BG51160A3 - METHOD FOR OBTAINING RIBOSOMES - Google Patents

Abstract

The method is applicable in genetic engineering laboratories for obtaining ribosomes, necessary for creating means for the treatment of viral diseases in plants, animals and humans. It is characterized by high efficiency and reproducibility. It represents a combination of genetic engineering operations providing for the inclusion in the overall nucleotide structure of a hybridization region containing one or more branches formed by a single-stranded RNA complementary to at least part of a target RNA, its association with a catalytic region capable of degrading the target RNA, after which the structure undergoes stabilization. 4 claims, 17 figures

Description

(54) METHOD FOR OBTAINING RIBOSOMES

The invention relates to a method for producing ribosomes having highly specific endoribonuclease activity for the needs of genetic engineering.

Ribosomes are known to exist in nature, for example in avocado solar mosaic virus (A5VU), tobacco mosaic virus satellite ribonucleic acids (TMBV) and alfalfa temporal stripe virus (ALSV).

Their disadvantage is that, although they can be obtained ready-made, they undergo self-catalyzed degradation, which is an essential and unique part of their life cycle. The self-catalyzed degradation reactions of RNA are due to the well-known fact of the influence of divalent metal ions and neutral or high pH, and as a result, RNA is obtained with a terminus having a 5'-hydroxyl and 2', 3' cyclic phosphate groups /1/. The self-degradation reactions are catalyzed by the ribonucleic acids themselves, probably as a result of the conformational attraction of the reactive groups in close proximity. The sites of self-catalyzed degradation in naturally isolated RNAs are localized in narrowly specific regions of the RNA secondary structure /2/. The ribosomes found in nature in Tetrahymena thermofolia (known as 1Y8 or L-19 1Y8 RNA) and which have been described in detail in /3/, have an eleven base active site that hybridizes to the target RNA sequence, subject to further degradation, requiring free guanosine or guanosine derivatives. The fragments obtained after degradation contain 5'-terminal phosphate and 3'-terminal hydroxyl groups. The limited number of nucleotides capable of hybridization to the RNA substrate determines the efficiency or productivity of the Sech endoribonuclease in general, oligonucleotides containing less than 12 nucleotides hybridizing poorly to the target sequence. It has also been found that a certain number of nucleotides in the active site of the Sech endoribonuclease must be preserved for the efficiency of the endoribonuclease activity. This limits the number of active site sequence permutations that can be designed to efficiently hybridize to the target sequence, making the restriction site region of the RNA target sequence suitable for digestion with Sech endoribonuclease. Sech ribonuclease also modifies DNA by adding a free guanosine nucleotide to the 5'-end of the digested RNA.

The object of the invention is to create a method for producing ribosomes having highly specific endoribonuclease activity, which is highly efficient and reproducible and which avoids the self-catalytic decomposition of natural products.

This problem is solved by constructing ribosomes from a corresponding DNA sequence (DNA which, upon transcription, produces a ribosome, synthesized according to known methods for DNA synthesis [10]), linked to an RNA polymerase promoter, such as, for example, a promoter for T7 RNA polymerase, P6 RNA polymerase or... RNA polymerase. A DNA sequence corresponding to the ribosomes according to the invention can be linked into a DNA transfer vector, such as a plasmid or bacteriophage DNA. When the transfer vector contains an RNA polymerase promoter linked to DNA corresponding to the ribosomes, the ribosome is usually obtained by incubation with RNA polymerase. Therefore, ribosomes can be produced in vitro by incubation of RNA polymerase with the RNA polymerase promoter linked to DNA corresponding to the ribosome, in this case ribonucleotides. In vivo, prokaryotic or eukaryotic cells (including mammalian or plant cells) can be transfected with a suitable transfer vector containing genetic material corresponding to the ribosomes of the invention, which is linked to an RNA polymerase promoter such that the ribosome is transcribed in the host cell. In this case, the transfer vector can be a bacterial plasmid or viral RNA or DNA. The nucleotide sequences corresponding to the ribosomes are placed under the control of strong promoters, such as lac, sU40 lac, sU40 eagu, methionine or promoters. The ribosomes can be directly transcribed in vivo from the transfer vector or conversely - they can be transcribed as part of a longer RNA molecule. Thus, DNA corresponding to a ribosomal sequence can be linked to the 3' end of the carrier gene, e.g. after the translational stop signal. Larger RNA molecules can help stabilize ribosomal molecules against nuclease digestion inside cells. Upon transcription, the carrier gene can lead to a protein whose presence can be directly detected, e.g. by an enzymatic reaction. For example, the carrier gene can encode an enzyme.

According to the invention, it is also possible to create a DNA transfer vector which contains a DNA sequence corresponding to a ribosome linked to a promoter to ensure transcription of the ribosome. To obtain the ribosomes, two synthetic oligonucleotides of complementary sequences, created in a known manner /4/, are hybridized together, one of the oligonucleotides encoding the desired ribosome. The respective ends of the hybridized oligonucleotides correspond to different restriction enzyme sites, called EcoK.1 at one end and P3P at the other end. After digestion with appropriate restriction enzymes (EcoK1 and P3P in the above example), the double-stranded DNA fragment can be cloned into a transfer vector. Where the plasmid vector contains an RNA polymerase promoter following the DNA sequence corresponding to the ribosomes of the invention, the RNA transcripts corresponding to the ribosomes can be generated both in vivo and in vitro. Where the ribosome is generated from two halves carried by a base pair between complementary nucleotides, each of the ribosomes can be produced according to the above techniques, and the halves incubated together to form the ribosomes.

In this case, it is preferable that the ribosomes have a hybridization region comprising one or more arms formed by single-stranded RNA and having a sequence complementary to at least a portion of the target RNA. These one or more arms are associated with a catalytic region capable of degrading the same RNA machinery. Where the hybridization region comprises one arm, it comprises at least 9 nucleotides, and where the hybridization region comprises 2 or more arms of RNA, the sum of the nucleotides is greater than 9.

According to one embodiment of the invention, the resulting ribosomes have the formula:

3' 5 ' / X /„ X x / x / _g /I/ A C /I/

A C & 1 _A C /II/

C · & c· A

5* 3* in which X is any ribonucleotide and each X residue may be the same or different; the sum of η and η is greater than 6 and η and η may be the same or different; the sign / + / represents pairs of pairs between complementary ribonucleotides; X and X are oligonucleotides of complementary sequences along at least a portion of its length to allow the formation of base pairs between the oligonucleotides? or X and X together form a single-stranded DNA sequence, at least a portion of this sequence including the stem formed by the base pairs between the complementary nucleotides; it is optional, but possible, in formula /1/ after 1a to insert a nucleotide selected from one of A, C, C or and.

Region /I/ of formula /1/ represents the arms or flanking sequences of the ribosome that hybridize to the corresponding portions of the target sequence. The arms may hybridize along the entire length of the target sequence or a portion thereof. Such a catalytic region in formula /1/ is depicted as region /II/. The catalytic region may contain one or more additional nucleotides that do not adversely affect catalytic activity. The catalytic region may also form part of the hybridization region.

Oligonucleotides X' and X can include up to 5,000 or more nucleotides.

Another specific embodiment of the invention is illustrated by a ribosome formula having the form:

A ^{A η} in s » x/x/ _n , ^T A ^{& 5} 6 & A X X X X * X /X/ * X X v i in which: X represents any fish-

nucleotide and each X residue may be the same or different; the sign / + / represents the base pairs between complementary ribonucleotides; η and n' are the same as defined in formula /1/; m and m' are one or more or may be the same or different; B represents the 20 bond, base pair, ribonucleotide or oligonucleotide containing at least 2 ribonucleotides; after 1a, one additional nucleotide selected from any of A, O, C or u, in formula 25 /2/ could be included.

The ribosomes of the invention contemplate a target RNA sequence that comprises the sequence XθυΥ, in which X° is any ribonucleotide, and is uracil and Y 30 is the flanking region that is not base-paired. It is preferred, but not exclusive, that X° is guanidine and XθυΥ is OiC or OiA. Any RNA molecule comprising these sequences can be digested by ribosomes of the present invention. As an exception, the sequence of the RNA transcript comprising X°ΌΥ is determined. The branches of the ribosomal sequence can be synthesized so as to be complementary to, and thus hybridizable with, the target sequence flanking the XθυΥ sequence. Upon hybridization of the ribosome flanking arms with the target RNA sequence flanking the X°ϋY sequence, the catalytic region of the ribosome cleaves the target RNA within the X°υY sequence. RNA cleavage is facilitated in this case by magnesium 50 or another divalent cation at a pH of approximately 8.0.

According to the invention, ribosomes can be designed to cleave any RNA whose sequence is known. The high repeatability of residues cleaved by ribosomes in RNA means that (1:64 for C1C in RNA with random and uniform repeatability of the base distribution) a large number of potential ribosomal cleavage sites can be predicted in any target RNA.

According to another aspect of the invention, there is provided a method for inactivating a target RNA sequence, which comprises reacting said target RNA sequence with ribosomes constructed according to the present invention.

In vivo, i.e. inside the cell or cells of an organism, a transfer vector such as a bacterial plasmid or viral RNA or DNA, which encodes one or more ribosomes, can transfer them into the cells /5/.

Inside the cells, the transfer vector can be replicated and transcribed by cellular polymerases to produce ribosomal RNAs that then inactivate a desired target RNA. In contrast, a transfer vector containing one or more ribosomal sequences can be transferred into cells or introduced into cells by micromanipulation techniques, such as microinjection, so that the transfer vector or a portion thereof is integrated into the host cell genome. Transcription of the integrated material reaches ribosomes, which act to inactivate a desired target RNA.

The ribosomes obtained according to the invention can be extensively therapeutically and biologically applicable. For example, human and animal diseases caused by viruses can be inactivated by administering to infected subjects ribosomes according to the invention adapted to hybridize with degraded RNA transcripts of the virus. Such ribosomes can be delivered by parenteral or any other means of delivery to the body. In contrast, a subject infected with a virus can be provided with an avirulent virus, such as a vaccine or adenovirus, which is genetically engineered to contain DNA corresponding to ribosomes linked to a promoter, so that the ribosomes are transcribed in the host cells, carried by a specially engineered virus to cause degradation and/or inactivation of the small RNA transcript of the disease-causing virus. The ribosomes obtained according to the invention have particular application for viral diseases, such as e.g. herpes simplex virus (H8V) or the AIDS virus (H1V).

The ribosomes of the present invention also have particular application for inactivating RNA transcripts in bacteria or other prokaryotic cells, plants or animals. In bacteria, RNA transcripts from e.g. bacteriophages, causing the death of bacterial transcripts from e.g. bacteriophages, can be inactivated by transfection of cells with a DNA transfer vector capable of producing ribosomes according to the invention that inactivate phage DNA. Conversely, ribosomes can be introduced into or removed from bacterial cells to cause degradation of phage RNA.

RNA transcripts in plants can be inactivated using ribosomes encoded by a transfer vector such as the T1 plasmid from Ao. When such vectors are transfected into plant cells, the ribosomes are produced under the action of RNA polymerase and can cause degradation of a specific target sequence. Accordingly, plant viruses whose RNA sequence is known, or RNA transcripts from plant genes, can also be inactivated using ribosomes.

Endogenous genes and transcripts in plants, animals or other cell types can be inactivated using ribosomes of the present invention. Accordingly, various undesirable genotypes or characteristics can be modulated. It is possible, for example, with the use of ribosomes constructed according to the invention to remove pits from fruits or to treat hereditary diseases in humans caused by the products of harmful proteins or the overproduction of specific proteins.

The invention is illustrated by the following figures.

Figure 1 shows the degraded sites of wild-type and mutant RNAs; an electrophoretic profile showing products of self-catalyzed RNA degradation.

a/ Summarizing the conserved structures associated with naturally isolated RNA cleavage sites in A8VU, satellite DNA from newt, and satellite RNAs from 3TovKU, LT8U, tobacco mosaic virus, and sunflower mosaic virus. Nucleotide sequences that are conserved among these structures are shown, while others are represented as X. Base pairs are represented by + and RNA cleavage sites - by an arrow.

b/ Shows the nucleotide sequences associated with the /+/ strand of TovKU RNA. The cleavage site is indicated by an arrow.

c/ In vitro mutant of TovKU containing an 11 nucleotide inertia along with flanking duplicates of three nucleotides (residues 7 to 9).

d) Subcloned Hae III fragments from wild-type TovKU and D-51 in vitro mutants, each transcribed in the / + / and / - / orientation and labeled transcripts fractionated by polyacrylamide electrophoresis. The positions of the digested 159 and 170 bases of transcripts from wild-type TovKU (AUT) and mutant (D-51) sequences are marked with an arrow. The sizes of the digestion products are shown.

Figure 2 shows the nucleotide sequence of the ribosome and ribosomal degradation products separated by gel electrophoresis.

a/ The nucleotides included in mutant D-51 contain a BamH1 restriction endonuclease site (Fig. 1c). BamH1 was used to digest the mutant DNA and two sequences were subcloned and transcribed in vitro. The RNA transcripts are shown schematically, with potential base pairs between RNAs marked with + . The fragment containing the digestion site marked with an arrow refers to -RNA; the fragment containing the ribosome is designated as Kζ -RNA.

b/ ³² P/ -Κζ -RNA /101 bases/ was incubated alone (lane 1), and with unlabeled 8 -RNA (lane 2). Ρ -8-RNA was incubated alone (lane 3), and with unlabeled and ³² P -labeled ΚΖ -RNA (lanes 4 and 5 respectively).

Figure 3 shows a schematic model of a ribosome according to one embodiment of the present invention. Region A represents the cleavage sequence within the target RNA. Region B represents the catalytic region and regions C represent the branches of the ribosome.

Figure 4 shows the design of ribosomes that are targets for CAT (chloramphenicol acetyl transferase) gene transcription. The ribosomes, designated KzCAT-1, 2 and 3, are directed against a site within 835 bases transcribed in vitro from the CAT gene. Relative locations of the cleavage sites of the transcripts are shown schematically with flanking bases numbered a. The three ribosomal sequences (a to c) are shown with their target sequences. The amino acid sequences of the CAT genes are numbered, the predicted sites for RNA cleavage are indicated by arrows. Kz-CAT-1 and 3 contain 24 base sequences derived from the / + / chain of 3TovKU (region B, Fig. 3), while Kz-CAT- contains a single i-A change in this region.

Figure 5 shows the results of CAT RNA fragmentation with ribosomes KgCAT-1 to 3.

a/ RNA labeled with Pi, designated as /P/CAT, was subjected to gel fractionation after incubation alone with one of the three ribosomes KgCAT-1 to 3 (lanes 1, 2 and 3 respectively). The localization of the complete transcript is indicated by arrows.

b/ 5'-terminal base analysis. The 3'-fragments produced by ribosomal cleavage of CAT mRNA are subjected to kinase, gel purification, complete nuclease digestion and terminal fractionation at pH 3.5 polyacrylamide gel electrophoresis. The 5'-terminal nucleotides, determined by reference to markers (lane M), are A, and C for the fragments produced by P CAT-1 to 3 (lanes

1,2 and 3 respectively).

Figure b shows the time course of catalytic activity of the ribosome KgCAT-1 against CAT RNA. The amount of degradation products at the 139th nucleotide is determined and plotted. The inset shows the accumulation of the 139 base fragment over time after polyacrylamide gel electrophoresis.

Figure 7 shows the relative extent of CAT RNA degradation under different temperature conditions. The substrate RNA is represented by a solid line. In each case, the degradation products are shown by a dashed line.

Figure 8 shows three ribosomes corresponding to KgCAT-2 that have branches or flanking sequences of different lengths.

Figure 9 shows the scheme of preparation of ribosomes corresponding to KzCAT-2, which include catalytic nonsense RNAs containing each of the degraded regions of KzCAT-1 to 3.

Figure 10 shows ribosomes hybridizing to short sequences containing SCA (10a) and Clu (10b), essential in CAT mRNA.

Figure 11 shows the sites of self-catalyzed RNA cleavage in citrus outer envelope virus (CEV) and its complements.

Figure 12 shows the ribosome KgCEU 25x/ + / hybridized with target CEU RNA /a/ and gel electrophoretic profile of /+/CEU RNA and complementary /-/ CEU RNA incubated with KgCEU 25x/ + / , /b line respectively. The cleavage products are indicated by arrows.

Figure 13 shows the KgSAT-2 ribosome hybridizing to its target sequence (a) and the Kg3SMoU ribosome (c). The catalytic region in each ribosome is highlighted. The differences in the catalytic regions of Kg3SMoU compared to KgSAT-2 are highlighted.

Figure 14 shows the ribosome of KrCEU-2 hybridizing to a target sequence of the citrus outer envelope virus (CEU) RNA. The cleavage site corresponds to nucleotide 336 in the CEU RNA sequence. The alternative sequence in the catalytic region, compared to the catalytic region of ZTovKU, is circular (a). Figure 14c shows an electrophoretic profile of a control strand of CEU/RNA, lane 7, and a /+/ strand of CEU RNA, lane 8, after incubation with P CEU ₂ .

Figure 15 shows the KgCAT-2/a ribosome compared to the KgCAT-2B/c ribosome. The catalytic regions are shown in bold. The changes in the catalytic region of KgCAT-2B compared to PCAT-2 are also highlighted.

Figure 16 shows the map of plasmid ρ135Ν.

Figure 17 presents graphically averaged results from four experiments on the inhibition of CAT expression in plants (tobacco protoplasts).

Reactions and manipulations involving DNA such as ligation, restriction enzyme digestion, bacterial transformation, DNA sequence alignment, etc., are carried out according to standard techniques described, for example, by Masha et al. /6/.

Manipulations involving RNA were also performed according to standard techniques /8/.

The advantages of the method according to the invention are that it is highly efficient and reproducible, and the resulting ribosomes are of wide applicability in biotechnological practice.

The invention is illustrated by the following exemplary embodiments:

Example 1. Self-catalytic cleavage of mutant TovRU RNA.

The regions associated with natural sites in AAVU, satellite DNA transcripts from newt and satellite DNAs from 3TovKU, LTZU, tobacco mosaic virus (TMV) and alfalfa Mediterranean mosaic virus (LMV) are aligned as shown in Figure 1a. Nucleotide sequences conserved between these structures are shown, while non-conserved sequences are represented as X. An extra i is positioned after residue 1A of the LTV/+/ strand.

The region associated with the self-catalytically cleaved / + / chain of ZTOvKU was studied to determine the enzyme substrate activity within this region. First, the ZTOvKU cloned DNA was mutated using an oligonucleotide linker (BamH1) insertion protocol.

Construction of a vector for in vitro expression of TovKU.

A 160 bp Tag 1-5pe1 fragment of 3TovRU with DNA was isolated from p3P 653 (17) and ligated with Acc1 3pe1, cut and treated with phosphatase pCEM-b to modify the Acc1 site. The resulting clone was linearized with Acc1, treated with phosphatase and a 359 bp Tag 1 fragment of 3TovRU with DNA was inserted. The resulting clones were screened for the presence of a cyclic 520 bp 3TovRU with a DNA sequence containing terminal residues 277 to 81 (pTT). The TovRU sequence is flanked by the promoters for T7 and P6 RNA polymerases and transcription, carried out in vitro, results in either a + or - orientation, which contains two self-cleavage sites.

In vitro mutagenesis.

Plasmid pTT3/50u£/ was linearized with BamH1, treated with 31 nuclease and religated to remove the unique BamH1 site. The resulting construct, pTT8-B, was treated with 2x10 ⁴ units of deoxyribonuclease 1 in 20 mM Tris-HCl pH 7.0;15 mM MgCl for 10 min at 37°C. The resulting linear DNA was equilibrated and/or filled in using T4 DNA polymerase and purified by 0.7% LCT agarose gel electrophoresis and extraction. The kinased BamH1 linker with the sequence (CCOATCCC) was ligated to the linearized plasmid for 24 hours at room temperature in the presence of 5% polyethylene glycol. Subsequently, the reactions were carried out with BamH 1 and the linear plasmid DNAs were repurified on 0.7% LCT agarose gel electrophoresis (here it was found necessary to remove the last small amounts of circular plasmid together with the unligated linkers). The plasmids were recircularized using T4 DNA ligase and transformed into E. coli DNA1. Colonies (greater than 1000) were picked from the agar plates, grown in liquid cultures to saturation and then mixed plasmid DNA cultures were prepared. The mixed 3TovRU with DNA inserts were cut with restriction enzymes at the flanking EcoRI and Psm1 sites, purified on 1% LCT agarose gel electrophoresis and subcloned into the EcoRI-Psm1 fragment treated with pCEM 4. The resulting transformants were re-pooled, cultured in liquid culture, and plasmid DNA was prepared. Plasmid DNAs were treated with BamH1 to degrade only those plasmids containing BamH1 linker sequences, and linear forms were re-purified twice by 0.7% LCT agarose gel electrophoresis, re-circularized with T4 DNA ligase, and transformed into E. coli DN-1. Individual transformants were screened for terminal positions of the inserted BamH1 linkers within the TovKU sequence by restriction enzymes, subcloned into M13, and aligned by the dideoxynucleotide chain termination technique.

A library of ZTovKU mutant residues and nucleotide sequence analyses showed that each mutant contained a BamH1 linker sequence that was inserted (CCCATCCC) along with a flanking or deleted ZTovKU sequence. The mutants were transcribed in vitro and the RNAs were tested for their ability to undergo cleavage. From these experiments, a 52-nucleotide sequence was identified as containing both the substrate and cleavage sites of ZTovKU RNA. This 52-nucleotide sequence, shown in Fig. 1c, contains the region of conserved sequences required for the self-cleavage of other RNAs (Fig. 1a). One mutant, designated D-51, contained an eleven-nucleotide BamH1 linker sequence interspersed between three duplicated ZTovKU nucleotides, numbered 7 to 9. This mutant underwent self-catalyzed RNA cleavage.

Fragments of 97 and 108 base pairs of HaeIII containing a 52-nucleotide sequence from the digestion of wild-type and mutant type D-51 RNAs (as shown in Fig. 1c and 1c) were excised from plasmid clones. The fragments were ligated into the 3ma1 site of pCEMd and screened for both orientations of the insert. Plasmids were linearized using EcoK1 and / + / and / - / RNA strands of 159 to 170 bases in length were transcribed using 200 units/ml T7 RNA polymerase in 50 mM Tris-HO, pH 7.5, 10 mM NaOH, mM _{MgCl 2,} 2 mM spermidine, 1000 units/ml ribonuclease, 500 uM ATP, STF and OTP with 200 uM aP/iTP. The ribonucleic acids were fractionated by electrophoresis on a 10% polyacrylamide, 7 molar urea, 25% formamide gel and autoradiographed.

As shown in Fig. 1, unspliced transcripts from the /-/ strand of RNA were examined. As expected, the /-/ strand does not contain a site for self-catalyzed degradation. With the /+/ strands of the wild-type and D-51 sequences, degradation can occur, with degradation of the D-51 RNA occurring less efficiently than that of the wild-type (Fig. 1ά). This experiment shows that the region of the double-stranded loop at the right-hand 52-nucleotide sequence involved in self-catalyzed RNA degradation is not essential.

Separation of enzyme and substrate activity

Using the BamH1 restriction endonuclease site included in D-51, flanking 5 H.coH1-BamH1 and Bam-H.coH1 fragments were obtained and each was subcloned into an E.coH plasmid suitable for in vitro transcription. This resulted in the elimination of the mutant single-stranded loop from the self-cleavage region, splitting the region into two RNA segments (Fig. 2a). The smaller H.coH1-BamH1 fragment contained nucleotides 321 to 9, including the active cleavage site, and was designated the 3 fragment. The BamH1-H.coH1 fragment, containing nucleotides 7 to 48, was designated the ribosome or Kζ fragment. The E.coH plasmids used for in vitro transcription were pOEM3 and pOEM4 /10/.

These expression plasmids contain: a/a type of replication; b/a selective gene, drug resistance (AMP); c/a multiple cloning site flanked by RNA polymerase promoters that can be used for in vitro production of transcripts.

T7 DNA polymerase treated, 25 kb digested Kz-pOEM3 and Xva1-ligated 8-pCEM4 were transcribed using 3Pb RNA polymerase under the same conditions as used above.

As shown in Fig. 2, both ribonucleic acids 3 and P showed negligible degradation when incubated alone (Fig. 2c, lanes 1 and 3) under conditions suitable for highly efficient self-degradation (50°C, 20 mM MgCl ₂ , pH 8.0). Labeled Kζ-PHK also appeared unchanged after incubation with

5-RNA (Fig. 2c, lanes 2 and 5). However, when 8-RNA was mixed with κζ-RNA, efficient cleavage of 3-RNA occurred (Fig. 2c, lanes 4 and 5), producing two fragments. The products were formed by cleavage of 3-RNA (84 bases) at a common site between nucleotides 359 and 1 to give 5 and 3 proximal fragments of 67 and 17 nucleotides, respectively. This indicates that 8-RNA acts as a substrate for ribonucleolytic cleavage by κζ-PHK, which operates under catalytic conditions.

A model of a ribosome based on the catalytic region of a 3TovKU RNA is shown in Fig. 3. The ribosome has two branches or flanking sequences of single-stranded RNA, shown at C, hybridized to complementary sequences on the substrate RNA, i.e.

RNA is cleaved. Each flanking sequence, shown at C, contains 8 ribonucleotides. Some of the nucleotides included in the C region are not critical. However, sufficient nucleotides must be present to allow the ribosomes to hybridize to the target RNA. Four nucleotides in each C region are required to represent the minimum number for hybridization.

The catalytic region B contains sequences that are well conserved in naturally occurring cleavage sites (see Fig. 1a). Compared to cleavage sites from known sequences, the length of the base pairs of stem II is not significant, as is the presence of an associated loop at one end.

The cleavage site within the target RNA is depicted in A (Fig. 3) as C1C. Based on our experiments (not shown), and those of Kozlup (PEB5 LET 288; 229-230, 1988; and PEB5 LET 239; 285-288, 1988) in the cleavage of naturally occurring RNAs, the sequences cyl, cis, cis, lys and uiC also act as cleavage sites within the RNA.

Example 2. Demonstration of the design, synthesis and activity of ribosomes with novel and highly specific endoribonuclease activity.

As an illustration of the invention, three ribosomes are described which are directed against transcripts of a widely used indicator gene derived from a bacterium, Tp9 Chloramphenicol Acetyl Transferase (CAT), which encodes the synthesis of antibiotic resistance in bacteria, plants and animals and which can be readily assayed. These ribosomes, described as Kζ CAT -1 to 3, correspond to potential Cb1C sites in CAT RNA at positions 139-140, 494-495 and 662-663 respectively. The sequences of these ribosomes are shown in Figure 4. In each case, the flanking sequences of the ribosome which hybridizes to the target CAT RNA are 8 nucleotides in length. The catalytic region is chosen to correspond to those of the 8TovU RNA shown in Figure 3.

The CAT gene was obtained from pCM4 and subcloned as a BamH1 fragment into pCEM-32 (from Proteda, MacHinson, JT, et al.). This plasmid was linearized with H1παIII and CAT gene transcripts were prepared using T7 RNA polymerase with 220 32 m/a - P/uTP. Ribosomal sequences were synthesized as oligonucleotides, Kζ CAT-1, 2 and 3 respectively. These were kinased, ligated by phosphatase treatment, cut with EcoK1-P3I for pCEM4 and incubated with the K1epo fragment of DNA polymerase 1 prior to bacterial transformation. The EcoK1-linearized plasmids were transcribed with T7 RNA polymerase to produce ribonucleic acids. Ribosomes were incubated with CAT transcripts in 50 mM Tris-HCl pH 8.0, 20 mM MgCl ₂ at 50°C for 60 min and the products fractionated by 5% polyacrylamide 7M urea, 25% formamide gel electrophoresis, followed by autoradiography.

When the 840-nucleotide CAT transcript was incubated with each of the three ribosomes, efficient and highly specific cleavage occurred (Figure 5), producing 2 RNA fragments in each reaction. The size of the fragments was determined by the cleavage sites (i.e., the 139 and 696,494 and 341,662 and 173 base fragments were the 5'- and 3'-products of the KgCAT-1 to 3-catalyzed cleavage, respectively). The conditions required for this ribosome-catalyzed cleavage were analogous to those observed in natural cleavage reactions (3, 11), with more efficient cleavage occurring at elevated pH, temperature, and divalent ions (data not shown). When present in molar excess, the three ribosomes catalyzed complete and complete degradation of the CAT RNA substrate after 60 min in 50 mM Tris HCl, pH 8.0, 20 mM MgCl ₂ at 50°C. Under analogous conditions with 0.1 μM substrate and 3 μM ribosomes, the T ι/ ₂ of the CAT mRNA substrate was 3.5, 3.5, and 2.5 min in the presence of KgCAT -1 to 3, respectively. The ribosomal sequences were inactive against complements of the substrate RNA (i.e., the + strand), and in the form of oligonucleotides (data not shown). The 3'-terminal cleavage fragments from each ribosome-catalyzed reaction were isolated and the 5'-terminal was treated with kinase ^P32 (50 mM Tris HCl, pH 9, 10 mM _MgCl2 , 10 mM DTT with 50 and Cl- ³² P ATP and 5 units of T4 polynucleotide kinase for 30 min at 37°C). The efficient kinase action of the fragments indicated that they possessed 5'-terminal hydroxy groups analogous to those produced in naturally occurring cleavage reactions.

The terminal nucleotides of the fragments obtained by cleavage of the CAT sequences with KgCAT -1 to 3 were determined. Specifically, the radiolabeled fragments were purified on a 5% polyacrylamide gel and treated with an equivalent amount of 500 units per ml ribonuclease T1, 25 units per ml ribonuclease T2 and 0.125 mg/ml ribonuclease A in 50 mM ammonium acetate, pH 4.5 for 120 min at 37°C. The products were fractionated on a 20% polyacrylamide gel containing 25 mM sodium citrate, pH 3.5 and 7 molar urea. Figure 5c shows that cleavage of the CAT sequence from K/CAT -1 to 3 occurs before nucleotides A, and and C, respectively.

The terminal sequence of the CAT gene fragment was determined directly by partial enzymatic digestion according to a known procedure /12/, using base-specific and partial ribonucleolytic digestion. The sequence of the fragments confirms that the digestion occurs at the expected locations within the CAT RNA (not shown).

Enzyme catalysis.

To demonstrate that these ribosomes catalyze the cleavage of the CAT mRNA substrate, each was incubated with a molar excess of substrate, under conditions that should promote simultaneous and efficient cleavage and dissociation of the products. Figure 6 shows the results of an experiment in which, after 75 min at 50°C, pH 8.0 in 20 mM MgCl, 10 rmol of Kζ CAT-1 catalyzed the specific cleavage of 163 rmol of the cleaved CAT mRNA (of 173 bases) used as substrate to yield 5 and 3 fragments of 139 and 34 bases, respectively. On average, each ribosome was involved in more than ten cleavage units. After 75 min at 50°C, some nonspecific RNA degradation was noted due to the extreme conditions, but 70% of the remaining intact RNA (163 rmol) was accumulated as a 139 base fragment. Similar results were obtained for Kζ CAT-2 and 3 (data not shown), and thus each acts as an RNA enzyme.

Example 3.

Effect of temperature on ribosomal activity

The effect of temperature on ribosomal activity in vitro was tested.

A series of reactions were performed to test the effect of ribosomes Kζ CAT -1 to 3 on CAT RNA substrate at 37°C and 50°C.

In this experiment, the reactions for each ribosome were performed in duplicate using the ribosomal fragmentation reaction conditions established in Example 2. One reaction was incubated at 37°C and the other at 50°C. Samples were removed after 90 min and the extent of the reaction was analyzed by denaturing gel electrophoresis. Figure 7 shows the reaction time for each of the ribosomes KgCAT-1 to 3 at 37°C and at 50°C. The extent of the reaction for each ribosome increased with increasing reaction temperature.

The time taken for 50% (μl/g) degradation of CAT RNA is set forth in Table 1.

Table 1

KgSAT -1 KgSAT -2 KgSAT -3 11/2 /minutes/ 50°C 3.5 3.5 2.5 37°C 55, 70.0 65.0

As can be seen from Table 1, the rate of ribosome reaction at 37°C is approximately 20 times lower than that at 50°C.

Example 4. Influence of the length of ribosomal branches (or flagellar sequences) on ribosomal catalytic activity.

The branching or flagellar sequences of ribosomes (region I of formula

1) hybridizes the ribosome to the target RNA where the RNA is fragmented. In this experiment, the effect on the rate of degradation of the target sequence was investigated by comparing the size of the complementarity and the corresponding base pair sequence of the ribosomal branches to the target sequence.

Ribosomes were produced with 4, 8 and 12 base complementarity to the target sequence KrCAT-2 on each strand (Figure 8a). Ribosomes were prepared according to Example 2. Ribosomal activity was determined by incubating ribosomal RNA with CAT RNA as described above.

A ribosome with 4 base complementarity on each strand does not cleave the substrate RNA. A ribosome with 8 base complementarity on each strand cleaves the CAT substrate as well as ribosomes with 12 base complementarity. Ribosomes with 12 base complementarity cleave target RNA more efficiently than is assessed by in vitro reactions with ribosomes with fewer base complementarities. Even though more than four base complementarity is required, increasing the length of the hybridization region of the ribosome increases its reactivity.

In the second experiment, the efficiency of ribosomes having a/ complementarity to the full length of the CAT transcribed target RNA and b/ multiple catalytic domains were examined.

Four CsC target sites in CAT RNA sequences were selected. Ribosomal catalytic regions against these sites were inserted into a complete nonsense /-/ sequence for the CAT transcript and catalytic activity was tested.

The four sites selected are the three precisely defined by KgSAT -1 to 3, described earlier, and further sites, which can be presented as follows:

New CAT sites * 192

H18 H18 A1a Ua1 Dry Azr S1u

5' 3' sli eli sss sis isio odi sos where 192 refers to amino acid 192 of the CAT polypeptide and refers to the cleavage site.

Oligoribonucleotides containing ribosomal catalytic regions and covering each of these cleavage sites were used for M13 mutagenesis experiments to produce a sequence containing the full complement of the CAT sequence, but with the four ribosomal catalytic regions inserted into them. M13 mutagenesis was performed by ligation of the oligonucleotides containing the ribosomal inserts to single-stranded M13 DNA containing uracil, followed by synthesis of complementary DNAs containing the insert. The complementary DNAs were recovered following cloning into a known strain of Escherichia coli (13).

The resulting double-stranded DNA was cloned into an in vitro expression vector to produce ribosomal RNA using the T7 polymerase transcription system. Ribosomal activity was determined by incubation of ribosomal RNA with CAT transcription, followed by gel electrophoresis of the reaction mixture after glyoxal treatment to denature nucleic acids.

Autolytic cleavage is observed at all of the expected sites of CAT transcription. Accordingly, flanking sequences from the branching or ribosomal regions may extend along the entire length of the RNA transcript that undergoes cleavage.

Figure 9 schematically shows the production of catalytic nonsense RNAs containing each of the four ribosomes. Catalytic nonsense RNAs contain approximately 900 bases.

Under the above reaction conditions, ribosomal and target sequences form high molecular weight complexes, probably through extensive base pairing. A strong denaturing treatment, such as glyoxal treatment, is recommended to break down the reaction products during electrophoresis.

Example 5. Target sequences for ribosomal fragmentation.

The CiA motif in mRNA was tested to determine whether the ribosome would affect DNA degradation at this sequence.

The specific site in the CAT mRNA containing the Cia motif (Figure 10a) was selected and a corresponding ribosomal sequence was prepared and tested for activity. Ribosomes contain branches of 8 ribonucleotides.

Synthetic oligonucleotides corresponding to the ribosomes of Figure 10 were prepared according to Example 2 and the double-stranded cDNA was cloned into an in vitro expression vector (pOEM 4, see above) in E. coli to produce ribosomal RNA using the T7 polymerase transcription system. Ribosomal activity was determined by incubation of ribosomal RNA with CAT mRNA, followed by gel electrophoresis of the reaction mixture as described above.

Ribosomes induce cleavage of the Cia target site (not shown). Accordingly, the Cia motif in RNA is a substrate for ribosomes of the present invention. This is not entirely unexpected since a naturally occurring site in satellite RNA from an alfalfa virus requires recognition of the Cia site.

Similarly, a Cii motif in the CAT RNA target sequence was tested with a suitable ribosome (see Figure 10c) and caused cleavage.

Example 6. Ribosomal degradation of viral RNA.

Viral RNA, particularly from citrus rind virus, is fragmented using the ribosomes of the present invention.

Two CiS target sites were selected from citrus peel virus (CWV). One site from the complementary strand sequence was also selected. Ribosomes were prepared against all of these sites and tested for activity. The ribosomes were designated CWV9x/ + /, CWV9x/-/ and CWV25x/ + /. Figure 11 shows the three cleavage sites in CWV RNA for each of these ribosomes.

Ribosomes were prepared according to previous methods. Ribosome KgCEU25x/+/ is shown in Figure 12. The ribosome cleaves the OiS motif at nucleotide 116 of CEU RNA. Figure 126 shows cleavage of CEU RNA by ribosome P KgCEU9x/+/. No cleavage was observed with ribosome KgCEU9x/-/.

The experiment shows that ribosomes are active against target RNA sequences from a variety of sources. This is expected, as all RNAs are formed from the basic ribonucleotide building blocks of adenine, guanine, cytosine and uracil, regardless of their origin from an animal, plant or microorganism.

Example 7. Examples and ribosomes possessing different catalytic regions.

The ribosome targeting the CAT-2 site was constructed using the catalytic region of a satellite RNA sequence from Mediterranean clover spot disease virus (MCSV). Twelve-base complementarity from the ribosomal flanking sequence was incorporated into the structure of the KgMSV ribosome. The _K2 CAT-2 and KgMSV ribosomes are shown in Figure 13a and 13b, respectively. The loop region of KgMSV contains 5 nucleotides having the sequence AAAAiC, in contrast to the loop of KgSAT-2, which contains four nucleotides having the sequence ACAO. In addition, KmMSV contains a C in the catalytic region, in place of the and in KgSAT-2. The different sequences in KgMSV when compared to KgSAT-2 are highlighted.

KzSOMon were produced according to Example 2. KzSOMon was active, producing two cleavage products as expected.

In another experiment, citrus peel virus (CEV), which has a target site at nucleotide 336 in its complementary RNA, was cleaved using a ribosome (CevCEV) that has the sequence shown in Figure 14a. The loop region labeled b in Figure 14 contains 6 nucleotides that have the sequence 3'-CSTATA-5'. This is different from the CevCEV region, which contains four nucleotides that have the sequence 3'-AOA-5'. This ribosome cleaves the CEV target complementary RNA at position -336, as shown in the electrophoretic profile of Figure 14a.

This experiment shows that the number of nucleotides and nucleotide sequences of the loop region are important for ribosomal activity. In these experiments, the ribosome was prepared according to the methods described above in the specification.

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

A modified ribosome corresponding to KgCAT-2, but containing four additional base pairs, has been constructed and studied. In Figure 15a, the sequence of the KgCAT-2 ribosome is shown hybridized to the target CAT RNA. The ribosome is shown as 156, with the additional base pair. The ribosome is tested to show comparable activity to that of KgCAT-2. This means that the base pair region of the catalytic region can be of different lengths without affecting catalytic activity.

The stable in vivo forms (data not shown) of the 3TovRU and RNA transcripts expressed in transgenic plants are predominantly circular, probably due to the joining of the 5' and 3' ends. Therefore, the use of two autolytic cleavage sites flanking a desired sequence in an in vivo RNA transcript seems to lead to circularization of the product, which may confer higher stability than linear transcripts. This approach has led to a novel method for the in vivo stabilization of ribosomal sequences. It is termed circularization.

Example 8. Activity of ribosomes in vivo.

Plasmids containing anti-CAT (CAT -chloramphenicol acetyltransferase) or a combined anti-CAT (ribosomal gene, see below) were introduced into tobacco protoplasts in the same amount and proportions relative to each other, together with another plasmid containing a functional CAT gene construct. CAT activity was measured and compared to the level of gene activity bases.

Materials and methods, a/ Electroporation and CAT assays. They were performed as described in /14/. Specifically, protoplasts of L. cochlear implant line T5 were prepared from a two-day suspension culture, suspended in 10 mM HEPE, pH 7.2, 150 mM NaCl, 0.2 M mannitol and supplemented to a density of 3.10 ^b /ml. Electroporation was performed by applying a direct current for 50 m at 250 V. The protoplasts were diluted tenfold and cultured for 20 h at 26°C in the dark. They were disrupted by sonication and an extract was obtained from them. The extracts were normalized for protein content and assayed for CAT activity in vitro using ¹⁴ C-chloramphenicol and acetyl CoA. The reaction products were separated by thin layer chromatography and visualized by autoradiography. The extent of the reaction was assessed by the production of radioactive product obtained from the Chloramphenicol matrix.

b/ Genius constructions.

Gene constructs were introduced into 0.1 ml of protoplast suspension as plasmid DNA, which was purified from bacteria by extraction and centrifugation twice with ClCl in an equilibrium density gradient. They were resuspended in 10 mM Tris/1 mM EDTA/pH = 7.5 for use.

The active CAT gene construct was derived from a plasmid designated pCAT7+. It was isolated by fusing the CAT gene sequence from plasmid pCM4 /15/ into plasmid ρ1353Ν, derived from ρ355Ν /16/, so that this gene construct is:

358 SAT N05

355 refers to the 355CaMU (cauliflower mosaic virus) promoter, N08 to the bespalin synthetase polyadenylation signal, T/C to transcription.

Various gene constructs were added to pCAT7+ with 0.2 µg as described below. The gene constructs are contained within plasmids with the following designation.

P.Ι358Ν - This is a vector plasmid, the map of which is shown in Figure 16, containing the 358 CaMU promoter and a plant bespalin synthetase 3 polyadenylation signal, which can be represented as follows:

358 N08

pCAT7 - This plasmid contains the CAT gene sequence inserted into ρ1358Ν, such that transcription results in the production of nonsense CAT RNAs, which can be represented as follows:

358 SAT - N05

pCAT7 - This plasmid contains the CAT gene sequence inserted into ρ1355Ν, such that transcription results in the production of nonsense CAT RNAs, which can be represented as follows:

355 SAT N05

pCAT19 - It contains the CAT gene with four catalytic ribosomal regions included therein (see Example 4 and Figure 9), inserted into pΙ358Ν, such that transcription results in the production of nonsense CAT RNAs containing four catalytic ribosomal regions, represented as follows:

5’ 358 SAT N08 a*-·····

electroporation. Activity is expressed as the percentage of converted chloramphenicol substrate in a one-hour sample.

Results. The following table shows the relative CAT activities in cells 20 hours after 5

Table 2

Electroporated plasmid

Treatment pCAT7+ p1358Ν

1A

1B

2A 0.2 18

2B 0.2 18

FOR 0.2 9

ZV 0.2 9

4A 0.2

4V 0.2

5A 0.2 9

5V 0.2

6A 0.2

6B 0.2 (for each treatment A and B are duplicates)

The following conclusions can be drawn from these results.

The introduction of the CAT gene construct resulted in remarkable CAT activity - comparing 2A,B with 1A,B. There is variability between the duplicates. From the trend seen in the other samples (see the following conclusions), it is like those of 2A and shows above-normal activity.

The concomitant introduction of a nonsense gene construct results in an increase in the level of activity - compare 3A,B and 4A,B with 2B. The magnitude of the increase is directly related to the level of the nonsense gene construct added as a plasmid - compare 3A,B and 4A,B.

Concomitant introduction of combined nonsense/ribosomal gene constructs results in an increase in gene activity compare 5A,B and 6A,B with 2B. Moreover, the increase is more pronounced than the corresponding level of the nonsense gene construct - compare 5A,B with 3A,B and 6A,B with 4A,B.

The average results for four in vivo experiments are shown in Figure 17. In these experiments, pCAT7-pCAT19 % conversion

28

32

26

19

19

22

14

16 turns of control means impact 2, meaningless impact 4, catalytic impact 6 and impact level 1.

The catalytic ribosome inhibits CAT activity by an average of 47%, compared to an average of 34% for a nonsense ribosome.

Introducing a ribosome-derived gene into a plant cell inhibits the activity of the genes it targets. What's more, the inhibition is higher than that for the corresponding nonsense RNA molecule.

These results indicate that ribosomes will be active in plant, animal, or microbial cells against a specific range of target RNA molecules.

The mechanisms of action of ribosomes in this example are not clear. For example, a nonsense ribosome may irreversibly hybridize to a target RNA and catalyze the cleavage of a phosphodiester bond at one or more selected target sites along the target RNA. Conversely, cellular enzymes may cleave nonsense RNA from their target sequence, such that the target sequence is cleaved into two or more fragments.

Example 9. In vivo activity of ribosomes in animal cells.

This example demonstrates the activity of ribosomes in inactivating target RNA in animal (mammalian) cells.

Materials and Methods. Active gene constructs encoding ribosomes were transferred into wild-type competent cells of the monkey cell line CO31 by electroporation. In this method, 3.10 ⁶ /ml CO31 cells suspended in a buffer solution with 10% PC8 (fetal calf serum) were contacted with the various gene constructs and an electrical charge was applied to induce electroporation of DNA into the cells. The transfected cells were incubated at 37°C for 48 hours in culture medium before assaying for CAT and luciferase activity.

The CAT gene construct was derived from a plasmid designated pTK CAT /18/. This plasmid was obtained by inserting the CAT gene sequence into plasmid pZU2 so that it was under the control of the herpes simplex virus thymidine kinase promoter.

The gene constructs encoding ribosomes were prepared based on a plasmid p8Y232A (19) containing the luciferase gene fused to the 8Y40 early promoter. The DNA encoding ribosomes was ligated into a Hoa1 site 3' from the luciferase gene according to standard methods and MataH3 el a1 (7).

The following constructions were made using standard MashaNa e!.a1./8irga/ techniques:

pPC58 - this plasmid vector contains DNA encoding the ribosome KrCAT-1, fused 3'-terminally from the luciferase gene in a non-functional orientation. This can be represented as follows:

3321 YT40 eifu ' |(shPegae 1| r,sat -h) 4th I Ps^ 23YA

with 232A referring to the p8Y232A sequence. 8Y40 early refers to the early promoter of 8Y40 and small T is the DNA encoding the small T sequence derived from 8Y40. This construct results in the production of an RNA molecule encoding luciferase and the ribosome KrCAT-1, later oriented as expected to be catalytic.

pPC1-6 - This plasmid is the same as pPC58 except that KxCAT-1 is replaced with KgCAT-3 in the sense orientation (5'-3').

pPC20 - This plasmid is the same as pPC16 except that KgCAT-3 is replaced with KgCAT-2, which has eleven nucleotide flanking sequences.

pPC12 - This plasmid is the same as pPC20 except that the ribosome KζΟAT-2 contains twelve nucleotide flanking sequences.

pPC50 - This plasmid contains the CAT gene with four catalytic ribosomal regions incorporated into it (see example 4 and example 9) in the sense orientation (5'-3'), which upon transcription results in an inactive ribosome.

This plasmid can be represented as follows:

ZU40 late CAT gene containing ribosomal region Poly A ⁺

non-catalytic RNA pPC54 - This plasmid is the same as pPC50, except that the CAT gene and ribosomal regions are in a nonsense (3’-5’) active orientation.

pPC64 - This plasmid possesses the ZU40 promoter and polyadenylation signals with pPC50 and contains the wild-type CAT gene with uninserted ribosomal regions. This gene is in a nonsense orientation and thus does not produce CAT protein.

pPC65 - This plasmid is the same as pPC64 except that the wild-type CAT gene is in the sense (5'-3') orientation and is thus productive of the CAT protein.

Research.

Luciferase activity was assayed according to the methods of Oe \Ue1 e1 a1. (Spirga). Specifically, CO3 cells were lysed 48 hours after transfection and the cell lysates were incubated with luciferase, a substrate for luciferase, and counted in a scintillation counter. CAT activity was also measured using CO cell lysates (cell lysates were divided into two and each portion was assayed for luciferase or CAT activity) according to the method of Spirga, ML. /20/.

In vivo assays showed that pPC58 and pPC4 did not affect CAT activity in transfected cells. This activity was referred to as 100% CAT activity and 0% CAT suppression. CAT activity in cells transfected with other plasmids was measured relative to pPC58. The percentage of CAT suppression was measured as follows:

100 SAT \ ^test x 100

CAT / control ' normalized to luciferase activity. CATtest = CAT test results for the test constructs. CATcontrol* ⁼ CAT test results for the control constructs (pPC4 and pPC58).

Luciferase production is an internal control for electroporation and allows for measurement of ribosomal production within each individual tissue culture subjected to electroporation.

Results

Experiment I.

μ% plasmid electroporated /1.5.10 ⁶ cells

Treatment TXAT rPC58 rPC20 pRS1-6 rPC12 %CAT suppression 1 5 2 56 2 5 1 1 53 3 5 2 40 4 5 2 0

Experiment II.

μ 8 plasmid electroporation/1.5.10 ^b cells

Treatment RTXAT rPC-1-6 rPC20 rPC12 rPC4 %CAT suppression 5 5 2 75 6 5 2 75 7 5 4 62 8 5 1 1 70 9 5 4 51 10 5 2 0

Treatments 5 to 10 were performed in duplicate and the average values are given.

Experiment III.

μ β plasmid electroporation /1.5.10 ⁶ cells

Treatment RTXAT pRS1-6 rPC4 %SAT supersion 11 5 2 66 12 5 2 0

The treatments were performed five times and the average values are given.

Experiment IV.

μ £ plasmid electroporation Treatment RTXAT rPC50 rPC54 rPC64 rPC65 %SAT sup- resia 13 5 2 0 14 5 2 26 15 5 2 2 16 0 2 YES

Each of the treatments from 13 to 16 was performed four times.

Many experiments have been performed with the TK promoter from pTKSAT replaced with the human metallothionein promoter. When this SAT construct was co-transfected into CO1 cells with plasmids encoding one or more ribosomes, an increase in SAT activity was observed.

The above results clearly demonstrate the in vivo ribosome inactivating activity in animal cells.

Despite the lack of in vivo efficiency of ribosomes, which is believed to be caused by one or more catalytic regions that are capable of cleaving target RNA, the presence of such regions in the "nonsense" RNA type ribosome may not always lead to cleavage in vivo if the entire RNA/nonsense RNA molecule is not involved. Regardless of whether the molecule is involved or not, the above examples demonstrate the lack of inactivation effect on the target RNA. The invention is applicable to all ribosomes that have catalytic regions capable of causing cleavage and a hybridization region, regardless of whether cleavage actually occurs in the target RNA. That is, the hybridization region may be so large as to ensure the coexistence of the RNA (ribosome) combination and to prevent the cleavage of the target RNA into different components, even if the catalytic region is capable of causing cleavage.

Claims (20)

Patent claims 1. A method for producing ribosomes by incorporating a hybridization region complementary in nucleotide sequence to at least part of a target RNA and a catalytic region adapted to degrade said target RNA, characterized in that one or more branches formed by single-stranded RNA and having a sequence complementary to at least part of the target RNA are incorporated, the branches are associated with a catalytic region capable of degrading the target RNA, after which the entire structure is subjected to stabilization. 2. The method of claim 1, wherein the hybridization region of one or more branches is adjacent to the catalytic region. 3. A method according to claims 1 and 2, characterized in that the hybridization region is adjacent to a selected degradation site in the target RNA. 4. The method of claim 1, wherein the catalytic regions are connected by flanking sequences, each of which comprises a sequence complementary to the target RNA. 5. The method according to claim 1, characterized in that the ribosomes are obtained by transcription of an exogenous nucleic acid sequence including a DNA or RNA transfer vector inside the cell, in which the inactivation also takes place. 6. The method of claim 5, wherein the exogenous nucleic acid sequence is provided by transfection of a DNA or RNA transfer vector into the cell or by a precursor vector. 7. The method according to claim 6, characterized in that the transfection is followed by genomic integration of the same exogenous nucleic acid sequence. 8. A method according to claims 1 to 7, characterized in that the cell is eukaryotic. 9. The method according to claim 8, characterized in that the eukaryotic cell is plant or animal. 10. A method according to claims 1 to 7, characterized in that the cell is prokaryotic. 11. The method according to claims 1 to 10, characterized in that the target RNA is endogenous to the cells. 12. The method of claim 11, wherein the target RNA encodes a protein characteristic of a genetic disease. 13. The method according to claims 1 to 10, characterized in that the target RNA is exogenous to the cells. 14. The method of claim 13, wherein the target RNA is viral. 15. The method according to claims 1 to 14, characterized in that the nucleic acid sequences of appropriate length and complementarity represent an RNA sequence that acts as nonsense with respect to the target RNA. 16. A method according to any one of the preceding claims, characterized in that the branches of the single-stranded RNA of appropriate length and complementarity that hybridize to the target RNA at a selected cleavage site comprise 9 or more nucleotides. 17. The method of claim 16, wherein the nucleic acid sequence comprises 16 or more nucleotides. 18. The method of claim 1, wherein the cleavage site in the target RNA is located immediately after the sequence X°υΥ, where X° is any ribonucleotide, and is uracil and Y is adenine, cytosine and uracil. 19. A method according to claim 18, wherein X° is C, Y is C or A. 20. A method according to any one of the preceding claims, wherein the ribosomes comprise catalytic regions connected by flanking sequences, and any flanking sequence complementary to the target RNA is capable of being degraded. Appendix: 17 figures