RU2144080C1 - Ribosime, method of inactivation of rna-target, method of ribosime preparing - Google Patents

Abstract

FIELD: molecular biology, biochemistry. SUBSTANCE: invention relates to synthetic single-stranded RNA molecules showing highly specific endonuclease activity. Ribosime has hybridizing and catalytic regions. Hybridizing region has one or more branches of a single-stranded RNA. The preferable ribosime has a catalytic region including the sequence AAGC*GAGXAGUC in "hairpin" N₁* N₂ structure where * means bases pairing between complementary nucleotides, X means any ribonucleotide and N₁ and N₂ mean ribonucleotides with complementary sequences, at least, in part of their length. Ribosime splits RNA-target after triplet xUy where x is any ribonucleotide, U means uracil and y is adenine, cytosine or uracil. The nucleotide sequence encoding the required ribosime is obtained followed by its ligation with a suitable transfer vector containing DNA or RNA or their combination. The ligated sequence is transcribed using RNA-polymerase. RNA-target into the cells is subjected by ribosime effect that is able to carry out the specific cleavage of indicated RNA-target in the site taken so manner that the indicated RNA-target is hybridized into the cell with ribosime to induce inactivation of RNA-target. Invention ensures to obtain ribosimes that are able to hybridize effectively with the broad raw of target RNA-sequences but without modifying the cleavage RNA-targent. EFFECT: improved method of ribosime preparing. 11 cl, 38 dwg, 9 ex

Description

 The invention relates to a class of molecules of synthetic RNAs and their derivatives, and in particular, to ribozymes having highly specific endoribonuclease activity.

 A number of naturally occurring RNA molecules, for example, such as the avocado sunspot viroid (ASBV), containing the RNA satellite of the tobacco ring spot virus / STOBRV / and the passing alfalfa streak virus / SLTSV /, are subject to self-catalyzed cleavage. Obviously, this cleavage is the main and only part of the life cycle of these and other RNAs.

 Self-catalyzed RNA cleavage reactions require divalent metal ions and a neutral or higher pH and result in RNA production at the ends having a 5'-hydroxyl group and 2 ', 3'-cyclic phosphate groups / Prody et al., Science 231: 1577-1580 / 1986 / and Buzayan et al., Virology 151: 186-199 / 1986 //. The cleavage reactions are catalyzed by the RNAs themselves, which is probably due to the conformation of bridging reactive groups in close proximity. Sites of self-catalyzed cleavage in natural RNAs are located in highly conserved regions of RNA secondary structure / Buzayan et al., Proc. Natl. Acad Soc. USA 83: 8859-8862 / 1986 / and Forster, A.C. and Symous, R.H. Cell, 50: 9-16 / 1987 //.

 The experiments of the present invention were carried out on a tobacco ring-spotting satellite virus / STORV / containing RNA, which was used to construct new endoribonucleases / hereinafter referred to as "ribozymes" /, i.e., enzymes including RNAs that catalyze the specific cleavage of target molecules RNA

 The term "ribozyme", as used herein, refers to molecules entirely consisting of RNA for its derivatives.

 The ribozymes of the present invention differ from the RNA endoribonuclease that is found naturally in Tetrahymena Thermophila / known as IVS or L-19IVS RNA / and which is described in detail in: Thomas Cech et al., (Zang, AT et al., Science / 1984 / 224: 574-578; Zang, AT and Cech, TR Science / 1986/231: 470-475; Zang, AT, et al., Nature / 1986/324: 429-433; Publication of International Application N 88/04300 University Patents Inc. The endoribonuclease described by Cech has an 8 bp active site that hybridizes to the target RNA sequence, followed by cleavage of the target RNA, subject to the presence of free guanose and derivatives thereof. Fragments resulting from cleavage contain terminal 5'-phosphate and 3'-hydroxyl groups. A limited number of nucleotides suitable for hybridization with an RNA substrate limits the efficiency or productivity of the endoribonuclease described by Cech. Oligonucleotides containing less than 12 nucleotides, poorly hybridize to target sequences. It is also obvious that a certain number of nucleotides in the active site of the Cech-endoribonuclease may be required to maintain the effective activity of the endoribonuclease. This limits the number of possible permutation of sequences of active sites that could be carried out to increase effective hybridization with target sequences, which, in turn, limits the number of target RNA sequences cleaved by Cech-endoribonuclease. Cech-endoribonuclease also modifies RNA by adding the free guanosine nucleotide to the 5 ′ end of the cleaved RNA.

 In contrast, the ribozymes of the present invention hybridize efficiently with a wide range of target RNA sequences without modifying the cleaved target RNA.

 The ribozymes of the present invention include a hybridizing region that is complementary to the nucleotide sequence of at least a portion of the target RNA, and a catalytic region that is adapted to cleave the target RNA. The hybridization region contains 9 or more nucleotides.

 In a preferred embodiment of the present invention, ribozymes have a hybridization region comprising one or more branches formed from single-stranded RNA and having a sequence complementary to at least a portion of the target RNA; wherein said one or more branches associated with the catalytic region are capable of cleaving said target RNA; and the hybridization region contains one branch of the indicated RNA branches, which consists of at least 9 nucleotides, while this hybridization region includes 2 or more RNA branches, and the sum of the nucleotides in these branches is more than 9 nucleotides.

где X представляет собой любой рибонуклеотид и каждый из остатков X могут быть одинаковыми или различными;
сумма n и n' превышает 6, и n и n' могут быть одинаковыми или различными;
/*/ означает пару оснований между комплементарными рибонуклеотидами;.In one embodiment of the present invention, the preparation of ribozymes of formula 1 is contemplated:

where X is any ribonucleotide and each of the X residues may be the same or different;
the sum of n and n 'is greater than 6, and n and n' may be the same or different;
/ * / means a base pair between complementary ribonucleotides ;.

X 'and X''are oligoribonucleotides of a complementary sequence along at least part of their length, so that the bases between the oligoribonucleotides mate; or X ′ and X ″ together form a single RNA sequence, wherein at least a portion of said sequence includes a stem formed by base pairing between complementary nucleotides; and optionally, in the formula / I / after ^I A, an additional nucleotide selected from A, G, C or U may be introduced.

 The region / I / of the formula / 1 / represents the shoulders or flanking sequences of the ribozyme that hybridize with the corresponding regions of the target RNA sequence. These arms can hybridize along the full length of the target RNA or part thereof. The catalytic region of RNA is depicted in the region / II / of the formula / 1 /. The specified catalytic region may contain one or more additional nucleotides that do not adversely affect the catalytic activity. These additives can be easily tested for ribozyme activity in a manner that does not require complex experiments, which will be described below. The catalytic region may also form part of a hybridization region.

 Oligoribonucleotides X ′ and X ″ may contain up to 5000 nucleotides or more.

где X представляет собой любой рибонуклеотид и каждый из остатков X могут быть одинаковыми или различными;
/*/ означает пару оснований между комплементарными рибонуклеотидами;
n и n' определены выше;
m и m' имеют значение 1 или выше и могут быть одинаковыми или различными;
"B" представляет собой связь, пару оснований, рибонуклеотид или олигорибонуклеотид, содержащий, по крайней мере, 2 рибонуклеотида;
и необязательно, в формуле /2/ после ^IA может быть введен добавочный нуклеотид, выбранный из A, G, C или U.In another embodiment of the present invention, the preparation of ribozymes of formula 2 is contemplated:

where X is any ribonucleotide and each of the X residues may be the same or different;
/ * / means a base pair between complementary ribonucleotides;
n and n ′ are as defined above;
m and m 'have a value of 1 or higher and may be the same or different;
“B” is a bond, a base pair, a ribonucleotide or an oligoribonucleotide containing at least 2 ribonucleotides;
and optionally, in formula / 2 / after ^I A, an additional nucleotide selected from A, G, C or U may be introduced.

 The ribozymes of the present invention can be obtained by methods known per se, used for the synthesis of RNA molecules. / For example, in accordance with the guidelines recommended by Promega, Madison, WI USA /. In particular, the ribozymes of the present invention can be obtained from the corresponding DNA sequence (DNA, which upon transcription produces a ribozyme and which can be synthesized by methods commonly used by specialists in DNA synthesis), actively linked to the RNA polymerase promoter, for example, the RNA- promoter polymerase T7 or SP6. The DNA sequence corresponding to the ribozyme of the present invention can be ligated into a DNA transfer vector, for example plasmid or phage DNA. When the transfer vector contains an RNA polymerase promoter actively linked to the DNA corresponding to the ribozyme, the ribozyme can be produced during incubation with the RNA polymerase. Therefore, ribozymes can be produced in vitro by incubating an RNA polymerase with an RNA polymerase promoter actively linked to the DNA corresponding to the ribozyme in the presence of ribonucleotides. In vivo prokaryotic or eukaryotic cells (including mammalian and plant cells) can be transfected with an appropriate insertion vector containing the genetic material corresponding to the ribozyme, which according to the present invention is actively linked to the RNA polymerase promoter so that said ribozyme is transcribed in the host cell. Transfer vectors can be bacterial plasmids or viral RNA or DNA. The nucleotide sequence corresponding to the ribozymes is mainly controlled by strong promoters such as lac, the late promoter SV 40, the early promoter SV 40, metallothionine, or λ-promoters. Ribozymes can be transcribed directly in vivo from a transfer vector, or alternatively, they can be transcribed as part of a larger RNA molecule. For example, DNA corresponding to ribozyme sequences can be ligated to the 3'-end of the carrier gene, for example, after a translation stop signal. Large, RNA molecules can help stabilize ribozyme molecules against nuclease digestion in cells. In translation, a carrier gene can contribute to the formation of a protein, the presence of which can be directly estimated using an enzymatic reaction. The host gene, for example, can encode an enzyme.

 In yet another embodiment of the present invention, the preparation of a DNA transfer vector that contains a DNA sequence corresponding to a ribozyme actively linked to a promoter is provided in order to ensure transcription of the ribozyme.

 One of the preferred methods for producing ribozyme is that two synthetic oligonucleotides of a complementary sequence are obtained by standard procedures and hybridized, for example, using a DNA synthesizer Model 380A / Applied Biosystems Inc. , Foster City, CA 94404 /. One of the oligonucleotides encodes the target ribozyme. The corresponding ends of the hybridized oligonucleotides correspond to various restriction sites, for example Eco R1 at one end and Pst1 at the other end. After cleavage with suitable restriction enzymes (for example, Eco R1 and Pst1), a double-stranded DNA fragment can be cloned into a transfer vector. If the plasmid vector contains an RNA polymerase promoter upstream of the DNA sequence corresponding to the ribozyme of the present invention, then RNA transcripts corresponding to the ribozyme can be obtained either in vitro or in vivo. If the ribozyme consists of two halves that are held together by base pairing between complementary nucleotides, then each half of the ribozyme can be produced in accordance with the above methods, after which the two halves are incubated together with the formation of the specified ribozyme.

Preferred ribozymes of the present invention cleave the target RNA, which contains the sequence X ^o UY, where X ^o is any ribonucleotide, U is uracil, and Y is adenine, cytosine or uracil. X ^o U forms part of a pair of bases flanking the region, and Y is not a paired base. Preferably, but not necessarily, if X ^o = guanidine and X ^o UY = GUC go CUA. Any RNA molecule containing these sequences can be cleaved by the ribozymes of the present invention. After the RNA transcript sequence containing the X ^o UY sequence has been determined, the shoulders of the ribozyme sequence can be synthesized, which is complementary / and therefore hybridizable / RNA to the target sequence flanking the X ^o UY sequence. When hybridizing the ribozyme arms with the target RNA sequence flanking the X ^o UY sequence, the catalytic region of the ribozyme cleaves the target RNA within the X ^o UY sequence. RNA cleavage is facilitated in the presence of magnesium or another divalent cation at a pH of approximately 8.0.

 Accordingly, preferred ribozymes of the present invention can be designed to cleave any RNA whose sequence is known.

 The high frequency of residues cleaved by ribozymes in RNA / 1: 64 for GUC in RNA with a random or equal base distribution frequency / means that the number of potential sites for ribozyme cleavage can be predicted with a fairly high degree of reliability in any given target RNA.

 In a further embodiment of the present invention, there is provided a method of inactivating a target RNA sequence, the method comprising: said target RNA sequence being reacted with a ribozyme of the present invention.

 A transfer vector, such as a bacterial plasmid or viral RNA or DNA encoding one or more ribozymes, can be transfected in vivo, i.e., into a cell or cells of the body / for example, Leewellyn et al., F. Mol. Biol. / 1987/195: 115-123; Hanahan et al., T. Mol. Biol./1983/166/. Once in the cell, the transfer vector can replicate and be transcribed by cell polymerases to form ribozyme RNAs, which then inactivate the desired target RNA. Alternatively, a transfer vector containing one or more ribozyme sequences can be transfected into cells or introduced into cells using micromanipulation techniques, such as microinjection, so that the transfer vector or part of it integrates into the genome of the host cell. Transcription of integrated genetic material leads to the production of ribozymes that inactivate target RNA.

 The ribozymes of the present invention have wide therapeutic and biological applications. For example, viruses that cause diseases in humans and animals can be inactivated by introducing into the object infected with the virus, the ribozyme of the present invention, which is adapted for hybridization and cleavage of RNA transcripts of the virus. These ribozymes can be administered parenterally or by other routes of administration. Alternatively, a non-virulent virus (such as a vaccine or an adenovirus) can be introduced into an object infected with a disease causing virus, which can be genetically engineered to contain DNA corresponding to the ribozyme actively linked to the RNA promoter, as a result of which the ribozyme is transcribed in the cells of an animal host transfected with the engineered virus, and cleaves and / or inactivates the transcript of the target RNA of the virus that caused the disease. In particular, the ribozymes of the present invention can be used to treat diseases caused by viruses such as the herpes simplex virus / AHV / or the AIDS virus / HIV /.

 The ribozymes of the present invention can also be used to inactivate RNA transcripts in bacteria or other prokaryotic cells, in plants and animals. In bacteria, RNA transcripts, for example, a bacteriophage that causes the death of bacterial cells, can be inactivated by transfection of the cell with a DNA transfer vector capable of producing the ribozyme of the present invention, which inactivates phage DNA. Alternatively, the ribozyme itself can be introduced into the bacterial cell to effect cleavage of phage DNA.

 RNA transcripts in plants can be inactivated using ribozymes encoded by a transfer vector, such as a Ti plasmid derived from Agrobacterium tumefaciens. When such vectors are transfected into a plant cell, ribozymes are produced under the action of RNA polymerase, which cleave a specific sequence of the target RNA. Accordingly, plant viruses whose RNAs are known, or RNA transcripts of plant genes can be inactivated using these ribozymes.

 Foreign gene transcripts in plants, animals, or other types of cells can be inactivated using the ribozymes of the present invention. As a result, unwanted phenotypes or traits can be modulated. It is possible, for example, to remove seeds from fruits with ribozymes or to treat hereditary diseases in humans caused by the production of a harmful protein or the excessive production of a specific protein.

 The present invention is illustrated by examples and the accompanying drawings, and neither one nor the other does not limit possible embodiments of the present invention.

 In FIG. 1 shows self-cleavage sites of wild-type RNA and mutated RNA; as well as electrophoresis profiles of products of self-catalytic cleavage of RNA.

 / a / shows the preserved structures associated with naturally occurring cleavage sites in ASBV transcripts of the DNA of the satellite Triton and RNA-containing satellite STOBRV, LTSV, tobacco mosaic virus, black nightshade mosaic virus / solanum nodiflorum / and underground clover mosaic virus. In FIG. the nucleotide sequence between these structures is shown, while others are denoted by X. Base pairing is indicated by / • /, and RNA cleavage sites are indicated by an arrow.

 / b / shows the stored nucleotide sequence associated with the cleavage of the + + / - STOBRV RNA strand. The cleavage site is indicated by an arrow.

 (c) shown in vitro - the STOBRV mutant containing an insert of eight nucleotides / circled / along with flanking duplication of three nucleotides / UGU residues 7-9 /.

/ d / shows subcloned Hal III fragments of wild-type STOBRV and in vitro mutant D-51, each of which is transcribed in both / + / and / - / orientations, as well as radioactively labeled transcripts fractionated by polyacrylamide gel electrophoresis. The positions of the unsplit transcripts of bases 159 and 170 from the wild-type sequences / WT / and mutant D-51 are indicated by an arrow;
In FIG. Figure 2 shows the nucleotide sequence of ribozyme and ribozyme cleavage products isolated by gel electrophoresis.

 (a) Nucleotides inserted in the D-51 mutant and containing the restriction endonuclease Bam H1 site are shown. Bam H1 was used to digest mutant DNA, and the two sequences were subcloned and transcribed separately in vitro. RNA transcripts are shown schematically with potential base pairing between the indicated / • / RNAs. Fragments containing the arrow site for cleavage indicated by S-RNA; and the ribozyme containing fragment is designated Rz-RNA.

/ b / [ ³² P] -R _z -PHK / 101 base / were incubated separately / lane 1 / and with un-labeled S-RNA / lane 2 /. [ ³² P] -S-RNA was incubated separately / lane 3 / with both unlabeled and [ ³² P] -labeled R _z -PHK / lanes 4 and 5, respectively /.

 In FIG. 3 shows a schematic model of a ribozyme in accordance with one embodiment of the present invention. Region A is a cleavage sequence in the target RNA. Region B represents a catalytic region, and region C represents the shoulders of the ribozyme.

In FIG. 4 shows the construction of ribozymes directed against the CAT gene transcript / chloramphenicol acetyltransferase /. Ribozymes designated R _z CAT-1, R _z CAT-2 and R _z CAT-3 are directed against three sites located in an in vitro transcript from 835 bases of the CAT gene. The relative locations of the cleavage sites on the transcript are schematically shown together with flanking numbered bases / a /. Also shown are // b / - / d // three ribozyme sequences with their target sequences. The amino acid sequences of the CAT gene are numbered, and the predicted sites for RNA cleavage are indicated by arrows. R _z CAT-1 and R _z CAT-3 contain 24-base sequences derived from / + / STOBRV strands / region B, FIG. 3 /, a R _z CAT-2 contains one UA substitution in this area.

In FIG. Figure 5 shows the results of CAT-cleavage of RNA with ribozymes R _z CAT-1 - R _z CAT-3.

/ a / [ ³² P] -CAT-RNA was subjected to gel fractionation after incubation both separately / - /, and with one of the three ribozymes R _z CAT-1 - R _z CAT-3 / lanes 1, 2 and 3, respectively / . Localization of the complete transcript is indicated by an arrow.

/ b / Analysis of 5'-terminal bases is shown. 3'-fragments produced by CAT-RNA ribozyme cleavage were digested with [5'- ³² P] kinase, gel purified, digested with complete nuclease, and the isolated terminal residues were fractionated by polyacrylamide gel electrophoresis. The 5'-terminal nucleotides identified by the markers / lane M / were A, U, and G for fragments produced using R _z -CAT-1 - R _z CAT-3 / lanes 1, 2, and 3, respectively /.

In FIG. 6 shows the time cycle of the catalytic activity of the ribozyme / R _z CAT-1 / against CAT-RNA. The amount of cleavage product (139 nucleotides) was calculated and a diagram was constructed. The inset shows the accumulation of the fragment in 139 bases over time after polyacrylamide gel electrophoresis.

 In FIG. 7 shows the relative rates of CAT-RNA cleavage as a function of various temperature conditions. The substrate RNA is represented by a solid line. In each case, the cleavage product is shown by a dashed line.

In FIG. Figure 8 shows three ribozymes / corresponding R _z CAT-2 / having arms or flanking sequences of different lengths.

In FIG. 9 shows a production scheme of a ribozyme including antisense RNA containing each of the cleavage regions R _z CAT-1 - R _z CAT-3.

 In FIG. 10 shows the hybridization of ribozymes with sequences containing GUA / 10a / and GUU / 10b / in messenger CAT-RNA.

 In FIG. 11 are shown for the self-catalytic cleavage of an exocortis citrus viroid / CEV RNA and its complement.

In FIG. 12 shows the ribozyme R _z CEV 25x / + / hybridized with the target CEV-RNA / a /, and the profile of gel electrophoresis of CEV-RNA and complementary / - / CEV-RNA incubated with R _z CEV25x / + / / b, lanes 1 and 2, respectively. The cleavage product is indicated by the arrow /.

In FIG. 13 shows the ribozyme R _z CAT-2 hybridized with its target sequence / a / and the ribozyme R _z SCMoV / b /. The catalytic region of each domain is framed. The differences / circled / catalytic region of R _z SCMoV compared to R _z CAT-2 are also shown.

In FIG. 14 shows the ribozyme R _z CEV-2 hybridized with its target sequence of citrus exocortis viroid RNA / CEV /. The cleavage site corresponds to nucleotide 336 in the CEV RNA sequence. Alteration in the nucleotide sequence in its catalytic region compared with the catalytic region of STOBRV is circled / a /. In FIG. 14 / b / shows the electrophoresis profile of control RNA [/ - / CEV strand], / lane 7 / and RNA / + / CEV strands / lane 8 /, after incubation with R _z CEV2.

In FIG. 15 shows the ribozyme R _z CAT-2 / a / in comparison with the ribozyme R _z CAT-2B / b /. The catalytic regions are framed. Changes in the catalytic region of R _z CAT-2B compared to R _z CAT-2 are also framed;
In FIG. 16 depicts a map of plasmid pT35SN;
In FIG. 17 shows a graph representing the activity / average of 4 experiments / inhibition of CAT expression in plants / tobacco protoplasts /.

 The following are examples illustrating the implementation of the present invention, but not limiting its possible options.

 DNA reactions and manipulations, such as ligation, digestion with restriction enzymes, bacterial transformation, DNA sequencing, etc., were carried out in accordance with standard techniques, such as, for example, described by Maniatis et al., / Molecular Cloning, Cold Spring Harbor, 1982. RNA manipulations were also carried out by standard methods, such as described, for example, by Uhlenbeck / Nature 328, 596-600 / 1987 / and Haseloff and Gerlach (Nature 334, 585-591 / 1988 /).

Example 1. Self-catalyzed cleavage of mutated RNA STOBRV
The consensus of regions associated with the cleavage sites of natural RNAs in ASBV, transcripts of satellite DNA of triton, and RNA of satellite STOBRV, LSTV, tobacco mosaic virus / VMoV /, black nightshade mosaic virus / SNMV / and underground clover mosaic virus is shown in FIG. 1a. In FIG. the nucleotide sequences stored between these structures are shown, and the unsaved sequences are denoted by X. Additional U are located after residue ^I A in the / + / LTSV strand.

 Regions associated with the self-catalytic cleavage of the / + / STOBRV strands were investigated for enzyme substrate activity in this region. First, the cloned STOBRV cDNAs were mutagenized by insertion of the oligonucleotide linker / Bam H1 / in accordance with the guidelines for the use of this method.

Construction of a vector for the expression of STOBRV in vitro:
160 bp TaqI - SpeI - STOBRV cDNA fragment was isolated from PSP653 / Gerlach et al., 1985, Virology 151: 172-185 and ligated with AccI-SpeI - cut and treated with pGEM 4 phosphatase to form the Acc I. site. The resulting clone was lanenzed with Acc I , treated with phosphatase and injected 359 on. TagI fragment of STOBRV cDNA. The resulting clones were screened for the presence of circularly permuted 520 bp the STOBRV cDNA sequence containing 277-81 terminally excess residues / pTTS /. The STOBRV sequence is flanked by promoters for T7 and SP6 RNA polymerases, and as a result of in vitro transcription, RNAs with a / + / or / - / orientation were obtained that contained two sites for self-cleavage.

 In vitro mutagenesis.

Plasmid pTTS / 50 μg / was linearized with BamH1, treated with SI nuclease and ligated again to remove the unique BamH1 site. The resulting construct, pTTS-B, was processed using 2 • 10 ^-4 units. DNase I in 29 mM Tris-HCl (pH 7.0), 15 mM MgCl ₂ for 10 min at 37 ^° C. The obtained linear DNAs were customized and / or treated with T4 DNA polymerase to fill the ends and purified by electrophoresis on an agarose gel of 0.7% LGT and by extraction. The kinase-treated BamH1 linker sequence (CGGATCGG) was ligated with a linearized plasmid overnight at room temperature in the presence of 5% polyethylene glycol. After that, the reactions were digested with BamH1, and linear plasmid DNAs were again purified by 0.7% LGT agarose gel electrophoresis (this was necessary to remove the last residues of the ring plasmid together with non-ligated linkers). Plasmids were again shorted using T4 DNA ligase and transformed into E. coli DN-1. Colonies (over 1000) were scraped from agar plates, cultured in liquid medium until saturated, and mixed plasmid DNA populations were prepared. Mixed STOBRV DNA inserts were excised by restriction enzyme digestion at the flanking EcoR1 and Pst1 sites, purified by 1% CT agarose gel electrophoresis, and subcloned into EcoR1-Pst1 cut and treated with pGEM 4 phosphatase. The resulting transformants were again collected, cultured in liquid medium and received plasmid DNA. Plasmid DNAs were treated with BamH1 to cleave only those plasmids that contain a BamH1 linker sequence, and then the linear forms were again purified by two electrophoresis on 0.7% LGT agarose gel, again closed with T4 DNA ligase and transformed into E. coli DN- 1. Individual transformants were screened for approximate localization of the inserted BamH1 linker in the STOBRV sequence by restriction enzyme digestion, subcloned into M13 mp19 and sequenced using a standard dideoxy termination method.

 The result was a library of STOBRV mutants. Analysis of the nucleotide sequence showed that each mutant contained an inserted BamH1 linker sequence / CGGATCGG / together with flanking duplicated or deleted STOBRV sequences. Mutants were transcribed in vitro, and RNA was examined for their ability to cleave. As a result of these experiments, the 52-nucleotide sequence was identified as containing a substrate and STOBRV RNA cleavage sites. This 52 nucleotide sequence depicted in FIG. 1b, contains the region of the stored sequence necessary for self-cleavage of other RNA / Fig. 1a /. One mutant designated D-51 contained an 8-nucleotide BamH1 linker sequence inserted between the three STOBRV duplicated nucleotides designated 7-9. RNA of this mutant was subjected to self-catalytic cleavage.

97 and 108 bp Hal III — fragments containing the 52-nucleotide cleavage sequence of wild-type and D-51-RNA / as shown in FIG. 1b and 1c / were removed from sequenced plasmid clones. Fragments were ligated into the Sma1 site of pGEM 4 and screened to determine both insert orientations. Plasmids were linearized using EcoRI, and RNAs with / + / and / - / strands of 159 and 170 bases in length were transcribed using 200 U / ml T7 RNA polymerase in 50 mM Tris-HCI, pH 7.5, 10 mM NaCl 6 mM MgCl ₂ , 2 mM spermidine, 1000 u / ml aranazine, 500 μM ATP, CTP and GTP with 200 μM O ³² PTP. RNA was fractionated by electrophoresis in 10% polyacrylamide, 7 molar urea, 25% formamide gel, and autoradiography was performed.

 As shown in FIG. 1d, no transcription of / - / RNA strands was observed. This was the expected result because the / - / thread did not contain a self-catalytic cleavage site. As for the / + / strands of both wild-type and D-51 sequences, in this case, cleavage took place; moreover, the cleavage of D-51-PHK was slightly less effective than wild-type RNA / Fig. 1d /. This experiment showed that the region of the single-stranded loop on the right side of the 52-nucleotide sequence involved in the self-catalytic cleavage of RNA does not play a major role.

Separation of enzyme and substrate activities:
Using the BamH1 restriction endonuclease site introduced in D-51, flanking Hal III - BamH1 and BamH1 - Hal III fragments were obtained and each of them was subcloned into an E. coli plasmid suitable for in vitro transcription. This leads to the elimination of the mutated single-stranded loop from the region of self-cleavage and cleavage of the region into two RNA fragments / Fig. 2a /. The smaller Hal III - BamH1 fragment contained nucleotides 321-9, including the actual cleavage site, and was designated as the S fragment. BamH1 - Hal III - fragment containing STOBRV - nucleotides 7-48, was called a ribozyme or R _z fragment. E. coli - plasmids used for in vitro transcription were pGEM3 and pGEM4 / Promega, Madison, WI, USA /.

These expression plasmids contained:
a / start of replication;
b / gene for selective drug resistance / Amp ^r /;
c / repeatedly cloning site flanked by promoters of RNA polymerase, which can be used for the production of transcripts in vitro.

Fragments of R _z -pGEM3 and S-pGEM4 treated with T7 DNA polymerase and digested with KpnI and XbaI were transcribed with RNA polymerase under the same conditions as described above.

As shown in FIG. 2, S-RNA and R _z -RNA did not show significant degradation upon their separate incubation / Fig. 2b /, lanes 1 and 3 / under conditions that promoted highly efficient self-cleavage / 50 ^° C., 20 mM MgCl ₂ , pH 8.0 /. Obviously, labeled R _z -RNA also remained unchanged after incubation with S-RNA / Fig. 2b, lanes 2 and 5 /. However, when S-PHK was mixed with R _z -RNA, there was an effective cleavage of S-RNA / Fig. 2b, lanes 4 and 5 /, with the formation of two fragments. The sizes of the products upon splitting of S-RNA / 84 bases / at the normal site between nucleotides N 359 and N 1 with the formation of fragments located at the 5'-end and 3'-end were 67 and 17 nucleotides, respectively. This means that S-RNA acts as a substrate for ribonucleolytic cleavage by R _z -RNA, which plays a catalytic role.

 A ribozyme model based on the catalytic region of STOBRV RNA is shown in FIG. 3. This ribozyme has two arms or flanking sequences of single-stranded RNA (shown in C), hybridizing with complementary sequences on substrate RNA, ie cleavable RNA. Each flanking sequence shown in C contains 8 ribonucleotides. The number of nucleotides contained in region C is not critical. However, this number should be sufficient for the hybridization of the ribozyme with the target RNA. Obviously, at least 4 nucleotides must be present in region C to accomplish this hybridization.

 Catalytic region B contains sequences that are highly conserved in cleavage regions of natural origin / cm. FIG. 1a /. Compared with the cleavage regions of known sequences, the length of stem II is of little importance, as is the presence of an associated loop at one end of it.

 The cleavage site in the target RNA is indicated at A / Fig. 3 / as a GUC. Based on the experiments of the present invention / not shown / and other experiments Koizumi / FEBS LETT 288; 228-230 / 1988 /; and FEBS LETT 239; 285-288 / 1988 /, it is obvious that at the sites in RNAs of natural origin, the sequences of GUA, GUC, CUC, AUC and UUC also act as binding sites within RNA.

Example 2. Description of the construction, synthesis and activity of ribozymes with new and highly specific endoribonuclease activity
To illustrate the present invention, three ribozymes were constructed that are directed against the transcript of a commonly used indicator gene derived from the bacterium, Tn 9 / CAT chloramphenylcolacetyltransferase, which reports antibiotic resistance in bacteria, plants, and animals and is easy to analyze. These ribozymes designated R _z CAT-1 - R _z CAT-3 correspond to potential GUC cleavage sites in CAT-RNA at positions 139-140, 494-495 and 662-663, respectively. The sequences of these ribozymes are depicted in figure 4. In each case, the flanking ribozyme sequences that hybridize to the target CAT RNA were 8 nucleotides in length. Catalytic regions were selected according to the STOBRV RNA regions shown in FIG. 3.

The CAT gene was obtained from pGM4 and was subcloned as a BamH1 fragment into pGEM-32 from Promega, Maclison, N 1. This plasmid was linearized by Hind III and CAT gene transcripts were obtained using T7 RNA polymerase with 220 mM [ α- ³² P] UTP. Ribozyme sequences were synthesized as oligonucleotides, R _z CAT-1, 2 and 3, respectively. They were treated with kinase, then they were ligated with phosphatase treatment, pGEM4 was cut with EcoRI-Pst1 and incubated with the Klenov fragment of DNA polymerase 1, and then transformed into bacteria. The EcoRI-linearized plasmid was transcribed with T7 RNA polymerase, resulting in ribozyme RNA. Ribozymes were incubated with a CAT transcript in 50 mM Tris-HCl, pH 8.0, 20 mM MgCl ₂ at 50 ^° C for 60 minutes, and the resulting products were fractionated by electrophoresis on 5% polyacrylamide, 7M urea, 25% formamide gel , and then performed autoradiography.

Upon incubation of the 840-nucleotide CAT transcript with any of the three ribozymes, highly efficient specific sequence cleavage took place / Fig. 5 / with the formation of 2 RNA fragments in each reaction. The sizes of the fragments corresponded to the predicted sites for cleavage / i.e. fragments with bases 139 and 696, 494 and 341, 662 and 173 were 5'- and 3'-products from the cleavage catalyzed by R _z CAT-1 - R _z CAT-3, respectively. The conditions required for these ribozyme-catalyzed cleavages are similar to those observed for natural cleavage reactions / Foster, AC and Symons, RH, Cell 49: 211-220 / 1987 / and Foster, AC and Symons, RH, Cell. 50: 9-16 / 1987 / /, moreover, more efficient cleavage was observed at elevated pH, temperature and concentrations of divalent cations / data not shown /. In the presence of a molar excess, these three ribozymes complete the complete cleavage of the CAT-RNA substrate after 60 minutes in 50 mM Tris-HCl, pH 8.0, 20 mM MgCl ₂ at 50 ^° C. Under similar conditions, using 0.1 μM substrate and 3 μM ribozymes of T _1/2 CAT mRNA substrate was 3.5; 3.5 and 2.5 minutes in the presence of R _z CAT-1 - R _z CAT-3, respectively. Ribozyme sequences were inactivated with respect to the complement of substrate RNA / t. e., / + / filament / and in the form of oligonucleotides / data not shown /. The 3'-terminal cleavage fragments from each ribozyme-catalyzed reaction were isolated, and the 5'-terminal was treated with [ ³² P] kinase / 50 mM Tris-HCl, pH 9, 10 mM MgCl ₂ , 10 mM DTT with 50 μCi γ- ³² P-ATP and 5 units. polynucleotide kinase T4, at 37 ^o C for 30 minutes /. Effective processing of the fragments with kinase has shown that they have 5'-terminal hydroxyl groups similar to those formed during cleavage reactions that occur in nature. Then, the terminal nucleotides of the fragments produced by cleavage of the CAT sequences using R _z CAT-1 - R _z CAT-3 were determined. In short, the radiolabeled fragments were purified on a 5% polyacrylamide gel and digested with equal volumes of 500 u / ml RNase T1, 25 u / ml RNase T2 and 0.125 mg / ml RNase A in 50 mM ammonium acetate, pH 4.5, at 37 ^° C for 120 minutes. The resulting products were fractionated on a 20% polyacrylamide gel containing 25 mM sodium citrate, pH 3.5 and 7 molar urea. From FIG. 5b it is seen that the cleavage of CAT sequences by R _z CAT-1 - R _z CAT-3 occurs exactly in front of nucleotides A, U and G, respectively.

 The end sequences of fragments of the CAT gene were determined directly using the partial enzyme digestion technique / Donis - Keller et al. , Nucleic Acid. Res. 4: 2527-2538 / 1980 / using basic specific ribonucleotic cleavage. The sequence of the obtained fragments confirmed that the cleavage in CAT-RNA occurs in the expected areas / not shown /.

 Enzymatic catalysis.

To demonstrate that the ribozymes of the present invention cause the CAT-mRNA substrate to be cleaved in a catalytic manner, each of these ribozymes was incubated with a molar excess of the substrate under conditions that would favor efficient cleavage and dissociation of the products. In FIG. 6 shows the results that were obtained after specific catalytic cleavage of 70 min at 50 ^o C, in 20 mm MgCl ₂ , pH 8.0, 10 rM R _z CAT-1 truncated / 173 bases / CAT-RNA substrate with the formation of 5 ' - and 3'-fragments with 139 and 34 bases, respectively. On average, each ribozyme was involved in more than 10 cases of cleavage. After 75 minutes at 50 ^° C, to some extent, non-specific RNA cleavage was observed due to extreme conditions, but 70% of the remaining intact RNAs / 163 rM / accumulated as a fragment with 139 bases. A similar result was obtained for R _z CAT-2 and R _z CAT-3 / not shown /, as a result of which we can conclude that each ribozyme acts as an RNA enzyme.

 Example 3. The effect of temperature on the activity of ribozyme.

 Studies have been conducted on the effect of reaction temperature on the degree of ribozyme activity in vitro.

The reaction was carried out using ribozymes R _z CAT-1 - R _z CAT-3 with a substrate of CAT-RNA at 37 ^o C and 50 ^o C.

In this experiment, the reaction for each ribozyme was duplicated using the reaction conditions for the ribozyme-catalyzed cleavage, as described in Example 2. One reaction was incubated at 37 ^° C and the other at 50 ^° C. Samples were removed exactly after 90 minutes, after which the reaction was analyzed. by polyacrylamide gel electrophoresis. In FIG. 7 shows the reaction time for each ribozyme R _z CAT-1 - R _z CAT-3 at 37 ^o C and 50 ^o C. The reaction rate of each ribozyme increased with increasing reaction temperature.

The time taken for 50% / t _1/2 / digestion of CAT-RNA is shown in table 1.

As can be seen from table 1, the reaction rate of ribozymes at 37 ^o C is approximately 20 times less than at 50 ^o C.

Example 4. The effect of differences in the lengths of the shoulders of ribozymes / or flanking sequences / on the catalytic activity of ribozymes
The shoulders of the flanking ribozyme sequences (region / I / of the formula I) hybridize the ribozyme with its target RNA, after which RNA cleavage takes place. In this experiment, we studied the change in the rate of cleavage of the target sequence with a change in the degree of complementarity, and then the length of the complementary pairing of the bases of the shoulders of the ribozyme with the target sequence.

Ribozymes were produced with complementarity of 4, 8 and 12 bases to the target sequence R _z CAT-2 on each arm / Fig. 8a /. Ribozymes were prepared as described in Example 2. Ribozyme activity was determined by incubating ribozyme RNA with CAT-RNA, as described previously. Ribozyme, having a complementarity of 4 bases on each arm, did not cleave the substrate RNA. A ribozyme with a complementarity of 8 bases on each arm cleaved the CAT substrate in the same way as a ribozyme with a complementarity of 12 bases. Ribozymes with complementarity of 12 bases cleaved target RNA more efficiently than ribozymes with fewer complementary bases. Even with complementarity of more than 4 bases, the reaction rate increases with increasing length of the hybridization region.

 In a second experiment, the reactivity of a ribozyme having / a / complementarity to the full length of the target CAT RNA transcript and / b / multiple catalytic regions was investigated. For this, four CUC target sites in CAT-RNA sequences were selected. The catalytic regions of the ribozyme with respect to these sites were inserted into the complete antisense / - / sequence for the CAT transcript, after which the catalytic activity was determined.

The four selected sites were three precisely defined by R _z CAT-1 - R _z CAT-3 and the sites described previously, and another site can be represented as follows:
New CAT Site
N 192
5' $\genfrac{}{}{0ex}{}{\text{His}}{\text{CAU}}$ $\genfrac{}{}{0ex}{}{\text{His}}{\text{CAU}}$ $\genfrac{}{}{0ex}{}{\text{Ala}}{\text{Gcc}}$ $\genfrac{}{}{0ex}{}{\text{Val}}{\text{Guc}}$ $\genfrac{}{}{0ex}{}{\text{Cys}}{\text{Ugu}}$ $\genfrac{}{}{0ex}{}{\text{Asp}}{\text{<figure-callout id="3" label="GAU Gly Ggc" filenames="00000013.png,00000021.png" state="{{state}}">GAU </figure-callout>}}$ $\genfrac{}{}{0ex}{}{\text{Gly}}{\text{Ggc}}$$\genfrac{}{}{0ex}{}{}{}$ 3 ′
where "192" refers to amino acid 192 in the CAT polypeptide, U refers to a cleavage site.

 Oligonucleotides containing ribozyme catalytic regions and including each of these cleavage sites were used for M13 mutagenesis in order to produce a sequence containing the full complement of the CAT sequence with 4 catalytic ribozyme regions introduced. M13 mutagenesis was carried out by binding oligonucleotides containing ribozyme insertions to single-stranded M13 DNAs containing uracil, followed by the synthesis of complementary DNAs containing insertion. After cloning in the corresponding strain of Escherichia coli, complementary DNAs were isolated /T.A. Kunkel 1985, Prog. Natl. Acad Sci. USA 82: 488-492 /. The resulting double-stranded cDNA was cloned in vitro in an expression vector in order to produce ribozyme RNA using a T7 polymerase transcription system. Ribozyme activity was determined by incubation of a ribozyme RNA with a CAT transcript followed by gel electrophoresis of the reaction mixture, carried out after processing the mixture with glyoxal to denature nucleic acids.

 Autolytic cleavage took place at all expected sites on the CAT transcript. Accordingly, the flanking sequences of the shoulders or ribozyme can be located along the entire length of the cleaved RNA transcript.

 In FIG. 9 schematically illustrates the preparation of catalytic antisense RNA containing each of the four ribozymes. Catalytic antisense RNA contains about 900 bases.

 Under the above conditions, the ribozyme and target sequences form high molecular weight complexes, probably due to extensive base pairing. Strong denaturing treatment, such as glyoxal treatment, is necessary to dissolve the reaction products during electrophoresis.

 Example 5. Target sequences for ribozyme cleavage.

 In order to verify whether the ribozyme actually performs cleavage in this sequence, we examined the GUA motif in mRNA.

 A specific site was selected in CAT mRNA, FIG. 10, including a GUA motif, and the corresponding ribozyme sequence was obtained, after which activity testing was performed. Ribozyme had shoulders with a length of 8 ribonucleotides.

 Then, as described in Example 2, synthetic oligonucleotides corresponding to the ribozyme shown in FIG. 10, and the double-stranded cDNA was cloned into an in vitro expression vector pGEM 4 / cm. above / in E. coli in order to produce ribozyme RNA using a T7 polymerase transcription system. Ribozyme activity was determined by incubating the ribozyme RNA with CAT mRNA followed by gel electrophoresis of the reaction mixture, as described previously.

 Cleavage with ribozyme was carried out at the target site GUA / not shown /. From this it can be concluded that the GUA motif in RNA is a substrate for the ribozymes of the present invention.

 This conclusion is not completely unexpected, since one of the natural sites of RNA cleavage of the satellite virus of the transient alfalfa streak requires recognition of the GUA site.

 A similar study of the GUU motif in the target CAT-RNA sequence was carried out using the corresponding ribozyme / cm. FIG. 10b / showed splitting.

 Example 6. Ribozyme cleavage in viral RNA.

 The viroid RNA, namely the citroid exocortis viroid, was digested using the ribozyme of the present invention.

Two target sites were selected in the RNA of the viroid of exocortis citrus / CEV /. And one site was also chosen in a complementary strand sequence. Ribozymes were obtained against these sites, after which activity was examined. These ribozymes were designated CEV9x / + /, CEV9x / - / and CEV25 / + /. In FIG. 11 shows three cleavage sites in CEV-RNA for each of these ribozymes. All ribozymes were prepared as previously described. Ribozyme R _z CEV25x / + / is shown in FIG. 12. This ribozyme cleaves the GUC at nucleotide 116 CEV-RNA.

In FIG. 12 / b / shows the cleavage of CEV-RNA with the ribozyme R _z CEV9x / + /. With ribozyme R _z CEV9x / - / cleavage was not observed.

 This experiment showed that ribozymes are active against RNA sequences originating from various sources. This result could be expected, since all RNAs are based on ribonucleotides containing adenine, guanine, cytosine and uracil, i.e. the bases that make up all RNAs, regardless of whether they are of animal, plant or bacterial origin.

 Example 7. Examples of ribozymes having variable catalytic regions.

Received a ribozyme directed to the CAT-2 site using a sequence with a catalytic region derived from the satellite RNA of the clover mosaic virus underground / SCMoV /. The flanking shoulder sequence of the ribozyme with a complementarity of 12 bases was introduced into the designed ribozyme R _z SCMoV. The ribozymes R _z CAT-2 and R _z SCMoV are shown in FIG. 13a and 13, respectively. The loop region of the R _z SCMoV ribozyme contained 5 nucleotides having the AAAUC sequence. This is not so different from the region of the loop of the ribozyme R _z CAT-2, which contains 4 nucleotides having the AGAG sequence. In addition, R _z SCMoV contains C in the catalytic region instead of U ^* , which is present in R _z CAT-2. Sequences that differ when comparing R _z SCMoV and R _z CAT-2 were labeled.

R _z SCMoV was prepared as described in Example 2. As expected, R _z SCMoV was active to form two cleavage products.

In another experiment, the target CEV site at -336 nucleotide in cRNA was digested with the ribozyme R _z CEV-2 having the sequence shown in FIG. 14a. The loop area indicated by "L" in FIG. 14 contained 6 nucleotides having the 3'-CCTATA-5 'sequence, which is different from the STOBRV loop region, which contains 4 nucleotides with the 3'-AGAG-5' sequence. This ribozyme cleaves the target complementary CEV-PHK at position -336, as shown in the electrophoresis profile in FIG. 14b.

 This experiment showed that the number of nucleotides and the nucleotide sequence of the loop region do not play a significant role in ribozyme activity. In these experiments, ribozymes were obtained by the methods described above.

 In another experiment, the effect of base pairing in the catalytic region (stem region) on ribozyme activity was investigated.

For this, a modified ribozyme was obtained that corresponded to R _z CAT-2, but contained 4 extra base pairs, which was then tested. In FIG. 15a shows the hybridization of the ribozyme sequence with the target CAT-RNA. The test ribozyme is shown in FIG. 15b, where additional base pairs are framed. The activity of the studied ribozyme corresponded to the activity of the ribozyme R _z CAT-2. This means that the site of the paired bases of the catalytic region of the ribozyme can have different lengths, which does not significantly affect the catalytic activity.

 It was found (data not shown) that the stable in vivo form of STOBRV RNA transcripts expressed in transgenic plants is mainly circular, which is probably due to ligation of the 5'- and 3'-ends. Therefore, the use of two autolytic cleavage sites flanking the desired sequence in an in vivo RNA transcript is most likely to produce a ring product that may have better stability than linear transcripts. This method, obviously, can serve as the basis for obtaining stabilized in vivo ribozyme sequences. This method relates to the closure of molecules.

 Example 8. In vivo - activity of ribozymes.

 This example describes in vivo studies of ribozyme activity in plant cells.

Experiment Log:
Plasmids containing anti-CAT / CAT = chloramphenicolacetyltransferase / or combined anti-CAT gene constructs / ribozyme / cm. below /, were introduced into protoplasts in the same amount and proportions relative to each other together with another plasmid that contained a functional CAT gene construct. Then CAT activity was measured and compared with the main level of gene activity.

Materials and methods:
/ a / Electroporation and CAT analysis
The method was carried out as described in Llewellyn et al. , T. Mol. Biol. / 1987/195: 115-123. In short, T5 Nicotiana plumbaginifolia protoplasts were obtained from suspension two days after the subculture was suspended in 10 mM HEPES pH 7.2, 150 mM NaCl, 0.2 M mannitol and adjusted to a density of 3 x 10 ⁶ ml. Electroporation was carried out using one 50 ms pulse at 250 V. Protoplasts were diluted 10 times and cultured for 20 hours in the dark at 26 ^° C. They were then destroyed by sonication and extracts were obtained. The extracts were normalized for protein content and analyzed for in vitro CAT activity using ¹³ C-chloramphenicol and acetyl CoA. The reaction products were separated by thin layer chromatography and visualized by autoradiography. The reaction limit was calculated using the production of derivatives of a radioactive product from a ¹⁴ C-chloramphenicol matrix.

Gene constructs
Gene constructs were introduced into 0.1 ml protoplast suspensions as plasmid DNA, which were purified from bacteria by extraction and two cycles of equilibrium centrifugation with a density gradient of CsCl. After that, resuspension was performed in 10 mM Tris / 1 mM EDTA / pH = 7.5 for further use.

где 35S относится к 35S CaMV-промотору /CAMV- вирус мозаики цветной капусты, NOS относится к сигналу полиаденилирования нопалинсинтетазы, T/C к транскрипции.Active CAT gene constructs were introduced into the plasmid designated pCAT7 / + /. It was obtained by fusion of the CAT gene sequence / from plasmid pGM4, see TT Close and P. Rodriguez, 1982, Gene 20: 305-316 / with plasmid pT35SN / derived from plasmid p35SN, WL Gerlach et al., 1987, Nature 328: 802-805 /; thus, the design of the active gene was

where 35S refers to the 35S CaMV promoter / CAMV cauliflower mosaic virus, NOS refers to the polyadenylation signal of nopaline synthetase, T / C for transcription.

pCAT7 - Эта плазмида содержит последовательность CAT-гена, инсертированную в pT35SN так, чтобы транскрипция приводила к продуцированию антисмысловой CAT-РНК, указанная плазмида может быть схематически изображена следующим образом:

pCAT19 - Эта плазмида содержит CAT-ген с четырьмя каталитическими областями рибозимы /см. пример 4 и фиг.9/, инсертированный в pT35SN так, чтобы транскрипция приводила к продуцированию антисмысловой CAT-РНК, содержащей 4 каталитические области рибозима; указанная плазмида может быть схематически изображена следующим образом:

Результаты:
В таблице 2 представлены относительные CAT-активности в клетках через 20 часов после электропорации. Активность выражали как процент конверсии хлорамфениколового субстрата за 1 час анализа.Along with 0.2 μg pCAT7 +, various gene constructs were added in excess, as described below. Gene constructs were contained in plasmids having the following structures:
pT35SN - This vector plasmid, a map of which is depicted in FIG. 16 contains a 35S CaM promoter and a 3'-polyadenylation signal of plant nopaline synthase and which can be schematically represented as follows:

pCAT7 - This plasmid contains the CAT gene sequence inserted in pT35SN so that transcription leads to the production of antisense CAT-RNA, the plasmid can be schematically depicted as follows:

pCAT19 - This plasmid contains the CAT gene with four catalytic ribozyme regions / cm. example 4 and FIG. 9 / inserted in pT35SN so that transcription leads to the production of antisense CAT-RNA containing 4 catalytic regions of the ribozyme; the specified plasmid can be schematically depicted as follows:

Results:
Table 2 presents the relative CAT activity in cells 20 hours after electroporation. Activity was expressed as the percentage conversion of chloramphenicol substrate for 1 hour of analysis.

Based on the results obtained, the following conclusions can be drawn:
/ a / Introduction of a CAT gene construct leads to significant CAT activity - cf. 2A, B with IA, B. There are also differences between duplicates. It can be seen from the results that, as in other samples / cm. items "o" and "c" below /, 2A show an abnormally low activity.

 / b / Concomitant administration of an antisense gene construct leads to a decrease in the level of activity - cf. 3A, B and 4A, B with 2B. The degree of this decrease is directly related to the level of the added antisense gene in the form of a plasmid - cf. 3A, B and 4A, B.

 / s / Concomitant administration of the combined gene construct of the antisense gene / ribozyme leads to a decrease in gene activity - cf. 5A, B and 6A, B with 2B. In addition, this decrease is more noticeable for the corresponding levels of antisense gene constructs - cf. 5A, B with 3A, B and 6A, B with 4A, B.

 The average results for four in vivo experiments are shown in FIG. 17. In this drawing, the column "control" represents the processing 2; the “antisense” column represents processing 4; the catalytic column represents processing 6; and "background" - processing 1.

 Catalytic ribozyme inhibits CAT activity by an average of 47% and an average of 37% for antisense ribozyme.

 The introduction of ribozygous genes into plant cells inhibits the activity of the genes against which they are directed. Moreover, this inhibition is higher than for the corresponding antisense RNA molecules.

 These results show that these ribozymes can be active in animal, plant or microbial cells against a number of target RNA molecules.

 The mechanisms of action of ribozymes in these examples are not entirely clear. For example, the antisense ribozyme can irreversibly hybridize with the target RNA and catalyze the cleavage of the phosphodiester bond at one or more target sites along the target RNA. Alternatively, cell enzymes can unwind antisense RNA from its target sequence so that the target RNA is split into two or more fragments.

Example 9. In vivo - activity of ribozymes in animal cells
This example illustrates the activity of ribozymes in the inactivation of target RNA in mammalian cells.

Materials and methods:
The constructs of active genes encoding ribozymes were transfected into practically accessible COSI from the monkey kidney cell line by electroporation. In this method, 3 × 10 ⁶ / ml of COSI cells were suspended in saline buffer containing 10% FC / amniotic calf serum /, subjected to interaction with various gene constructs, and electric discharge was applied to effect electroporation of DNA in the cells. Transfected cells were incubated at 37 ^° C. for 48 hours in culture medium, after which analysis for CAT and luciferase activity was performed.

 Gene CAT constructs were introduced into the plasmid designated pTK CAT / Miksicek et al., Cell. 46: 283-290, 1986 /. This plasmid was formed by introducing the CAT gene sequence into the plasmid so that it was under the control of the herpes simplex virus thymidine kinase promoter.

 Gene constructs encoding ribozymes were introduced into plasmid pSV 232A (De Wet et al., Molecular and Cell Biology 7: 725-737, 1987) containing the luciferase gene fused to the early promoter SV 40. DNA encoding ribozymes were ligated to the Xbal site at the 3'-end of the luciferase gene in accordance with standard methods described by Maniatis et al., (Molecular Cloning, a Laboratory Manual, Cold Spring Harbor, 1982 /.

Using standard techniques, Maniatis et al., / See above / the following designs were obtained:
pFC58 - This plasmid vector contained DNA encoding the R _z CAT-1 ribozyme fused to the 3'-end of the luciferase gene in a non-functional orientation.

где 232A относится к последовательностям pSV232A, ранний SV40 означает ранний промотор SV40 и малая T означает ДНК, кодирующую малую промежуточную последовательность T промотора SV40. Эта конструкция позволяет продуцировать молекулу РНК, кодирующую люциферазу и рибозим R_zCAT-1, причем последний находится в ориентации, при которой он не имеет каталитического действия.The scheme of this plasmid can be represented as follows:

where 232A refers to the pSV232A sequences, early SV40 means the early SV40 promoter and small T means DNA encoding the small intermediate sequence T of the SV40 promoter. This design allows the production of an RNA molecule encoding a luciferase and ribozyme R _z CAT-1, the latter being in an orientation in which it has no catalytic effect.

pFC4 - This plasmid is the same pFC58 except that instead of R _z CAT-1 produce R _z CAT-3.

pFC1-6 - This plasmid is similar to pFC58 except that the ribozyme R _z CAT-1 is replaced by the ribozyme R _z CAT-3 in the sense orientation / 5'-3 '/.

pFC20 - This plasmid is similar to pFC1-6 except that R _z CAT-3 is replaced by the ribozyme R _z CAT-2 having 8 nucleotide flanking sequences.

pFC12 - This plasmid is similar to pFC20 except that the ribozyme R _z CAT-2 contains 12 nucleotide flanking sequences.

pFC54 - Эта плазмида аналогична pFC50 за исключением того, что CAT-ген и области рибозима находятся в антисмысловой /3'-5'/ активной ориентации.pFC50 - This plasmid contains the CAT gene with 4 catalytic ribozyme regions / cm. Example 4 and FIG. 9 / in the semantic orientation / 5'-3 '/, which upon transcription gives an inactive ribozyme. This plasmid can be depicted schematically as follows:

pFC54 - This plasmid is similar to pFC50 except that the CAT gene and the ribozyme regions are in the antisense / 3'-5 '/ active orientation.

 pFC64 - This plasmid contains the SV50 promoter and polyadenylation signals that are common with pFC50, and also contains the wild-type CAT gene, but without ribozyme regions. This gene is in antisense orientation and therefore cannot produce CAT protein.

 pFC65 - This plasmid is similar to the pFC64 plasmid except that the wild-type CAT gene is in the 5/3/3 sense orientation and therefore has the ability to produce a CAT protein.

Tests:
According to the methods of De Wet et al., / See above / analyzed for luciferase activity. In short, COS cells were lysed for 48 hours after transfection, and the cell lysate was incubated with luficerin, a luciferase substrate, and luminescence was determined using a scintillation counter.

 CAT activity was also determined using a COS cell lysate / cell lysates were halved and each half was used either for luciferase assay or for CAT activity / according to Sleing, M.T. Anal. Biochem 156: 251-256 / 1986 /.

нормализованную по отношению к продуцированию люциферазы.In vivo assays showed that pFC58 and pFC4 did not affect CAT activity in transfected cells. This activity was designated as 100% CAT activity and 0% CAT inhibition. CAT activity in cells transfected with other plasmids was measured with respect to pFC58. The percentage of CAT inhibition was determined by the formula:

normalized with respect to the production of luciferase.

CAT _research = result of CAT analysis for the studied structures. CAT _{'roll pin.} = CAT analysis for control constructs / pFC4 and pFC58 /.

 The production of luciferase is an internal control for electroporation and allows measuring the production of ribozyme in each individual electroporated tissue culture dish. See results at the end of the description.

Claims (11)

1. A ribozyme for inactivating a selected endogenous or exogenous target RNA in a cell by efficiently specific cleaving it at a site selected in such a way as to cause inactivation of said target RNA, said ribozyme containing: 1) a catalytic region that is capable of cleaving the RNA- sequence targets and catalytic activity of which does not depend on the sequence of the selected RNA target; 2) a hybridizing region comprising a hybridizing region extending from the 5'-end of the catalytic region and a hybridizing region extending from the 3'-end of the catalytic region, the hybridizing regions having a length sufficient for stable hybridization with complementary target RNA sequences flanking the selected cleavage site, and has a structure

where X is any ribonucleotide that may be the same or different;
(X) _n and (X) _{n '} each represents an oligoribonucleotide with a predetermined sequence that is capable of interacting by base pairing with the target RNA sequence to be cleaved and not covalently linked to XAAGC and XCUGA sequences in the period, respectively;
n and n 'each represents an integer that determines the number of ribonucleotides in the oligonucleotide, provided that the sum of n + n' is sufficient to realize stable interaction of the ribozyme with the target RNA sequence by base pairing;
each * represents a base pairing between ribonucleotides located on one side or another;
each solid line represents a chemical bond providing covalent binding between ribonucleotides located on one or another side;
a = 0 or 1, and if a = 0, then A located at the 5'-end of (X) _a binds to G located at the 3'-end of (X) _a ;
m and m 'each represents an integer that is greater than or equal to 1;
b is 0 or an integer greater than 2;
each dashed line independently represents either (i) a chemical bond providing covalent binding between ribonucleotides located on either side of (X) _b when b is an integer greater than or equal to 2, or (ii) the absence of any chemical tissue communication when b = 0.  2. Ribozyme according to claim 1, characterized in that (n + n ') ≥ 14.  3. Ribozyme according to claim 1 or 2, characterized in that n and n 'are greater than 6. 4. Ribozyme according to claim 1, characterized in that the cleavage site in the target RNA is located immediately after the sequence X ⁰ -UY, where X ⁰ represents any ribonucleotide, U is uracil and Y is adenine, cytosine or uracil. 5. Ribozyme according to claim 4, characterized in that X ⁰ is G and Y is C or A. 6. Ribozyme according to claim 1, characterized in that it has a structure

where X each represents a ribonucleotide, which may be the same or different;
(X) _n-1 and (X) _n each oligoribonucleotide having a predetermined sequence that is capable of interacting by base pairing with the target RNA sequence to be cleaved and is not naturally covalently linked to CAAAGC and XCUGA sequences, respectively;
n and n 'each represents an integer that determines the number of ribonucleotides in the oligonucleotide, provided that the sum of n + n' is sufficient to realize stable interaction of the ribozyme with the target RNA sequence by base pairing;
each * denotes base pairing between ribonucleotides located on one or the other side;
each solid line represents a chemical bond providing covalent binding between ribonucleotides located on one or the other side;
a = 0 or 1 and, when a = 0, then A, localized at the 5'-end of (X) _a , binds to G, localized at the 3'-end of (X) _a ;
m and m 'each is an integer that is greater than or equal to 1;
b is 0 or an integer greater than or equal to 2;
each dashed line independently represents either (i) a chemical bond providing covalent binding between ribonucleotides located on either side of (X) _b when b is an integer greater than or equal to 2, or (ii) the absence of any such chemical bond when b = 0. 7. Ribozyme according to claim 1, characterized in that it has a structure

where each X is a ribonucleotide, which may be the same or different;
(X) _n-1 and (X) _n each is an oligoribonucleotide with a predetermined sequence that is capable of interacting by base pairing with the target RNA sequence to be cleaved and does not naturally covalently bind to CAAAGC and XCUGA sequences, respectively;
n and n 'each represents an integer that determines the number of ribonucleotides in the oligonucleotide, provided that the sum of n + n' is sufficient to realize stable interaction of the ribozyme with the target RNA sequence by base pairing;
each * represents a base pairing between ribonucleotides located on one or the other side;
each solid line represents a chemical bond providing covalent binding between ribonucleotides located on one or the other side;
m and m 'each is an integer that is greater than or equal to 1;
b is 0 or an integer greater than or equal to 2;
each dashed line independently represents either (i) a chemical bond providing covalent binding between ribonucleotides located on either side of (X) _b when b is an integer greater than or equal to 2, or (ii) the absence of any such chemical bond when b = 0. 8. Ribozyme according to any one of claims 1 to 7, characterized in that it has a structure
3 '- [- (Y) _r -Q- (Y) _S -] _Z -5',
where Q is a compound having a structure according to any one of claims 1 to 7;
Y each is a ribonucleotide, the same or different;
r and s each is an integer greater than or equal to 0;
Z is an integer greater than or equal to 1.  9. A method for producing a ribozyme according to any one of claims 1 to 6, characterized in that a nucleotide sequence encoding the desired ribozyme is obtained, ligated to a suitable transfer vector containing DNA or RNA, or a combination thereof, and the ligation sequence is transcribed using RNA polymerases.  10. The method according to claim 9, characterized in that the resulting ribozyme is purified.  11. A method of inactivating a selected endogenous or exogenous RNA target within a cell, comprising exposing a target RNA inside a cell to a ribozyme capable of specifically cleaving said target RNA at a site selected such that said target RNA hybridizes within the cell with a ribozyme so , to cause the inactivation of the specified RNA target, characterized in that the specified ribozyme is a ribozyme according to one of claims 1 to 8.

Images