled at the 5 'end.

EXAMPLE 6 BIOCHEMICAL CHARACTERISTICS OF THE REACTION CONDITIONS OF DIVISION IN TRANS The inventors have investigated the effects of various variables that would be expected to affect the ORN structure and that have been found to affect the cleavage rates of other ribozymes. An equirnolar ratio of S and Rz (0.05 μM of each) was used for most of the initial investigations; A more detailed analysis specifically under conditions of sustained state or under conditions of a single change, is described below. The rate of division increased with temperature until the optimum point was reached around 30 ° C and then abruptly decreased above 40 ° C (Figure 12). No reaction was observed in the absence of a divalent cation, and the reaction rate increased with increasing MgCl2, reaching a maximum at around 100 M, when magnesium was the only cation present. To determine if some of the MgCl2 was acting simply as a structural counter ion, the effects of spermidine (Figure 14), NaCl and KCl were investigated in the presence of a saturating concentration of MgC.1.2 (10 mM). In the presence of 10 mM MgCl2, spermidine at 1 mM or higher increased the cleavage velocity by about ten-fold, compared to the same reaction without spermidine (FIG. 14). Low KCl concentrations (less than 100 rnM) also stimulated the reaction rate, up to about ten times. Perhaps surprisingly, NaCl had almost no effect. These observations are similar to the effects of previously observed cations on the VSN ORN autodivision rate (Collins and Olive, 1993 supra). The reaction rate showed only a small pH dependence: the almost 100-fold increase in hydroxide concentration between the pH of 7.1 and 8.9 resulted in only an increase of twice the speed (Figure 13). The effect of pH specifically under conditions of a single change is described below. Finally, the effect of MgCl was analyzed again; 2 under "optimized" reaction conditions containing 50 rnM Tris, pH 8.0; 2 mM spermidine, 25 M KCl, and incubated at 30 ° C (Figure 15). Under these conditions, 10 mM MgCl2 allowed the same cleavage rate as the reaction containing 70 mM MgCl2, under suboptimal conditions. In this way, the combiner effects of temperature, pH and cations different than the magnesium salt, increased the division substantially. However, no reaction was observed in the absence of MgCl2, indicating that neither esperrnidine nor KCl could replace magnesium in the division.

EFFECTS OF pH UNDER CONDITIONS OF ONLY ONE CHANGE The transession reaction rate only showed a small pH dependence at equimolar concentrations of ribozirna and the substrate (Figure 13). However, these experiments were carried out at saturable concentrations of MgCl2 and probably were not under conditions of a single change. Consequently, it was possible that some step in the reaction, other than the actual division step, could have been the speed limiting step, thus masking the effect of the increased concentration of the hydroxide ion. To investigate this possibility, conditions of a single change under optimized reaction conditions were established empirically, measuring the initial trans-division rates of 0.13 rnM of substrate, increasing ribozyme concentrations. The initial rate of division increased with the ribozyme concentration to approximately 2.5 nm and subsequently leveled, suggesting that the reaction approached the conditions of a single change (FIG. 11). The rate of division, as a function of MgCl2 concentration, was again investigated using 0.13 μM of S and 5 μM of Rz, and it was found to be essentially the same as in Figure 15. One was selected. 25 M concentration of MgCl2 to ensure that magnesium was not limiting. Transdivision reactions, using 0.13 μM substrate and 5 μM ribozyme, on a pH scale, showed only a minor improvement in the reaction rate.

THE KINETICS OF SUSTAINED STATE REACTION To determine if the Ova ribozyme is capable of multiple changes, Rz was incubated with approximately a 20-fold molar excess of S (Figure 16). If each ribozyme molecule divided only? N only substrate, a maximum of 1/20 of S could be divided. In contrast, it was observed that the division continued at a constant speed until approximately 40% of S was divided, then it decreased slowly as the concentration of undivided S, available, decreased. This indicated that the ribozyme Ova behaved as a true enzyme, since it was capable of multiple rounds of division. In addition, as expected from an enzyme, the initial rate of division was directly proportional to the concentration of the ribozyme under conditions of excess substratum (Figure 16).

The trans-division reaction exhibited a saturation curve with respect to the substrate concentration that is typical for the Michaelis-Menten kinetics (Figure 16B). A KM of 0.13 μM and kCat of 0.7 minutes-* was obtained from these data. It had been observed that these values varied by about a factor of about two when the experiments were repeated with different ribozirna loads, over a period of two years. The inventors have modified the natural intramolecular autodivision reaction of the VSN ORN by constructing a ribozyme containing 144 nucleotides of VSN ORN, which is capable of an intermolecular trans-division reaction. This ribozyme acts as a true enzyme by dividing an ORN of substrate of 32 nucleotides. In the presence of excess substrate, the initial rate of division is proportional to the ribozyme concentration; and a single ribozyme molecule can divide multiple substrate molecules. The ribozyme is specific for dividing a single phosphodiester ligature, which is divided into the natural self-dividing reaction. The trans-division reaction exhibits Michaelis-Menten kinetics with Km = 0.13 μM and kcat = 0.7 in-1, approximately. Fedor and Uhlenbeck (1990 Proc. Nati, Ocad, Sci. USO 87, 168) have noted that kcat values on the 1 min-1 scale and Km values on the nano-olar scale are characteristic of many diverse ribozymes. The shorter contiguous region of VSN ORN functions as the substrate for the ribozyme described here contains a single nucleotide upstream of the cleavage site and 19 nucleotides downstream. The prior characterization of the inventors of the intramolecular autodivision reaction also showed that only one nucleotide upstream of the dividing site was required (Guo et al., 1993).; in this sense, VS is similar to VHD ribozymes, which also requires only a single upstream cleotide for autodivision or trane-division (Been, 1994, supra). The substrate consists mainly of a stem-loop structure, flanked by three nucleotides at the 5 'and 3' ends, some of which may be involved in a non-Uatson-Crick structure (Figure 6). This conclusion is based on the predictions of minimum free energy, aberrant electrophoretic mobility and the accessibility pattern to specific nucleases for a single filament. The alteration of some base pairs in the stem, by certain substitutions of a single base has little or no effect on the autodivisión. However, in some positions, the identity of one of the bases in a particular pair is critical: even when the compensatory substitution is made in the complementary position to reestablish the turn, division can not be restored. The inventors believe that the specific bases in the specific positions are more important than simply the presence of a stem structure.

The stem-loop structure of the VSN substratum does not leave long regions available for the formation of Uatson-Crick pairs with the ribozyme. The secondary structure of the VSN ORN of minimum self-division has been determined and a working model for the structure of the ribozyme has been proposed (figure 5). The ribozíma does not have regions of a single long filament (I say, of more than 5 nucleotides). This is in contrast to most trans action ribozymes, derived from hammerhead, hairpin, VHD and mtron group I ORNs, which have been designed to interact with single-filament regions of its substrates, by forming one or two inter-molecular spirals that flank the site to be divided. In addition to the formation of base pairs, it is known or suspected that tertiary interactions contribute to the attachment to the substrate of various ribozymes (P le and co-authors, 1992 Nature 350, 628). In fact, tertiary interactions alone are sufficient to allow a very weak binding (KM> 0.1 μM) but specify in the stem-curl Pl of a mtr-on of group I, to its catalytic core (Doudna and Zsostak, 1989 Nature 339, 519). RNase P also recognizes substrates that contain substantial secondary structure and have a very limited potential to form Uatson-Crick pairs with ribozyme (Guerrier-Takada and Altrnan 1993 Bioche ist 32, 7152). It was noted in the previous characterization of the VS RNA autodivision reaction, that the division rate was 5.1 affected essentially by the pH (Collins and Olive, 1993, s? pra). Consistent with that observation, the transdivision reaction described here also shows little dependence, if any, on the pH, even when examined under conditions of a single change. These observations differ from the results examining the speed of the chemical cleavage step of hammerhead ribozymes (Dah and coauthors, 1993 Biochernistry 32, 13040), RNase P (Guerrier-Takada and coauthors, 1986 Biochernistry 25, 1509; Smith and Pace, 1993 Biochernistry 32, 5273, Beebe and Fierke, 1994 Biochemist and 33, 10294) and the group I intron of Tet.rahy na (Herschlag and coauthors, 1993 Biochemistry 32, 8312). For these ribozirnas, the velocity of the division step was not found to increase with increasing pH. Failure to observe the dependence of pH on the ORN of VS could mean that OH is not involved in the division reaction, that the reaction proceeds by means of a novel mechanism or, more likely, that the reaction in VS trans is not limited by the speed of the chemical division step under those conditions, but rather by some step that precedes the actual division. An interesting candidate for said rate limiting step would be a change in the conformation of the substrate and / or the next binding to the ribozyme. 0 the saturating ribozirna concentration, the pseudo prirner speed constant order for the trans-division of S (approximately 0.6 rnin-1) z? it is approximately 10 voices greater than the autodivision rate of the 611 ORN under similar conditions (Collins and Olive, 1993, supra). Since it is contemplated that the trans-division reaction essentially recreates the same ORN conformation as in the autodivision reaction, the higher velocity suggests that the divisible conformation can be more easily reached when S (stem-ripple I; ) is not restricted by the covalent binding to the ribozirna nucleus. In support of this idea, it was also found that Gil's ORN aAddivision velocity could be increased several times by increasing the distance between stem-ripple I and the pbozima core. These observations are consistent with the idea that at least one conformational change involving the stem of the substrate occurs during the reaction. The optimal temperature for the transdivision reaction is substantially less than that of the autodivision reaction (30 ° C versus about 45 ° C) and the activity decays much more rapidly at higher temperatures (Collins and Olive, 1993 s? Pra). The retention of the activity at higher temperatures in the autodivision reaction indicates that the active site of the ribozyme does not begin to denature until at least 45 ° C. The optimum temperature of the trans-division reaction may reflect a decreased bond of the substrate at higher temperatures. The observation that the VS ribozyme can recognize a substrate containing a stable secondary structure can be useful from the perspective of the ribozyme engineering. Among the limitations for modifying the hammerhead, hairpin, or group I intron ribozymes to divide the non-native target RNAs, is the requirement that the target site be a single filament region to allow the recognition through the formation of base pairs with the ribozyme. Because the cleavage site for the VS ribozyme is adjacent to a stable secondary structure, the VS ribozyme may have unique properties that can be adapted to divide certain ORNs that are not accessible to the action of other ribozymes.

EXAMPLE 7 IMPROVEMENT OF THE REACTION OF RNA DIVISION, MEDIATED BY ANTIBIOTICS, CATALYZED BY THE RIBOZÍMA DE VS Several examples of the inhibition of the function of a ribozyme or of the interaction of ORN-protein have shown that certain antibiotics can interact speci? Cally with RNA (Yar? S, 1988 Science 240, 1751; Schroeder and co-authors, 1993 Science 260 , 1443). Small peptide antibiotics, such as violet, have been shown to inhibit the reactions of certain ORNs and certain complexes of ORN-protein (Liou and Tanaka, 1976 BBRC 71, 477, Uank and co-authors, 1993 3. Mol. Biol. 236, 1001). The Applicant has found that some peptide antibiotics (e.g., viomycin) increase the division of ORN catalyzed by the ribozyme VS. Antibiotics decrease, by an order of magnitude, the concentration of metal ions required for ribozyme activity. Orally, viomycin facilitates intermolecular interactions between VSN ORN molecules. Referring to figure 17, the ORN of VS with 100 mM of viomycin for 0, 1, 15 and 30 minutes before adding the reaction regulator (40 mM Tris-HCl pH 8.0, 50 nMM KCl and 10 mM MgCl2). The reaction is carried out at 37 ° C and aliquots are taken at regular intervals of time and the reaction is stopped by adding an equal volume of for amide retaining buffer. The reaction products are resolved on denaturing polyacrylamide gels. A graph of the fraction of the substrate divided as a fraction of time is carried out. The divided ORN fraction increased with an increase in pre-incubation time. The improvement mediated by the antibiotic in the division rates is observed in solutions that already contained optimal concentrations of magnesium and KCl. Referring to figure 18, the antibiotic mediated decrease in the requirement of divalent cation (Mg2 +) is discussed. The split reaction of catalyzed ORN > ) > The ribozirna of VS is analyzed by concentrations of magnesium chloride. The VSN ORN was preincubated with 75 rnM of viornicine for 30 minutes in the presence of 40 rnM Tps-HCl. The reaction was started at 37 ° C by adding varying concentrations of MgCl2 - A graph of the velocity (rmn-1) is shown, as a function of time. The presence of viornicin seems to significantly decrease the MgCl2 requirement in the reaction. The sequences mentioned in figures 6-9 are indicative that they are not limiting. Those skilled in the art will recognize that the vandals (base substitutions, omissions, insertions, mutations, chemical modifications) of the VS ribozyme can be easily generated using techniques known in the art, and are within the scope of the present invention.

USES IN DIAGNOSIS The methods of this invention can be used as diagnostic tools to examine genetic deviations and mutations within diseased cells, or to detect specific RNA molecules, such as the virus ORN. The intimate relationship between the ribozyme activity and the target ORN structure allows the detection of mutations in any region of the molecule that alters the formation of base pairs and the three-dimensional structure of the target ORN. By using multiple ribozymes described in this invention, the changes in nucieotide that are important for the structure of the ORN and the viral function, as well as in the cells and tissues, can be enhanced. The division of target ORNs with ribozymes can be used to inhibit gene expression and define the role (essentially) of the gene products specified in the progression of the disease. In this way, other genetic destinations can be defined as important mediators of the disease. These experiments will lead to the best treatment of the progression of the disease producing the possibility of combination therapies (for example, multiple ribozymes that are intended for different genes, ribozymes coupled with known small molecule inhibitors or intermittent treatments with combinations of ribozymes and / or other chemical or biological molecules). Other in vitro uses of the ribozymes of this invention are well known in the art and include detection of the presence of rnflRN associated with related condition. This ORN is detected when determining the presence of a division product after treatment with a ribozirna, using the common and current methodology. In a specific example, ribozymes that can only divide the wild-type forms or the mutant forms of the target RNA are used for the analysis. The first ribozyme is used to identify the wild type RNA present in the sample, and the second pbozyme will be used to identify the mutant ORN of the sample. As reaction controls, the synthetic substrates of both the wild-type ORN and the mutant will be split by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of division in the "non-target" ORN species. The splitting products of the synthetic substrates will also serve to generate size markers for the analysis of the wild-type and rNastic ORNs in the sample population. Osí therefore, each analysis will require two ribozímas, two substrates and an unknown sample that will be combined in six reactions. The presence of the cleavage products will be determined using the RNase protection analysis, so that the entire length and division fragments of each ORN can be analyzed on a polyacrylaminic geJ line. It is not absolutely necessary to quantify the results to obtain an introduction in the expression of the mutant ORNs and the putative risk of the desired phenotypic changes in the target cells. The expression of rnORN whose protein product is involved in the development of the phenotype is adequate to establish the risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of ORN levels will be adequate and will lower the cost of the initial diagnosis. The higher proportions of wild-type to wild-type will be correlated with a higher risk, whether the ORN levels are qualitatively or quantitatively compared.

TABLE I CHARACTERISTICS OF RIBOZYMS I troñes of group I Size: ~ 2Q0 a > 100 nucleotides Requires a U in the target sequence immediately 5 'of the division site. Unites 4-6 nucleotides in the. 5 'side of the division site. More than 75 known members of this class. It is found in the r-ORN of Tetrahyrnena therrnophila, the fungal mitochondria, chloroplasts, T4 phage, blue-green algae and others. ORN of ARNseP (MI RNA) Size: ~ 290 to 400 nucleotides. ORN portion of a riboprotein enzyme. Divide the tORN precursors to form tORN- mature. Approximately 10 known members of this group are all of bacterial origin. Hammerhead Ribozyme Size: ~ 13 to 40 nucleotides. It requires the destination sequence UH immediately 5 'with respect to the division site. Join a variable number of nucleotides on both sides of the dividing site. 14 known members of this class. It is found in many plant pathogens (vir? Soides) that use the ORN co or infectious agent (figures 1 and 2). Hair pin ribozirna Size: around 50 nucleotides. It requires the GUC sequence immediately 3 'with respect to the division site. Unites 4 to 6 nucleotides in the. side 5 'of the division site and a variable number on the 3' side of the division site. Only 3 members of the class are known. It is found in three plant pathogens (the ORN satellites of the tobacco ring virus, arabis mosaic virus and the mottled yellow chicory virus), which use the ORN as an infectious agent (figure 3). Ribozyme of hepatitis virus delta (VHD). Size: 50 - 60 nucleotides (currently). Recently demonstrated the division of the destination ORN. The sequence requirements have not been fully determined.

The binding sites and structural requirements have not been fully determined, although 5 'sequences are not required with respect to the division site. Only one member of this class is known. It is found in the human VHD (figure 4). Ribosome of ORN of Neurospora VS. Size: about 144 nucleotides. It is found in the Ne? Rospora VS ORN (figure 5O).

TABLE II EFFECT OF BASE SUBSTITUTIONS ON THE SPEED OF VS AUTODIVISION ORN Const ante of velocity of the mutant divided by the constant of velocity of Gil of the type silves- e. The velocity constant for Gil varied from 0.06 to 0. 08 rn? N-? . The division rate was not accurately measured, but similar to 1 wild type. These rulers were made in a vanante of Gil that contained two paree of different bases in the turn V (mutant see) Velocities were normalized using the mutant Ve as the relevant wild type. The division speed was not determined.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS I. Ribozyme capable of dividing an ORN molecule from a separate substrate, characterized in said ribozyme because it has three regions of base pairs, generally in an "I" configuration, wherein the regions of upper and lower base pairs, comprise between 4 and 80 bases inclusive, of which at least 50% are formed in pairs with each other, and wherein a connecting region between the upper and lower base pair regions comprises between 4 and 20 bases inclusive, of which at least about 50% are formed in pairs if "2.- The ribozyme in accordance with the claim 1, further characterized in that the connector region further comprises a region of a single filament, between 1 and 7 bases, inclusive. 3. The ribozyme according to claim 1, characterized in that said single-filament region is adjacent to the upper region of base pairs. 4. The ribozirna according to claim 1, further characterized in that the upper region comprises a left portion and a right portion, each of which has between 3 and 30 bases, inclusive. 5. The ribozyme according to claim 1, further characterized in that the lower region comprises a left portion and a right portion, each of which has at least 3 to 30 bases, inclusive. 6. The ribozin according to claim 1, further characterized in that the lower region comprises at least one protruding base. 7. The ribozyme according to claim 1, further characterized in that the connected region comprises at least one outgoing base. B. ~ The ribozyme in accordance with the claim 1, further characterized in that the region of higher base pairs - comprises bases not formed in pairs with other bases in the upper region of base pairs, which are available to form a base pair with a substrate ORN. 9. The ribozyme in accordance with the claim 8, further characterized because the bases that are not formed in pairs comprise at least 3 bases. 10. The ribozyme according to claim 1, further characterized in that the substrate for the ribozyme comprises a region of base pairs comprising at least two base pairs. 11. The ribozyme according to claim 10, further characterized in that the substrate comprises the sequence 3 'GONN 5', wherein the division by the ribozyme is between said N; and where each N is independently any base. 12. - The ribozyme according to claim 1, further characterized in that the region of base pairs comprises bases not formed to bases at its 5 'end, available to form base pairs with? N substrate RNA. 13. The ribozirna in accordance with the claim 1, further characterized in that the ribozyme makes contact with the ORN substrate only 3 'with respect to the division site. 14. The ribozirne according to claim 1, further characterized in that the ORN substrate is? N double-stranded ORN, wherein the nucleic acid molecule is able to make contact with the dual-filament ORN substrate only. 'with respect to the division site, and cause the division of the RNA substrate at the division site. 15. The pbozirna according to claim 1, further characterized in that the ORN substrate is an ORN of? N single filament, and wherein the ribozirna is able to make contact with the ORN substrate of? N single filament only 3 'with respect to the division site and cause division of the ORN substrate at the division site. 16. The ribozyme according to claim 1, further characterized in that the nucleic acid molecule is derived from Ne? Rospora VS. ORN. 17.- The pbozirna in accordance with the claim 1, characterized in that the ribozyme is active enzymatically active to cut an ORN duplex having at least two base pairs. 18. The ribozirna according to claim 1, further characterized in that the ribozyme is enzymatically active to cut 5 'with respect to the sequence NOGNnGUCNm, wherein each N is independently any base of nucleotide, where n and m are independently an integer between 3 and 20, inclusive; and wherein the sequence forms at least two internal base pairs. 19. The ribozirna in accordance with the claim 1, further characterized in that the ORN substrate binds to the ribozyme at a site distant from the division site. 20. - The ribozymes according to claim 1, further characterized in that the ribozirna is a circular molecule; wherein the circular molecule makes contact with a separate ORN substrate and causes the division of the ORN substrate at a dividing site. 21. The ribozyme according to claim 1, further characterized in that the ribozyme comprises ribonucleotides. 22. A cell characterized in that it comprises nucleic acid encoding the ribozyme of claim 1. 23. An expression vector, characterized in that it comprises nucleic acid encoding the ribozin of claim 1, in a manner that allows the expression of the ribozyme within a cell. 24. - A cell characterized in that it comprises an expression vector of claim 23. 25. ™ An expression vector according to claim 23, further characterized in that the ribozirna encoded by the vector is capable of dividing a separate ORN substrate molecule, selected from a group consisting of viral ORN, messenger ORN, pathogenic ORN and cellular ORN. 26. The ribozirna according to claim 1, further characterized in that the activity of the ribozirna is increased by a cofactor. 27. The ribozyme according to claim 26, further characterized in that the cofactor is selected from the group consisting of antibiotics and peptides. 28.- Method for dividing a separate ORN molecule, characterized in that it comprises contacting the molecule with a ribozirna of claim 1.