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WO1999036517A2 - Procede de selection de ribozymes capables de modifier par covalence les acides ribonucleiques, en trans, sur les groupes 2'-oh - Google Patents

Procede de selection de ribozymes capables de modifier par covalence les acides ribonucleiques, en trans, sur les groupes 2'-oh Download PDF

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Publication number
WO1999036517A2
WO1999036517A2 PCT/EP1999/000181 EP9900181W WO9936517A2 WO 1999036517 A2 WO1999036517 A2 WO 1999036517A2 EP 9900181 W EP9900181 W EP 9900181W WO 9936517 A2 WO9936517 A2 WO 9936517A2
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ribozyme
ribozymes
rna
vector
sequence
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PCT/EP1999/000181
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WO1999036517A3 (fr
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Andreas Jenne
Michael Famulok
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Andreas Jenne
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Publication of WO1999036517A3 publication Critical patent/WO1999036517A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • 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.
  • 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.
  • 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).
  • ribozymes 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.
  • RNAs modified in this way are no longer translatable.
  • 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:
  • 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.
  • 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) .
  • Phe-AMP phenylalanyl-2 '- (3') -AMP
  • Bio-Phe-AMP biotinyl-N-Phe-AMP
  • 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.
  • 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.
  • 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.
  • RNA is known, this should preferably be taken into account when designing the selection, as explained below.
  • active sequences are reverse transcribed.
  • 2 'modifications typically result in the reverse transcriptase enzyme being unable to produce DNA transcripts due to a stop at the modified position.
  • 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.
  • 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.
  • the selection process can be used again, whereby the partially randomized ribozyme sequence is advantageously used as the basis for the new selection becomes.
  • 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.
  • the present invention also includes vectors containing these DNA encoding ribozymes.
  • vector refers to a plasmid (eg pUC18, pBR322, pBlueScript), a virus genome or another suitable vehicle.
  • 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.
  • 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 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.
  • VA1 adenovirus
  • HSV herpes simplex
  • 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.
  • 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.
  • DNA encoding promoter ribozyme can be introduced into the cell directly or with the aid of a virus.
  • 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) .
  • 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).
  • the DNA is episomal (Flotte et al., Am. J. Respir. Cell. Mol. Biol. 11 (1994), 517-521).
  • 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
  • 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.
  • 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.
  • a suitable promoter for example an SP6, T3 or T7 promoter
  • 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).
  • 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.
  • RNA RNA-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.
  • ribozymes according to the invention not only represent an alternative to the hammerhead ribozymes mentioned, but also show advantages in certain applications:
  • transgenic organisms can be controlled by administration of the reactant.
  • 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.
  • the present invention relates to medicaments which contain the ribozyme according to the invention.
  • administration takes place in different ways.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • the ribozyme DNA sequences are also linked to 3 'DNA sequences, which ensure the termination of the transcription.
  • sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens.
  • 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.
  • plasmids in particular those plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells
  • binary plasmids or Ti plasmids from the Agrobacterium tumefaciens 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.
  • 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.
  • 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 ⁇ M radioactively labeled clone 11-RNA and 25 ⁇ M 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.
  • the aminoacylated substrate was generated as follows: 10 ⁇ M 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.
  • 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.
  • Usage exponential function of the aminoacylation reaction with 5 ⁇ M clone 11-RNA and 0.5 mM biotin-Phe-AMP 1.
  • 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.
  • Example 1 illustrates the invention.
  • Example 1 illustrates the invention.
  • 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”.
  • ribozymes and deoxyribozymes are capable of catalyzing a variety of chemical reactions (Pan, Curr. Opin. Chem. Biol. Vol. 1 No. 1 (1997), 17-25).
  • 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.
  • 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.
  • 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 Kunststoff, 1995.
  • 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 ⁇ l 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.
  • 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.
  • RNA was precipitated with ethanol, resuspended in 500 ⁇ l streptavidin coupling buffer (150 mM NaCl, 25 mM Na-P0 4 , pH 6.9) and with Incubate 250 ⁇ l of swollen streptavidin agarose (Pierce) for 30 minutes.
  • streptavidin coupling buffer 150 mM NaCl, 25 mM Na-P0 4 , pH 6.9
  • 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
  • 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
  • RNA library enriched in transesterases was cloned and sequenced after selection cycle 13 (Famulok, J. Am. Chem. Soc. 116
  • 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.
  • FIG. 4 Deletion analysis succeeded in restricting the full-length clon-11 ribyzy to a catalytically active minimal motif (see FIG. 4).
  • 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.
  • reaction site aminoacylated 2'-hydroxyl function within the RNA sequence
  • 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.
  • 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.
  • the sequencing pattern for the aminoacylated and non-aminoacylated oligonucleotide is identical from the 3 'end to 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).
  • Reaction conditions 5 ⁇ M 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 ).
  • reaction vessel was centrifuged at 12,000 g for 1 minute.
  • proportion of aminoacylated RNA was determined by measuring the radioactivity.
  • both the flow (wash fractions) and the streptavidin agarose in the scintillation counter were used.
  • [1] 0.2, 0.5, 1.0, 2.5, and 5.0 mM.
  • [I] inhibitor concentration
  • K ⁇ concentration at half maximum inhibition

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Abstract

L'invention concerne un procédé de sélection in vitro permettant de sélectionner des ribozymes capables de modifier par covalence, en trans, les groupes 2'-OH des acides ribonucléiques. L'invention concerne en outre les ribozymes obtenues suivant ce procédé. Par ailleurs, l'invention concerne des médicaments contenant celles-ci, pouvant être utilisés, de préférence, pour l'inhibition de l'expression des gènes, par exemple, en thérapie génétique. Les ribozymes conformes à l'invention peuvent être également utilisées pour la fabrication de mutéines et d'acides ribonucléiques résistant à la nucléase, par exemple, d'oligonucléotides "antisens" résistant à la ribonucléase.
PCT/EP1999/000181 1998-01-14 1999-01-14 Procede de selection de ribozymes capables de modifier par covalence les acides ribonucleiques, en trans, sur les groupes 2'-oh WO1999036517A2 (fr)

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DE19801153.9 1998-01-14
DE1998101153 DE19801153A1 (de) 1998-01-14 1998-01-14 Verfahren zur Selektion von Ribozymen, die Ribonucleinsäuren in trans an 2'-OH-Gruppen kovalent modifizieren können und durch dieses Verfahren erhältliche Ribozyme

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Cited By (4)

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EP1232285A1 (fr) * 1999-11-24 2002-08-21 The Research Foundation Of State University of New York Arn catalytiques a activite d'aminoacylation
US6794140B1 (en) 1999-04-30 2004-09-21 Andrew Simon Goldsborough Isolation of nucleic acid
EP1964916A1 (fr) * 2005-12-06 2008-09-03 The University of Tokyo Catalyseur d'acylation polyvalent et utilisation de celui-ci
US7622248B2 (en) * 2002-02-15 2009-11-24 The Research Foundation Of State University Of New York Ribozymes with broad tRNA aminoacylation activity

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Title
HAGER, A. ET AL.: "Ribozymes : aiming at RNA replication and protein synthesis" CHEMISTRY AND BIOLOGY., Bd. 3, September 1996 (1996-09), Seiten 717-725, XP002104957 ISSN: 1074-5521 *
IBBA M : "STRATEGIES FOR IN-VITRO AND IN-VIVO TRANSLATION WITH NON-NATURAL AMINO-ACIDS" BIOTECHNOLOGY & GENETIC ENGINEERING REVIEWS, (1996) VOL. 13, PP. 197-216., XP002109693 *
ILLANGASEKARE M ET AL: "AMINOACYL-RNA SYNTHESIS CATALYZED BY AN RNA" SCIENCE, Bd. 267, 3. Februar 1995 (1995-02-03), Seiten 643-647, XP002044710 ISSN: 0036-8075 *
JENNE A ET AL: "A novel ribozyme with ester transferase activity" CHEMISTRY & BIOLOGY, VOL. 5, NO. 1, PP. 23-34.,15. Januar 1998 (1998-01-15), XP002109695 *
LORSCH J R ET AL: "IN VITRO EVOLUTION OF NEW RIBOZYMES WITH POLYNUCLEOTIDE KINASE ACTIVITY" NATURE, Bd. 371, 1. September 1994 (1994-09-01), Seiten 31-36, XP002044711 ISSN: 0028-0836 in der Anmeldung erw{hnt *
PAN, T.: "Novel and variant ribozymes obtained through in vitro selection" CURRENT OPINION IN CHEMICAL BIOLOGY, Bd. 1, 1997, Seiten 17-25, XP002109694 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6794140B1 (en) 1999-04-30 2004-09-21 Andrew Simon Goldsborough Isolation of nucleic acid
US6867290B2 (en) 1999-04-30 2005-03-15 Cyclops Genome Sciences, Ltd. Modified polynucleotides and uses thereof
US7244568B2 (en) 1999-04-30 2007-07-17 Cyclops Genome Sciences Limited Isolation of nucleic acid
EP1232285A1 (fr) * 1999-11-24 2002-08-21 The Research Foundation Of State University of New York Arn catalytiques a activite d'aminoacylation
EP1232285A4 (fr) * 1999-11-24 2005-01-12 Univ New York State Res Found Arn catalytiques a activite d'aminoacylation
US7001723B1 (en) 1999-11-24 2006-02-21 The Research Foundation Of State University Of New York Catalytic RNAs with aminoacylation activity
US7622248B2 (en) * 2002-02-15 2009-11-24 The Research Foundation Of State University Of New York Ribozymes with broad tRNA aminoacylation activity
EP1964916A1 (fr) * 2005-12-06 2008-09-03 The University of Tokyo Catalyseur d'acylation polyvalent et utilisation de celui-ci
EP1964916A4 (fr) * 2005-12-06 2010-10-20 Univ Tokyo Catalyseur d'acylation polyvalent et utilisation de celui-ci
US8188260B2 (en) 2005-12-06 2012-05-29 The University Of Tokyo Versatile acylation catalytic RNAs and uses thereof
JP5119444B2 (ja) * 2005-12-06 2013-01-16 国立大学法人 東京大学 多目的アシル化触媒とその用途

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