WO1999036517A2

WO1999036517A2 - Method for selecting ribozymes which are capable of covalently modifying the ribonucleic acids on 2'-oh-groups in trans

Info

Publication number: WO1999036517A2
Application number: PCT/EP1999/000181
Authority: WO
Inventors: Andreas Jenne; Michael Famulok
Original assignee: Andreas Jenne
Priority date: 1998-01-14
Filing date: 1999-01-14
Publication date: 1999-07-22
Also published as: DE19801153A1; EP1060246A2; WO1999036517A3

Abstract

The invention relates to an in vitro selection method for selecting ribozymes which are capable of covalently modifying the ribonucleic acids on 2'-OH-groups in trans and to the ribozymes obtained using this method. The invention also relates to medicaments containing said ribozymes, said medicaments being preferably used for inhibiting gene expression - for example in gene therapy. The inventive ribozymes can also be used for producing muteins and nuclease-resistant ribonucleic acids, for example ribonuclease-resistant 'antisense' oligonucleotides.

Description

Process for the selection of ribozymes which can covalently modify ribonucleic acids in trans at 2 '-OH groups

The present invention relates to an in vitro selection method by means of which ribozymes can be selected which can transovalently modify 2'-OH groups of ribonucleic acids. The present invention also relates to ribozymes obtainable by this method. In addition, the present invention relates to medicaments containing ribozymes which can preferably be used to inhibit gene expression, for example in gene therapy. The ribozymes according to the invention can also be used to produce muteins and nuclease-resistant ribonucleic acids - for example ribonuclease-resistant "antisense" oligonucleotides.

Ribozymes are RNA molecules that are able to catalyze chemical reactions. The most prominent representative among the ribozymes is mechanistically and structurally very well-characterized hammerhead ribozyme that catalyzes the site-specific hydrolysis of phosphodiester bonds in RNA. This property opens up the possibility of using the hammerhead ribozyme for gene therapy, for example by inhibiting the expression of a particular gene by sequence-specific cleavage of an mRNA (Birikh et al., Eur. J. Biochem. 245 (1997), 1-6). So far, this has been achieved both in cell culture (Jones and Sullenger, Nature Biochem. Vol 15 (1997), 902-905) and in plants (Yang et al., Proc. Natl. Acad. Sei. USA 94 (1997) 4861-4865 ) and animal experiments (Lieber and Kay, J. Virol. 70 (1996), 3153-3158), the ribozyme either being supplied to the cells from outside (exogenous application) or being synthesized in the cells by transcription of a vector (endogenous application) . In general, such and similar ribozymes thus have great potential as inhibitors of gene expression in transgenic animals and plants, as tools for science and diagnosis, and as human therapeutic agents (Burke, Nature Biotech. Vol. 15 (1997), 414-415; Marschall et al., Cell. Mol. Neurobiol. 14 (1994), 523-538). Ribozymes have in common with "antisense" oligonucleotides the principle of acting at the level of the gene, in contrast to most drugs that target protein functions. However, due to their catalytic properties, the ribozymes are given a greater role for the mentioned fields of application than the "antisense" oligonucleotides (Woolf, Antisense Res. Dev. 5 (1995), 227-232).

The repertoire of naturally occurring ribozymes is limited to the cleavage and ligation of specific phosphodiester bonds in ribonucleic acids. More recently, however, not only functionality has been achieved by techniques of "in vitro selection" (Tuerk and Gold, Science 249 (1990), 505-510; Ellington and Szostak, Nature 346 (1990), 818-822) to improve natural ribozymes, but also to produce a series of ribozymes with new properties (Pan, Curr. Opin. Chem. Biol. Vol. 1 No. 1 (1997), 17-25). By using the in vitro selection, for example, ribozymes with RNA ligase activity

(Bartel and Szostak, Science 261 (1993), 1411-1418), with polynucleotide kinase activity (Lorsch and Szostak, Nature 371

(1994), 31-36) and peptidyl transferase activity (Zhang and Cech, Nature 390 (1997), 96-100). So far, however, it has not been possible to generate synthetic ribozymes that sequence-specifically modify RNA molecules on internal 2'-hydroxyl groups. Ribozymes with such properties could, similar to hammerhead ribozymes, be used in therapeutic procedures and also lead to interesting applications in modern biotechnology.

Due to the relatively limited catalytic spectrum of the naturally occurring ribozymes, their applicability for influencing gene expression, for example in therapeutic processes, is limited. The effect of ribozymes currently being tested to inhibit gene expression is limited to the cleavage of target RNAs. The present invention is therefore based on the technical problem of providing ribozymes which are capable of influencing gene expression in another way.

This technical problem is solved by providing the embodiments characterized in the patent claims. It has surprisingly been found that the ribozymes obtainable by the selection method described in more detail below can sequence-specifically modify foreign RNA molecules on internal 2'-OH groups. RNAs modified in this way are no longer translatable. These ribozymes can therefore be used for a novel inhibition of gene expression, for example in gene therapy. The present invention thus relates to a method for the selection of a ribozyme which can covalently modify 2'-OH groups of ribonucleic acids in trans, the method being characterized by the following steps:

(a) incubation of a ribonucleic acid library with a reactant which can react with a 2'-OH group in ribonucleic acids,

(b) selection and isolation of ribonucleic acids which have covalently bonded with the reactant,

(c) Rewriting in cDNA and amplification in step

(b) ribonucleic acids obtained,

(d) iteratively running through steps (a) to (c),

(e) sequence determination of the selected ribonucleic acids,

(f) determination of the modified 2'-OH group within the selected ribonucleic acids,

(g) cleavage of the selected ribonucleic acid into a substrate part (I) and catalytic part (II = ribozyme), and

(h) Check whether the catalytically active part (II) can trans covalently modify the substrate part.

The sub-steps required for this selection process are known to the person skilled in the art from the relevant literature, for example described above. The procedure is described in detail in the example below.

In the present invention, the term “covalently modify” is understood to mean any desired covalent modification on a 2 ′ OH group of an RNA. These are preferably all types of addition and substitution reactions which lead to bond formation with the involvement of the 2 'hydroxyl group. Covalent modification via acylation is particularly preferred in the present invention.

The term "reactant" is understood to mean any molecule that can be transferred either as a whole or as a part to the 2'-OH group of RNAs. In principle, this includes all compounds that covalently bond with oxyanions or hydroxyl groups. lent react. Such compounds are, for example, carboxylic acid derivatives (esters, acid halides, etc.), strained ring systems (epoxies, etc.), compounds with multiple bonds (arenes, etc.) and compounds of the RX type, with R = alkyl or aryl and X = leaving group (halogen, tosyl Etc.) . A preferred reactant is amino acid AMP ester, which is preferably phenylalanyl-2 '- (3') -AMP (Phe-AMP), particularly preferably biotinyl-N-Phe-AMP (Bio-Phe-AMP) .

The term "iterative traversal" means the following: In order to enrich a population of catalytically active sequences, the steps of "selection" and "amplification" have to be carried out alternately several times in succession. Since it is not possible to quantitatively separate all non-specific (i.e., non-functional) RNAs from the functional sequences in a single selection step, several selection cycles must be carried out. As many cycles are preferably carried out until an accumulation of the desired activity can no longer be detected (generally after 6-20 cycles).

The modified 2 'OH group in the target RNA can in principle be determined by the following methods: primer extension (Ruskin et al., Cell 38 (1984), 317), MALDI-TOF sequencing with exonucleases (Smirnov et al., Analyt. Biochem. 238 (1996), 19-25), or as described in Example 1 by RNAse sequencing.

In principle, the cleavage of the selected ribonucleic acid into the substrate part and the catalytically active part can be carried out as follows: If the 2 'modification is known, the originally selected in cis-ribozyme is "cut" about 5 to 10 bases upstream or downstream of this position. The two RNA fragments (ribozyme and substrate part) are either by in vitro transcription of suitable DNA templates or by automated oligonucleotide Solid phase synthesis generated. The trans-ribozyme is then tested for its ability to convert the oligonucleotide substrate.

The present invention further relates to ribozymes which are obtainable by the process described above and described in the example. In a preferred embodiment according to the invention, the ribozyme is characterized in that it has the secondary structure and nucleic acid sequence from position 28 to 136 shown in FIG. 6a, or a sequence and / or secondary structure deviating therefrom, these deviations not resulting in the loss of the original catalytic activity to lead. These deviations relate to the addition, deletion and / or insertion of bases, the original sequence being retained at least 90%, preferably at least 95% and more preferably at least 98%. The secondary structure is preferably retained with these introduced changes. These deviations primarily concern the sequence from positions 28 to 40 and / or 68 to 84 in FIG. 6a, which must be selected according to the sequence of the selected target RNA (substrate), i.e. it must be sufficiently complementary to the target RNA to be able to bind and modify it. The “loops” shown in FIG. 6a should preferably be retained.

If the target RNA is known, this should preferably be taken into account when designing the selection, as explained below. In the amplification step of the in vitro selection, active sequences are reverse transcribed. As mentioned below, 2 'modifications typically result in the reverse transcriptase enzyme being unable to produce DNA transcripts due to a stop at the modified position. Thus, ribozymes are preferably selected which are 2 'modified within the 3' primer binding sites, since the 3 'primer is fed externally to the reverse transcription reaction as the initiator sequence for elongation. For the Introduction of the modification within a specific target sequence would therefore be chosen as the primer sequence.

The person skilled in the art can insert variations into the sequence / structure of the ribozyme using generally known techniques (for example by using renewed selection rounds of the selection method according to the invention, in vitro mutagenesis, etc.), which can also lead to a ribozyme whose catalytic activity is increased or that has a different substrate specificity. For example, if the ribozyme shown in FIG. 6a is to bind and modify an RNA which does not have the sequence of the substrate from positions 137 to 164, the sequence of the ribozyme required for hybridization must be between positions 28 to 40 and / or 68 to 84 as described above be changed so that it can bind and modify a substrate with the desired sequence. For example, in order to change the specificity of the ribozyme in such a way that a different reactant than the one used in the selection is also transferred to the target RNA, the selection process can be used again, whereby the partially randomized ribozyme sequence is advantageously used as the basis for the new selection becomes. According to standard methods, the person skilled in the art can also test whether a modified ribozyme still has the desired properties, for example by means of the methods explained in the example.

The present invention also relates to a DNA sequence which encodes the ribozyme according to the invention. The nucleic acid molecules according to the invention can also be inserted into a vector. Thus, the present invention also includes vectors containing these DNA encoding ribozymes. The term "vector" refers to a plasmid (eg pUC18, pBR322, pBlueScript), a virus genome or another suitable vehicle. In a preferred embodiment, the nucleic acid molecule according to the invention is functionally linked in the vector to regulatory elements which Allow transcription in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, for example a promoter, such vectors typically contain an origin of replication and specific genes which allow the phenotypic selection of a transformed host cell. The regulatory elements for expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GAL1 promoter in yeast, and the CMV, SV40 -, RSV-40 promoter, MMTV-LTR promoter, MLV-LTR promoter (adenovirus (VA1), herpes simplex (HSV); "immediate-early" 4/5 promoter, CMV or SV40 enhancer for expression in animal cells. Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable vectors include, for example, expression vectors based on T7 for expression in bacteria (Rosenberg et al., Gene 56 (1987), 125) , pMSXND for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263 (1988), 3521), and vectors derived from Baculovirus for expression in insect cells.

In a preferred embodiment, the vector containing the nucleic acid molecules according to the invention is a virus, for example an adenovirus, vaccinia virus or "adeno-associated virus" (AAV), which are useful in gene therapy. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV.

General methods known in the art can be used to construct expression vectors containing the ribozymes of the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as described, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).

DNA encoding promoter ribozyme can be introduced into the cell directly or with the aid of a virus. In the direct method, the DNA is bound, for example, to a Fab fragment via a poly-L-lysine and is absorbed by the cells carrying the corresponding antigen (Ferkol et al., J. Clin. Invest. 95 (1995), 493-502) . When a virus is used, the DNA packaged in it is introduced into the cell by means of the virus. If the promoter-ribozyme unit is flanked on the 5 'and 3' sides by viral "inverted terminal repeats", this unit can integrate into the genome (Goodman et al., Blood 84 (1994), 1492-1500). If this is not the case, the DNA is episomal (Flotte et al., Am. J. Respir. Cell. Mol. Biol. 11 (1994), 517-521). For example, the virus is adenovirus (Brody and Crystal, Ann. NY Acad. Sci. 716 (1994), 90-101), "adeno-associated-virus" (AAV) in combination with cationic liposomes (Philip et al., Mol. Cell. Biol. 14 (1994), 2411-2418), adenovirus in combination with retroviruses (Adams et al., J. Virol. 69

(1995), 1887-1894), Sendai viruses (von der Leyen et al., Proc. Natl. Acad. Sci. USA 92 (1995), 1137-1141), retroviruses (Rettinger et al., Proc. Natl. Acad. Sci. USA 91

(1994), 1460-1464) or vaccinia viruses (Lee et al., Cancer Res. 54 (1994), 3325-3328).

The present invention also relates to the host cells containing the ribozymes described above. These host cells include bacteria, yeast, insect, plant and animal cells, preferably mammalian cells. Preferred mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293 and WI38 cells. Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the nucleic acid molecules according to the invention using the vectors described above are known in the art. The present invention also comprises a method for producing the ribozyme according to the invention, which can be enzymatic or chemical methods. For example, the DNA sequence encoding the ribozyme can be inserted into a vector which is replicable in a prokaryotic host under the control of a suitable promoter, for example an SP6, T3 or T7 promoter, after the amplified plasmid has been obtained allows the host to in vitro transcribe the DNA sequence encoding the ribozyme and to obtain ribozyme RNA. Alternatively, the ribozyme can be synthesized in large quantities by a chemical method, for example, a method based on the phosphoramidite reaction (Sproat et al., Nucleosides & Nucleotides 14 (1995), 255-273).

In a further preferred embodiment, the present invention relates to a ribozyme which is modified in such a way that resistance to nucleases is obtained. This increases the residence time and thus the effectiveness of the ribozyme at the target site, for example in certain cells of a patient. In addition, the amount of the ribozyme to be administered and any side effects associated therewith can be reduced.

Examples of such modifications are the substitution of the 2 '-OH groups of the ribose by 2' -H-, 2 '-O-methyl-, 2 ¹ - O-allyl-, 2' -fluoro- or 2 '-amino Groups (Paolella, et al., EMBO J. 11 (1992), 1913-1919, and Pieken et al., Science 253

(1991), 314-317) or the modification of phosphodiester bonds, for example one or two oxygen atoms being replaced by sulfur (phosphorothioate or phosphorodithioate; Eckstein, Ann. Rev. Biochem. 54

(1985), 367-402, and Beaton et al. , in: Eckstein, F. (ed.) Oligonucleotides and analogues - A practical approach - Oxford, JRL Press (1991), 109-135) or against a methyl group (methylphosphonate; Miller, ibid., 137-154). Further Modifications include conjugation of the RNA with poly-L-lysine, polyalkyl derivatives, cholesterol or PEG. The ribozymes according to the invention preferably contain at least one of the phosphate modifications described above and / or at least one of the ribosemodifications described above.

The transcription of the DNA sequences encoding the ribozyme according to the invention leads to the synthesis of ribozymes which can inhibit the translation of the desired target RNA. Both the DNA sequences coding for the ribozyme and the ribozymes according to the invention are therefore themselves suitable as medicaments, preferably for inhibiting gene expression in vitro or in vivo.

The ribozymes according to the invention not only represent an alternative to the hammerhead ribozymes mentioned, but also show advantages in certain applications:

- The inhibition of gene expression in transgenic (i.e. expressing the ribozyme) organisms can be controlled by administration of the reactant. For example, a transgenic organism can produce a ribozyme that is capable of modifying a particular mRNA on a particular internal OH group with an external reactant. Only when the reactant is administered, for example at a certain development stage, does the ribozyme develop its catalytic activity and the expression of the target gene is prevented ("inducible knock-out").

- 2 '-modifying ribozymes could be used as sequence-specific gene probes if an easily detectable marker molecule is transferred to the target RNA (eg fluorescein or, as in example 1, biotin, molecules labeled with bio-tin detected via phosphatase-conjugated avidin can be). - 2 '-modifying ribozymes are particularly suitable for combating retroviruses, since the modification introduced hinders or even makes it impossible to rewrite the viral RNA genome into DNA (Lorsch et al., Nucl. Acids Res. 23 (1995), 2811- 2814). In this case too, the action of the ribozyme can be controlled by administering the reactant.

The present invention thus also relates to medicaments which contain the DNA encoding the ribozyme according to the invention or a vector comprising the DNA encoding the ribozyme according to the invention, optionally in combination with a pharmaceutically acceptable carrier.

In a further embodiment, the present invention relates to medicaments which contain the ribozyme according to the invention.

Depending on whether the ribozyme itself or the DNA sequence encoding the ribozyme is applied, possibly in a recombinant vector, the administration can take place in different ways. In the first case, administration takes place, for example, after coupling the 3 'ends of the ribozymes to poly- (L-lysine) using standard methods, as described, for example, by Leonetti et al. , in: Cohn and Moldave (ed.), Progress in Nucleic Acid Research and Molecular Biology 44 (1993), 143-146 (New York, Academic Press), by microinjection according to methods known to the person skilled in the art (see, for example, Leonetti et al ., PNAS USA 88 (1991), 2702-2706), after encapsulation in liposomes, for example by the method described by Farhood (Farhood et al., Ann. NY Acad. Se. 716 (1994) 23-34), after packaging in phages (Picket and Peabody, Nucleic Acids Res. 21 (1993), 4621-4626), or by peptide-mediated introduction into the cells, as described, for example, by Derossi et al. , J. Biol. Chem. 269 (1994), 10444-10450. Furthermore, by coupling the promoter-ribozyme DNAs to specific antibodies or other suitable ligands, a targeted one The ribozyme or the promoter-ribozyme-encoding DNAs can be introduced into desired organs, tissues or cells (Leonetti et al., PNAS USA 87 (1990), 2448-2451). The administration takes place, for example, systemically or locally, intravenously, intramuscularly, intraperitoneally, via catheter or by inhalation of aerosols.

In the second case, administration takes place, for example, via a transfection, for example via standard processes known to the person skilled in the art, such as calcium precipitation, electroporation, the DEAE dextran process, via cationic liposomes, for example lipofectin, polyamines, and the transferin polylysine Method or linkage of the DNA or the recombinant vector to a specific antibody or other ligand. Administration can be as described above.

The formulation of the active ingredient can optionally be carried out in combination with pharmaceutically acceptable carriers, for example a diluent, excipient, wetting agent, surfactant, binder, etc., depending on the type of administration.

The active ingredient is administered in an appropriate dose depending on the patient himself, the type and severity of the disease, etc. The required dose amount can be determined routinely by a person skilled in the art, also taking into account whether the administration takes place as a single dose or, over a certain period of time, through multiple doses.

Compounds present in the cell, preferably amino acid adenylates, acetylcholine, acetyl coenzyme A, cyclic AMP and nucleoside triphosphates and other cell molecules which can react with 2'-OH groups, serve as potential reactants. On the other hand, ribozymes can be selected which have a reactant which is not present in the cell need. These ribozymes offer the advantage that the reaction can be controlled from the outside - by adding the reactant. It is preferred to use suitable reactants already in the selection process, which are known to be well tolerated and easy to apply and, if appropriate, are readily absorbed by cells, ie are permeable to cell membranes.

The ribozymes according to the invention can also be used in gene therapy if the gene expression is negatively influenced due to incorrect splicing of the RNA. The 2'-OH modifications introduced by the ribozymes according to the invention suppress, for example, reactions at (wrong) positions which require a free hydroxyl function.

Another embodiment relates to the ribozymes according to the invention for the production of muteins. Since the ribozymes also catalyze the transfer of a biotinylated amino acid from the described modified substrate (28mer-Phe-Bio) to AMP (reverse reaction), this activity can be used to identify tRNAs at their 3 'ends with non-cognate amino acids, or other molecules, according to the following reaction:

Ribozyme amino acid AMP + tRNA> aminoacylated tRNA

With the help of "incorrectly loaded" tRNAs produced in this way, protein mutants can thus be produced in a targeted manner by in vitro translation according to standard methods (Mendel et al., Ann. Rev. Biophys. Biomol. Stmc. 24 (1995), 435-462).

Such ribozymes are particularly interesting for new applications in combinatorial chemistry (Roberts and Stostak, PNAS USA 94 (1997), 12297-12302) because they make substrates can be introduced site-specifically into proteins that are not determined by the genetic code.

Finally, the present invention relates to the use of the ribozymes according to the invention for the production of nuclease-resistant ribonucleic acids, since it has been possible to show that RNAs modified in this way are resistant to nucleases. These could therefore also use in gene therapy, for example as "antisense" -Oligonucleo- ^"tide, find when a long half-life of the RNAs used is desirable.

The ribozymes according to the invention or the vectors coding for them can also be used for the production of transgenic plants, for example in order to enrich desired metabolic products by inhibiting the expression of certain genes or to generate virus resistance. The present invention thus also relates to transgenic plants.

In order to ensure the expression of the ribozyme DNA sequences in plant cells, these can in principle be placed under the control of any promoter which is functional in plant cells. The expression of the said DNA sequences can generally take place in any tissue of a plant regenerated from a transformed plant cell according to the invention and at any time, but preferably takes place in those tissues in which an altered gene expression is advantageous either for the growth of the plant or for is the formation of ingredients within the plant. Promoters which ensure specific expression in a specific tissue, at a specific time of development of the plant or in a specific organ of the plant therefore appear to be particularly suitable. Suitable promoters are known to the person skilled in the art. The DNA sequences which encode the ribozymes described above are preferably linked, in addition to a promoter, to DNA sequences which ensure a further increase in transcription, for example so-called enhancer elements. Such regions can be obtained from viral genes or suitable plant genes or can be produced synthetically. They can be homologous or heterologous to the promoter used. Advantageously, the ribozyme DNA sequences are also linked to 3 'DNA sequences, which ensure the termination of the transcription. Such sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens.

The ribozyme DNA sequences which are introduced and expressed in plant cells according to the invention are preferably stably integrated into the genome in the plant cells according to the invention.

The transgenic plant cells according to the invention can in principle be cells of any plant species. Of interest are both cells of monocotyledonous and dicotyledonous plant species, in particular cells that store starch, oil, or agricultural crops, e.g. Rye, oats, barley, wheat, potato, corn, rice, rapeseed, pea, sugar beet, soybean, tobacco, cotton, sunflower, oil palm, wine, tomato etc.

The transfer of the DNA molecules containing the ribozyme DNA sequences takes place according to methods known to the person skilled in the art, preferably using plasmids, in particular those plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, for example binary plasmids or Ti plasmids from the Agrobacterium tumefaciens system. In addition to the Agrobacterium system, other systems for introducing DNA molecules into plant cells are also possible, such as the so-called biolistic method or the transformation of protoplasts (see Willmitzer L. (1993), Transgenic Plants, Biotechnology 2; 627-659 for an overview). Methods for transforming monocotyledonous and dicotyledonous plants are described in the literature and are known to the person skilled in the art.

The figures show:

Fig. 1: Different nucleophilic positions within the RNA library can react with the aminoacyl ester function of 1 (biotin-Phe-AMP): The attack of the amino function (1) leads to peptide bond formation. Reaction of internal (2) or terminal (3) hydroxyl groups with 1 results in RNA aminoacyl esters.

Fig. 2: Enrichment of aminoacylated RNA in the course of the selection. The ratio of RNA to substrate 1 and the incubation times of the respective cycle are given at the bottom of the graphic. A 5 'amino-functionalized RNA library (H ₂ N-Cys-Cit-SS-RNA) was used in cycles 1-7, while selection was carried out in all subsequent cycles without a coupled dipeptide. The proportion of aminoacylated RNA was less than 0.01% in the first six selection cycles. Selection cycle 10 was carried out under mutagenic conditions to allow the evolution of aminoacyltransferases of higher activity.

3: Sequences of the selected clones without constant primer binding sites. Mutations within individual sequences are underlined. Fig. 4: Proposed secondary structure of the clone 11 ribozyme. Deletion analysis showed that only the framed sequence is needed for the catalysis.

Fig. 5: The catalytic activity of the clone 11 ribozyme is dependent on the magnesium ion concentration. The reactions were carried out at room temperature with 5 μM radioactively labeled clone 11-RNA and 25 μM 1 in selection buffer, the Mg concentration being set to the stated value (x-axis). Aliquots were taken from the reaction mixture at six different times. To quantify the ribozyme activity, the aminoacylated RNA formed was coupled to streptavidin agarose.

Fig. 6: (a) Proposed secondary structures for the complex of ribozyme 28-136 and substrate oligonucleotide (position 137-3 'end). The aminoacylated position C147 is marked.

(b) RNAse sequencing of the "wild-type substrate oligonucleotide" (left) and the aminoacylated 28-mer oligonucleotide (right). The differences in the sequencing pattern show that base position C147 was modified by ribozyme 28-136. The aminoacylated substrate was generated as follows: 10 μM ribozyme 28-136 were incubated with a few picomoles of 5 ′ -labeled 28 mer oligonucleotide and 1 mM biotin-Phe-AMP 1 in selection buffer. After 120 minutes, the RNA was precipitated and coupled to streptavidin agarose. Non-biotinylated RNAs were removed by washing, while biotinylated oligonucleotides were eluted from the streptavidin matrix with 2 M 2-mercaptoethanol or biotin in excess. Sequencing with RNases was carried out according to a protocol of the Manufacturer (Pharmacia). The following RNases were used: T1 for lane G, RNase U2 for A, RNase Phy M for A / U and RNase B. cereus for U / C. OH: partial alkaline digestion of the oligonucleotide. R: Untreated control RNA. The fragments were separated electrophoretically on a 20% polyacrylamide gel and visualized by autoradiography. The result was reproduced using 3'- [ ³² P] -labeled 28-mer oligonucleotide.

Fig. 7: Representative MALDI-TOF analysis of the reaction product generated by ribozyme catalysis (28-mer Phe-biotin). 5 μm ribozyme 28-136 were incubated with 1 μM 28-mer oligonucleotide and 1 mM biotin-Phe-AMP 1 in selection buffer for 2 hours. The RNA was then precipitated, resuspended in water and injected into a dynamo mass spectrometer without further treatment. The molecular weight of the modified 28-mer substrate was determined as the average value of various measurements in the presence of the unmodified (unreacted) 28-mer oligonucleotide as an internal standard. The mass difference between unreacted and reacted 28 mer oligonucleotide was calculated to be ^{= 370} ' ^{5 Da} . The theoretical difference in mass between 28-mer and 28-mer

Biotin is ^Δ theoretical ^{= 372} ^{'5 Da} - ^{D: Le} -Abwei ^¬ monitoring of 2 Da, relative to the measured value is within the error limits of the measurement accuracy of the mass spectrometer.

Figure 8: Kinetic characterization of the intramolecular reaction catalyzed by the clone 11 ribozyme. The initial velocities v _{Q of} the reaction are plotted against different concentrations of substrate 1. The Michaelis-Menten parameters K.-. and k _cat was obtained by curve fitting the equation v _Q = (RNA] ₀ - [1] -k _cat / (K ^ + [1]) using KaleidaGraph (Abelbeck Software). Usage: exponential function of the aminoacylation reaction with 5 μM clone 11-RNA and 0.5 mM biotin-Phe-AMP 1. The rate constants k ₀ k _s were determined using the equation [P] _t = [RNA] - [RNA] • exp (-k _obs • t), where [P] _t = concentration of the aminoacylated RNA at time t; [RNA] = Kon ^¬ concentration of clone 11 RNA.

9: Analysis of the back reaction catalyzed by the ribozyme 28-136 (28-mer-Phe-biotin + AMP): 5'-

39

[P] labeled 28-mer Phe-biotin were purified by affinity chromatography on streptavidin-agarose, eluted with 2 M 2-mercaptoethanol and resuspended in selection buffer, (a) representative course of the reaction of the reverse reaction as well as negative control experiments at 2.0 mM AMP. t _Q , t _go 'and t _ς ^. show the decrease in 28-mer Phe-biotin oligonucleotide with simultaneous increase in the deacylated reaction product (unmodified 28-mer). In the absence of AMP or ribozyme ^ 5 ^

(without AMP) and t _Q (without AMP) or t _5j -_ (without Rib.) and t _Q (without Rib.)], no change in the 28-mer Phe-biotin concentration was found. (b) Reaction course of the reverse reaction as an exponential function at 2 mM AMP, 5 μM ribozyme 28-136 and 5 nM 28-mer-Phe-biotin.

10: Reaction course of the back reactions catalyzed by the ribozyme 28-136 (28-mer-Phe-biotin + ATP and 28-mer-Phe-biotin + tRNA ^fMet ): • 5 ′ - [ ³² P] labeled 28-mer- Phe-biotin were purified by affinity chromatography on streptavidin-agarose, eluted with 2 M 2-mercaptoethanol and resuspended in selection buffer. 5 nM 28-mer Phe-biotin were incubated with 10 mM ATP or 0.2 mM tRNA ^fMet and 5 μM ribozyme 28-136 in selection buffer. After the times indicated, aliquots were removed from the reaction mixture and analyzed on a 20% polyacrylamide gel. (Representative course of the reaction of the reverse reaction at 2.0 mM AMP). t _{Q 5j} -, to t _17j1 show the decrease in 28-mer Phe-biotin oligonucleotide with a simultaneous increase in the deacylated reaction product, ie unmodified 28-mer.

11: Ribozyme-catalyzed loading of tRNA-3 'ends with substrates R. (a) The ribozyme (shown in bold) catalyzes the transfer of R-CO, part of the reactant R-AMP, with elimination of adenosine-5 '- monophosphate on itself (on an internal 2'-hydroxyl function). (b) The acylated ribozyme transfers the ester R-CO to the 3 'end of a tRNA (terminal adenosine is shown). This reaction corresponds in principle to the reverse reaction - however, instead of AMP, the terminal adenosine of a tRNA occupies the binding pocket for the reactant. (c) The released ribozyme can react again with the reactant R-AMP ("multiple turnover"). For R = biotin-Phe and ribozyme = clone 11-ribozyme, it was shown that the proposed reaction is possible in principle (cf. FIG. 10). The clone 11-ribozyme acts as a general aminoacyl-tRNA synthase, since the specificity for the reactant is primarily determined by the AMP part (cf. Example 1f, inhibition by AMP).

The following example illustrates the invention. Example 1 :

In Vitro Selection of RNA Molecules Using Ester Transferase

activity

(a) Introduction

By screening combinatorial nucleic acid libraries, RNA or DNA molecules with specific binding properties and novel catalytic functionalities can be isolated. The technique used for this is often referred to in the literature as "in vitro selection". Recent results in this area have shown that ribozymes and deoxyribozymes are capable of catalyzing a variety of chemical reactions (Pan, Curr. Opin. Chem. Biol. Vol. 1 No. 1 (1997), 17-25). In the following, the in vitro selection and characterization of a novel ribozyme is described which involves the transfer of the N-biotinylated aminoacyl ester of N-biotinylated phenylalanyl-2 '(3') -adenosine-5'-ester (Biotin-Phe- AMP) catalyzed to an internal 2 'hydroxyl group within the ribozyme sequence. The catalyzed reaction is hereinafter referred to as "ester transfer" or "aminoacylation". It was shown that the amino acylation is competitively inhibited by the AMP part of the acyl donor substrate (biotin-Phe-AMP), which suggests that a substrate-specific binding pocket is formed by the ribozyme. "Ribozyme engineering" succeeded in producing a ribozyme variant (ribozyme 28-136) which catalyzes the aminoacyl transfer to an external oligonucleotide (hereinafter referred to as "in trans reaction"):

Ribozyme 28-136 oligonucleotide + biotin-Phe-AMP → oligonucleotide-Phe-Bio + AMP

Furthermore, it was shown that the ribozyme 28-136 also accelerates the deacylation of the oligonucleotide (ie the back reaction) with high efficiency. If AMP was replaced by a tRNA, the oligonucleotide was deacylated and ter aminoacylation of the tRNA 3 'end. The ribozyme described here thus extends the repertoire of RNA catalysis to ribozymes which are able to catalyze and reverse-catalyze the aminoacyl transfer from RNA 3 'ends to internal 2' positions.

(b) Preparation of the DNA and RNA libraries

The RNA library for the first selection cycle was produced by in vitro transcription of a synthetic DNA library with the following sequence: 5'-GGG AGC TCT GCT CTA GCC AC-N30-GAC GGT TAG GTC GCA C-N30-GTG AAG GAG TGT C- N30-GGC ACC TGC CAC GAG TC-3 '(N stands for an equimolar mixture of nucleotides A, C, G, T; the underlined nucleotides are a 30% randomized citrulline aptamer motif (Famulok , J. Am. Chem. Soc. 116 (1994), 1698-1706) The DNA library was generated by automated oligonucleotide solid phase synthesis The sequences of the primers used were TCT AAT ACG ACT CAC TAT AGG GAG CTC TGGC TCT AGC CAC and GAT CCC TCG TGG CAG GTG CC (promoter sequence for the T7 RNA polymerase is underlined) The PCR amplification of the DNA library and the in vitro transcription using T7 RNA polymerase were carried out according to standard molecular biological protocols (Abelson ed., Meth. In Enzymology 267: 275-436 (1996) a detailed description can be found in: A. Jenne, diploma thesis from the Department of Biochemistry at the LMU Munich, 1995. About the 5 'amino-modified RNA library used in the first 7 selection cycles (H ₂ N-Cit-Cys- SS-RNA), the in vitro transcription was carried out in the presence of an 8-fold excess of guanosine 5 'phosphorothioate (Burgin and Pace, EMBO J. 9 (1990), 4111-4118) compared to the remaining nucleotide triphosphates. The resulting 5'-thiol-modified RNA library was then treated with a 50-molar excess of thiopyridyl-activated NH ₂ -cit-Cys (thipy) -COOK in coupling buffer (5 mM EDTA, 25 mM Na-PO ₄ , pH 7 , 7) reacted for 20 minutes. The complete The reaction was determined spectrometrically by measuring the thiopyridone released at 343 nm. The RNA library was then precipitated with ethanol and then washed twice with 150 μl of 70% ethanol. In the selection cycles 7-13, selection was made with a 5 'unmodified RNA library (without an introduced thio / amino function), since the coupled dipeptide had been shown to have no effect on the enriched aminoacyltransferase activity.

(c) Synthesis of the compounds biotin-Phe-AMP and NHn-Cit-Cvs (thipy) -COOH

The synthesis of the compounds used in the selection NH ₂ -Cit-Cys (thipy) -COOH and N-biotinylated phenylalanyl-2 '(3') -adenosine-5 '-ester (Biotin-Phe-AMP) are described in: A Jenne, diploma thesis at the LMU Munich from the Department of Biochemistry, 1995. Both compounds were characterized by NMR and mass spectrometry.

(d) performing the in vitro selection

The in vitro selection was carried out using an RNA library of approximately 5 × 10 different sequences. The RNA was denatured at 94 ° C. for 2 minutes in magnesium-free selection buffer (200 mM NaCl, 50 mM K-MOPS, pH 7.4). The concentration of Mg ions in the buffer was then adjusted to 5 mM and cooled to room temperature. The transesterification reaction was initiated by adding N-biotinylated phenylalanyl 2 '(3') adenosine 5 'ester (biotin-Phe-AMP). The reaction mixture was then incubated at room temperature and, after the times indicated in FIG. 2, filtered through a Sephadex G-50 column (Pharmacia, approx. 10 ml) in order to remove excess biotin-Phe-AMP. Those fractions that contained RNA were precipitated with ethanol, resuspended in 500 μl streptavidin coupling buffer (150 mM NaCl, 25 mM Na-P0 ₄ , pH 6.9) and with Incubate 250 μl of swollen streptavidin agarose (Pierce) for 30 minutes. The mixture was then transferred to a polyethylene column (Biorad) and each with 20 column volumes of wash buffer W1 (IM NaCl, 5 mM EDTA, 25 mM K-MOPS, pH 7.4), wash buffer W2 (4 M urea, 5 mM EDTA, adjusted to pH 7.4 with K-MOPS), washing buffer W3 (3 M guanidinium chloride, 5 mM EDTA) and finally washed with water in order to separate non-biotinylated RNA molecules. RNA molecules that were coupled to streptavidin-agarose via the biotin part were cleaved with the biotin-streptavidin interactions with 0.2 M 2-mercaptoethanol (selection cycles 1-6) or 2 M 2-mercaptoethanol (Selection cycles 7-13) eluted. The eluted RNA molecules were precipitated with ethanol, reverse transcribed, PCR-amplified and rewritten into RNA for the next selection cycle by in vitro transcription (Famulok, J. Am. Chem. Soc. 116

(1994), 1698-1706). In the first six selection cycles, 1 mM biotin-Phe-AMP was incubated with 20 μM [c-- ³² P] -UTP-labeled amino-functionalized RNA for 20 hours. In later selection cycles, the selection pressure was increased by shortened incubation times and reduced concentrations of RNA or biotin-Phe-AMP (see FIG. 2). The percentage of transesterases (ribozymes which catalyze the aminoacyl transfer) was determined via the proportion of radioactively labeled RNA which had bound to streptavidin agarose. In selection cycle 10, the PCR amplification was carried out under mutagenic conditions in order to enable the evolution of even more active ribozymes (Cadwell and Joyce, PCR Meth. Appl. 2 (1992) 28-33). The RNA library enriched in transesterases was cloned and sequenced after selection cycle 13 (Famulok, J. Am. Chem. Soc. 116

(1994), 1698-1706). General instructions for the selection steps described can be found in Abelson ed., Meth. In Enzymology 267 (1996), 275-436. (e) Selected ribozyme sequences

Sequences of 27 clones were obtained, which can be divided into three sequence families (I, II, II) (see FIG. 3). Members of a family differ only in point mutations, probably due to the additional diversity introduced in selection cycle 10. Clone 11 from sequence family III was subjected to a more detailed analysis.

(f) Deletion analysis of the clone 11-ribozyme, magnesium dependency and inhibition of the reaction

Deletion analysis succeeded in restricting the full-length clon-11 ribyzy to a catalytically active minimal motif (see FIG. 4). In order to determine the minimum sequence requirements of the ribozyme, various shortened versions of the clone 11 ribozyme were produced. All RNA constructs tested were obtained by in vitro transcription of corresponding DNA fragments, which in turn were generated by "nested PCR" using suitable primers. FIG. 5 shows the dependence of the ribozyme-catalyzed aminoacylation on the concentration of magnesium ions. Further analysis showed that the clone-11 ribozyme was competitively inhibited by AMP (Ki = 4.7 mM), whereas biotin is only a very weak inhibitor of the reaction.

(g) Determination of the reaction site in the clone 11-ribozyme and preparation of an in-trans ribozyme variant

The procedure for determining the reaction site (= aminoacylated 2'-hydroxyl function within the RNA sequence) was as follows:

1. Partial RNase T1 digestion of the aminoacylated clone-11

Ribozyme for determining the sequence area in which the reaction takes place. 2. Cleavage of the clone 11-ribozyme into a substrate part (28-mer oligonucleotide; base position 137-164) and into a catalytic part (ribozyme 28-136; base position 28-136) on the basis of those obtained under point 1 Data.

3. RNase sequencing of the aminoacylated 28-mer oligonucleotide for exact determination of the reaction site.

4. Mass spectroscopic examination and modification analysis of the ribozyme-catalyzed reaction to confirm the reaction site.

To 1.) In order to find out which 2'-hydroxyl function was aminoacylated in the clone 11-ribozyme, 5'- or 3 '- [ ³² P] -labelled ribozyme was incubated with biotin-Phe-AMP and the biotinylated RNAs were immobilized Streptavidin agarose. After the non-aminoacylated RNAs had been removed by washing the affinity matrix, the coupled aminoacylated RNAs were partially digested on guanosine residues with RNase T1. The non-aminoacylated RNA fragments resulting from this treatment were collected by rinsing the affinity matrix, whereas the aminoacylated fragments remaining on the agarose were eluted with 2 M 2-mercaptoethanol in the heat and collected separately. Both fractions (the aminoacylated and the non-aminoacylated fragments) were then analyzed on a denaturing 20% polyacrylamide gel. The analysis showed that the reaction site is located within the 3 'primer region (between the 3' end and position 137) because, unlike non-aminoacylated fragments, aminoacylated fragments are more retarded in the gel, ie migrate more slowly.

Re 2.) For the exact determination of the reaction site, the originally selected clone-11-ribozyme was split into a substrate part (28-mer oligonucleotide; base position 137-164) and into a catalytic part (ribozyme 28-136; base position 28- 136). The proposed secondary structure of the complex of ribozyme 28-136 and the oligonucleotide substrate is shown in Figure 6a. When the ribozyme 28-136 and biotin-Phe-AMP were incubated, a significant proportion of the 28-mer oligonucleotide used could be immobilized on streptavidin-agarose, which clearly showed that the ribozyme 28-136 catalyzes the aminoacylation of the oligonucleotide. To 3.) In order to determine which 2 'hydroxyl function had reacted within the 28-mer oligonucleotide, the aminoacylated 28-mer oligonucleotide was sequenced with base-specific lawns. The result is shown in Figure 6b. - The sequencing pattern for the aminoacylated and non-aminoacylated oligonucleotide is identical from the 3 'end to position C147. At position C147 the non-amino acylated 28-mer oligonucleotide shows the expected band in the U / C lane (circle in Fig. 6b), while no band is found with the amino-acylated 28-mer oligonucleotide (box in Fig 6b). The observed nuclease resistance at position C147 for the aminoacylated 28-mer oligonucleotide is obviously a result of the 2 'esterification. The sequencing bands which can be assigned to positions 148-164 show a reduced gel mobility for the aminoacylated oligonucleotide (relative to the non-aminoacylated oligonucleotide).

4.) The MALDI-TOF mass spectrum of the in-trans-aminoacylation and the analysis with modified 28-mer oligonucleotides summarized in Table II prove beyond doubt that the 2'-OH group at position C147 is the atomic position of the aminoacylation .

(h) Kinetic analysis of the ribozyme-catalyzed aminoacylation

Both the full-length clone-11 and the ribozyme variants described under (f) and (g) were characterized kinetically. The observed rate constants for the intramolecular aminoacylations are summarized in Table I. The Michaelis-Menten parameters for the Length-clone-11 catalyzed reactions were determined with kcat = 0.04 min ^-1 and Km = 119 μM (see Fig. 8). In the in-trans reaction (aminoacylation of a 28-mer oligonucleotide with biotin-Phe-AMP by the ribozyme 28-136) both the outward and the reverse reaction were examined. The quantification of the reverse reaction is shown in Fig. 9. The analysis showed that the equilibrium of the reaction with a logK of approximately -6 is strongly on the side of the starting materials (28-mer and biotin-Phe-AMP).

Table I Rate constants for intramolecular aminoacylation of truncated clone 11 constructs

Clone 11 construct (nucleotide boundaries) ^ _Q -_ _s [h ^-1 ] ^

1-164 1, 8 + 0, 4

16-164 2, 7 ± 0, 2

28-164 5, 6 + 0, 1 35-164 ^§ n. d. *

38-164 ^§ 3, 6 + 0, 3

43-164 0

48-164 0

1-160 0 1-154 0

1-145 0

38-160 0

38-154 1.0 ± 0.4

38-145 0

# Reaction conditions: 5 μM RNA were incubated with 50 μM 1 in selection buffer (12.5 mM MgCl ₂ ). At different times, aliquots were taken from the reaction mixture and coupled to streptavidin agarose. The speed constants k _0] -. _s were determined using the equation [P] _t = (RNA] - [RNA] ^• exp (-k _obs -t), where [P] _t = concentration of the aminoacylated RNA at time t; [RNA] = concentration of clone 11 RNA. * The response was too slow to calculate k ^ _g .

§ The values for k ^ _{g of} these constructs were determined in triplicate. The activities of the other clones were measured in duplicate. The length of the clones was confirmed by analysis using polyacrylamide gel electrophoresis.

Table II Rate constants of intermolecular aminoacylation using various 28-mer oligonucleotide

Substrates.

RNA 2 'modification base ₁₄₇ ^ ₀ _ _s [min ^-1 ]

28 OH c 0.018 ± 5-10 ^"4

28 -dC H c 0

28 -OCH-, OCH c 0

28-NH. NH. c 0

28-U OH u 0, 023 ± 1, 6 - 10

Reaction conditions: 5 μM ribozyme 28-136 were incubated with 10 nM modified 28-mer oligonucleotide substrate and 1 mM 1 in selection buffer (12.5 mM MgCl ₂ ).

Ribozyme kinetics

All aminoacylation reactions were carried out at room temperature in selection buffer with a MgCl ₂ concentration of 12.5 mM. Ribonucleic acids were analyzed according to standard protocols (Sambrook, J., Fritsch, EF & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual., 2nd ed. CSHL-Press,

3

Cold Spring Harbor) at the 5 'end [P] marked. Intramolecular reactions were carried out in 100 μl volumes with the concentrations of ribozyme and 1 indicated in FIG. 8 and Table I. 10 to 15 μl aliquots were taken from the reaction batches at 8 different times. After ethanol precipitation, the aminoacylated RNAs were coupled to streptavidin agarose according to the manufacturer's protocol (Pierce) by swelling the resuspended samples for 30 minutes with 50 μl Affinity matrix incubated. The streptavidin agarose was then transferred to a spin filter reaction vessel (Millipore) and washed thoroughly with the denaturing buffers W2 and W3. For this purpose, the reaction vessel was centrifuged at 12,000 g for 1 minute. The proportion of aminoacylated RNA was determined by measuring the radioactivity. For this purpose, both the flow (wash fractions) and the streptavidin agarose in the scintillation counter

(Beckman) quantified. The initial rates of the reactions were calculated from the measurement data obtained by "curve fitting" (cf. FIG. 8). Intermolecular reactions were carried out in 25 ul volumes. Concentrations of the forward reaction: [Ribozyme 28-136] = 5.0 μM, [28-mer] = 20 nM and

[1] = 0.2, 0.5, 1.0, 2.5, and 5.0 mM. Concentrations of the reverse reaction: [ribozyme 28-136] = 5.0 μM, [28-mer-Phe-biotin] = 5 nM, and [AMP] = 0.1, 0.25, 0.5, 1.0, 2.0, and 10.0 mM. 2 μl aliquots were removed at 10 different times, the reaction being stopped by adding 5 μl PAGE buffer (9.0 M urea, 20 mM EDTA) and cooling to -80 ° C. The samples thus obtained were then analyzed on a 20% polyacrylamide gel and on a phosphor imager

(Molecular Dynamics) evaluated (see. Fig. 9a and 10). The values for k _obs were determined using this data by "curve fitting" (cf. FIG. 9b). The equilibrium constant K of the intermolecular reaction was calculated according to:

[28merPheBio] x [AMP]

K =

[28mer] x [BioPheAMP]

Inhibition studies were carried out with 0.045, 0.45, 2.25, 4.5 and 11.25 mM AMP or biotin, 11 μM ribozyme 28-164 and 45 μM substrate 1. Aliquots were taken from the reaction batch at five different times and analyzed as described for the intramolecular kinetics. The values of Inhibition _constants K ^ were calculated according to k _orS k _Q / (1+ [I] / K _j _), with ₀ k _s = observed rate constant, k _Q = rate of the uninhibited reaction,

[I] = inhibitor concentration, and K ^ = concentration at half maximum inhibition.

Claims

P a t e n t a n s r u c h e

L. A method of selecting a ribozyme capable of modifying 2'-OH groups of ribonucleic acids in trans covalently, the method being characterized by the following steps:

(a) incubation of a ribonucleic acid library with a reactant which can react with a 2'-OH group in riconculinic acids,

(c) transcription in cDNA and amplification of the ribonucleic acids obtained in step (b),

(d) iteratively running through steps (a) to (c),

(e) sequence determination of the selected ribonucleic acids,

(g) cleavage of the selected ribonucleic acid into a substrate part (I) and catalytic part (II = ribozyme),

(h) Checking whether the catalytically active part (II) can covalently modify the substrate part.

2. The method according to claim 1, wherein the 2 '-OH group of the substrate part is acylated.

3. The method according to claim 2, wherein the donor for the amino acid part to be transferred to the 2 'OH group of the substrate part biotinyl-N-phenylalanyl-2' (3 ') -adenosine-5' - monophosphate (Bio-Phe-AMP ) is.

- .. Ribozyme obtainable by the process according to one of claims 1 to 3.

5. Ribozyme according to claim 4, characterized in that it

(a) has the secondary structure and nucleic acid sequence shown in Figure 6a from positions 28 to 136, or

(b) has a sequence and / or secondary structure deviating from (a), these deviations not leading to the loss of the original catalytic activity.

6. DNA sequence encoding the ribozyme according to claim 4 or 5.

7. Vector containing a DNA sequence coding for a ribozyme according to one of claims 1 to 5.

8. The vector of claim 7, wherein the DNA sequence is functionally linked to regulatory elements that allow transcription in prokaryotic or eukaryotic host cells.

9. The vector of claim 7 or 8, which is an RNA virus.

10. The vector of claim 9 which is a retrovirus.

11. Host cell containing a vector according to any one of claims 7 to 10.

12. A host cell according to claim 11 which is a bacterium, a yeast, insect, plant or animal cell.

13. The host cell of claim 12 which is a mammalian cell.

14. A process for producing the ribozyme according to claim 4 or 5, which is an enzymatic or chemical process.

15. Medicament containing the ribozyme according to claim 4 or 5, the DNA according to claim 6 or the vector according to one of claims 7 to 10.

16. Medicament according to claim 15 for inhibiting gene expression or for combating retroviruses in vitro or in vivo.

17. Ribozyme according to claim 4 or 5, DNA according to claim 6 or vector according to one of claims 7 to 10 for use as a sequence-specific gene probe.

18. Ribozyme according to claim 4 or 5, DNA according to claim 6 or vector according to one of claims 7 to 10 for the production of nuclease-resistant ribonucleic acids or for the production of transgenic plants.

19. Use of the ribozyme according to claim 4 or 5, the DNA according to claim 6 or the vector according to one of claims 7 to 10 for the manufacture of a medicament for the inhibition of gene expression or for the control of retroviruses in vivo or in vitro, for the production of nuclease-resistant ribonucleic acids or transgenic plants or as a sequence-specific gene probe.

20. Ribozyme obtainable by the method according to any one of claims 1 to 4, wherein only steps (a) to (f) are carried out.

21. Ribozyme according to claim 20 for use as a ribozyme which acylates tRNA-3 'ends.

22. Ribozyme according to claim 20 for use as aminoacyl tRNA synthetase.