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WO2006123537A1 - Nouvelle endoribonuclease - Google Patents

Nouvelle endoribonuclease Download PDF

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Publication number
WO2006123537A1
WO2006123537A1 PCT/JP2006/308981 JP2006308981W WO2006123537A1 WO 2006123537 A1 WO2006123537 A1 WO 2006123537A1 JP 2006308981 W JP2006308981 W JP 2006308981W WO 2006123537 A1 WO2006123537 A1 WO 2006123537A1
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WIPO (PCT)
Prior art keywords
polypeptide
sequence
nucleic acid
seq
rna
Prior art date
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PCT/JP2006/308981
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English (en)
Japanese (ja)
Inventor
Masamitsu Shimada
Masanori Takayama
Yoko Tatsumi
Kiyozo Asada
Ikunoshin Kato
Original Assignee
Takara Bio Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takara Bio Inc. filed Critical Takara Bio Inc.
Priority to JP2007516241A priority Critical patent/JPWO2006123537A1/ja
Publication of WO2006123537A1 publication Critical patent/WO2006123537A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a novel sequence-specific endoribonuclease useful in the field of genetic engineering.
  • mK is an endoribonuclease that recognizes a specific base of UAH (H is C, A or U) and cleaves mRNA (Patent Document 1, Non-Patent Document 10).
  • RelE and PemK family toxins may be endoribonucleases that cleave mRNA in a base-specific manner.
  • PemK family toxins may be endoribonucleases that recognize specific bases and cleave mRNAs independently of ribosomes.
  • Many PemK family toxins are present in prokaryotes, and their sequence comparison has been well studied (Non-patent Documents 1 and 11).
  • Patent Document 1 Pamphlet of International Publication No. 2004Z113498
  • Non-Patent Document 1 Journal 'Ob' Battereriol. (J. Bacteriol.), 182, p5 61-572 (2000)
  • Non-Patent Document 2 Science, No. 301, pl496-1499 (2003)
  • Non-patent Document 3 Molecular Microbiol., No. 48, pl389-1400 (2003)
  • Non-Patent Document 4 Cell, 122, 131-140 (2003)
  • Non-Patent Document 5 Journal 'Ob' Molequila 'Biology. Mol. Biol.), No. 332, p809-819 (2003)
  • Non-Patent Document 6 Moleculera 'Microbiology, No. 51, pl705-1717 (, 200 4)
  • Non-Patent Document 7 Molecular Cell, No.12, p913-920, 200 3)
  • Non-Patent Document 8 Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), No. 80, p3143-4150 (2005)
  • Non-Patent Document 9 FEBS Letters, No. 567, p316-320 (2004)
  • Non-Patent Document 10 Journal 'Ob' Biological 'Chemistry, Vol. 279, p20678 -20684 (2004)
  • Non-Patent Document 11 Journal 'Ob' Morekiyura 'Biotechnology. Mol. Biot enol.), No. 1, p295-302 (1999)
  • Non-Patent Document 12 Genome Biology, No. 4, R81 (2003)
  • Non-Patent Document 13 Method in Enzymology, No. 3 41, p28-41 (2001)
  • An object of the present invention has been made in view of the above-described prior art, and is to find a novel sequence-specific endoribonuclease, which is a novel sequence-specific endoribonuclease.
  • the purpose is to identify the specificity of the cleavage sequence and provide its use in genetic engineering. Means for solving the problem
  • the present inventors screened a sequence-specific endoribonuclease, and the NMB2038 homologue derived from Neisseria meningitides was a novel sequence-specific endoribonuclease.
  • the present invention provides: [1] The amino acid sequence described in SEQ ID NO: 4 in the sequence listing, or an amino acid sequence having at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the sequence, and the sequence A polypeptide having specific endoribonuclease activity, (2) a nucleic acid encoding the polypeptide of (1),
  • nucleic acid according to [2] having the base sequence set forth in SEQ ID NO: 3 in the sequence listing; [4] The nucleic acid according to [2] or [3] is capable of being hybridized under stringent conditions. And a nucleic acid encoding a polypeptide having sequence-specific endoribonuclease activity, (5) [2] to [4] V, a recombinant DNA comprising the nucleic acid according to item 1,
  • a method for producing a single-stranded RNA degradation product comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA;
  • a method for degrading single-stranded RNA comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA,
  • polypeptide of the present invention is represented by the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing, or an amino acid sequence having at least one deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence. And sequence-specific endoribonuclease activity.
  • the activity of the polypeptide of the present invention is a single-stranded RNA-specific endoribontale. It can hydrolyze the phosphodiester bond on the 3 ′ side of ribonucleotides in a single-stranded nucleic acid containing ribonucleotides as a constituent base.
  • the nucleic acid hydrolyzed by the activity has a 3 ′ end having a hydroxyl group and a 5 ′ end having a phosphate group, a 3 ′ end having a phosphate group and a 5 ′ end having a hydroxyl group, or a 2 ′ or 3 ′ site. This produces a 5 'end with click phosphate and hydroxyl groups.
  • the substrate of the polypeptide of the present invention may be a nucleic acid having at least one molecule of ribonucleotide, such as RNA, RNA containing deoxyribonucleotide, DNA containing ribonucleotide, and the like. It is not limited to these.
  • the substrate contains a nucleotide different from that contained in a normal nucleic acid within a range not inhibiting the action of the polypeptide of the present invention, such as deoxyinosine, deoxyuridine, hydroxymethyldeoxyuridine and the like. Well, okay.
  • the polypeptide of the present invention specifically acts on a single-stranded nucleic acid.
  • Double-stranded nucleic acids such as double-stranded RNA, RNA-DNA nobled, etc. cannot be cleaved! /.
  • the polypeptide of the present invention is characterized by having an activity of cleaving a nucleic acid in a specific base sequence.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is a sequence of 5′-GAACU-3 ′ in a single-stranded RNA molecule. It hydrolyzes the phosphodiester bond between two A residues. This activity can be confirmed by detecting the resulting RNA degradation product after allowing the polypeptide of the present invention to act on single-stranded RNA.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 4 has the 12th and 13th bases of the oligoribonucleotide. Preferentially hydrolyze the phosphodiester bond in between.
  • the above-mentioned polypeptides may contain 5′—GAZAUU—3 ′, 5′—GAZACC (or A) —3 ′, 5, —AA / ACU—3 ′ and 5, —GU / ACU.
  • the position indicated by “Z” in the sequence may also be cleaved. Since the end ribonuclease activity of the polypeptide of the present invention is expressed without the coexistence of ribosomes, the activity is independent of liposomes.
  • Single-stranded RNA-specific endoribonuclease activity of the polypeptide of the present invention Can be measured, for example, using single-stranded RNA as a substrate. Specifically, a single-stranded RNA transcribed with RNA polymerase in the shape of a DNA or a chemically synthesized single-stranded RNA is allowed to act on a polypeptide whose activity is to be cleaved. It can be measured by examining whether it occurs. RNA degradation can be confirmed, for example, by electrophoresis (agarose gel, acrylamide gel, etc.). If a suitable label (for example, a radioisotope, a fluorescent substance, etc.) is attached to RNA as a substrate, it becomes easy to detect degradation products after electrophoresis.
  • a suitable label for example, a radioisotope, a fluorescent substance, etc.
  • the polypeptide of the present invention has one or more amino acid residues in the amino acid sequence described in SEQ ID NO: 4 in the sequence listing as long as it exhibits endoribonuclease activity that hydrolyzes single-stranded RNA in a sequence-specific manner. It includes a polypeptide represented by an amino acid sequence having at least one of deletion, addition, insertion or substitution. Examples of the polypeptide having such a mutation include a polypeptide having 50% or more homology to the polypeptide of SEQ ID NO: 4, preferably a polypeptide having 70% or more homology, particularly preferably 90%. A polypeptide having the above homology is exemplified. Even if the polypeptide having these mutations recognizes and cleaves a sequence different from the polypeptide having the amino acid sequence described in SEQ ID NO: 4, it is encompassed in the present invention.
  • the above-described polypeptide may have a peptide region that is essential for its activity! /,
  • a peptide for improving the efficiency of translation, or purification of a previous polypeptide is easy.
  • the present invention provides nucleic acids that exhibit sequence-specific endoribonuclease activity.
  • the present invention is not particularly limited as the nucleic acid, the amino acid sequence described in SEQ ID NO: 4 in the sequence listing, or the deletion or addition of one or more, for example, 1 to 10 amino acid residues in the sequence, An amino acid sequence having at least one of insertion or substitution and encoding the polypeptide having the sequence-specific endoribonuclease activity described above Is mentioned.
  • the polypeptide described in SEQ ID NO: 4 includes 50
  • An amino acid sequence having at least% homology preferably an amino acid sequence having at least 70% homology, particularly preferably an amino acid sequence having at least 90% homology is exemplified.
  • the nucleic acid of the present invention includes a nucleic acid that can hybridize to the above-mentioned nucleic acid under stringent conditions and that encodes a polypeptide having sequence-specific endoribonuclease activity.
  • the stringent conditions are as follows: 1989, Cold 'Spring' Nova One 'Laboratory published, edited by J. Sambrook et al., Molecular ⁇ ⁇ ⁇ Cloning:' Laboratory ⁇ ⁇ ⁇ Examples include conditions described in the second edition of the manual (Molecular Cloning: A Laboratory Manual 2nd ed.). Specifically, for example, conditions of incubation with a probe at 65 ° C.
  • the nucleic acid hybridized to the probe can be detected after removing non-specifically bound probe by washing at 37 ° C. in 0.1 ⁇ SSC containing 0.5% SDS, for example.
  • nucleic acid encoding the polypeptide of the present invention can be obtained, for example, by the following means.
  • a gene having homology in the amino acid sequence to a toxin such as MazF or PemK having endoribonuclease activity that recognizes a specific base sequence and cleaves mRNA is a polymorphism having sequence-specific ribonuclease activity. It is a candidate nucleic acid encoding a peptide. Such candidate genes can be found, for example, from the bacterial genome.
  • Candidate genes can also be isolated from bacterial genomic strength by PCR using primers designed based on nucleotide sequence information, for example. If the entire base sequence is known, the entire sequence of the candidate gene can be synthesized using a DNA synthesizer.
  • Protein expression with candidate gene ability can be carried out in an appropriate host transformed with an expression vector incorporating the candidate gene, for example, E. coli.
  • Expression of sequence-specific ribonucleases that degrade host RNA may be lethal to the host and is induced Until now, the expression of candidate genes needs to be strictly suppressed.
  • an expression system such as a pET system (manufactured by Novagen) using a T7 polymerase promoter or a cold shock expression control system pCold system (manufactured by Takara Bio Inc.).
  • a peptide such as the histidine tag
  • an expression vector containing such a peptide coding region may be used.
  • the measurement of endoribonuclease activity can be carried out by the above-described method using single-stranded RNA as a substrate.
  • the cleavage site can be identified by a primer extension using a cleaved RNA as a saddle and a primer complementary to the RNA and reverse transcriptase. Since the extension reaction stops at the cleavage site in the first primer extension, the cleavage site can be identified by determining the length of the extended strand by electrophoresis.
  • an oligoribonucleotide having an arbitrary sequence is chemically synthesized, the candidate gene expression product is allowed to act, and then cleaved by denaturing acrylamide gel electrophoresis or the like. What is necessary is just to determine the presence or absence.
  • the polypeptide of the present invention is, for example, (1) purified from a culture of a microorganism producing the polypeptide of the present invention, or (2) capable of culturing a transformant containing a nucleic acid encoding the polypeptide of the present invention. It can be produced by a method such as purification.
  • the present invention is not particularly limited as a microorganism producing the polypeptide of the present invention!
  • bacteria belonging to the genus Neisseria are exemplified.
  • the polypeptide of the present invention can be obtained from N. meningitides, particularly preferably from N. meningitides ATCC No. 13090 strain.
  • the microorganism may be cultured under conditions suitable for the growth of the microorganism.
  • the target polypeptide produced in the bacterial cells or the culture solution can be obtained by methods commonly used for protein purification, such as disruption of bacterial cells, fractionation by precipitation (such as ammonium sulfate salting-out), and various chromatographic methods.
  • the polypeptide of the present invention can be obtained from the transformant transformed with the recombinant DNA containing the nucleic acid encoding the polypeptide of the present invention.
  • the recombinant DNA is preferably provided with a suitable promoter functionally connected upstream of the nucleic acid encoding the polypeptide. Since the polypeptide of the present invention may have a lethal effect on the host, the above promoter and the expression system including the promoter can transcribe nucleic acid that encodes the polypeptide of the present invention. It is preferable that it is strictly controllable. Examples of such a system include the pET system and the p Cold system.
  • the above recombinant DNA may be introduced as it is into a host cell, and may be introduced by being inserted into an appropriate vector such as a plasmid vector, a phage vector, or a virus vector. Furthermore, the above recombinant DNA may be integrated into the host chromosome. There are no particular limitations on the host to be transformed, such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plants, animals, plant culture cells, animal culture cells, and other hosts that are commonly used in the field of recombinant DNA. It is done.
  • the polypeptide of the present invention produced by these transformants can be purified using the purification method as described above.
  • the nucleic acid encoding the polypeptide of the present invention encodes a polypeptide to which a peptide for facilitating purification of the polypeptide is added, purification becomes very easy.
  • a purification method according to the added peptide for example, using a metal chelate resin for histidine-tag and a dartathione-fixed resin for daltathione S-transferase, respectively, A pure polypeptide can be obtained by a simple operation.
  • single-stranded RNA can be degraded to produce an RNA degradation product. Since the polypeptide of the present invention can cleave RNA in a base sequence-specific manner, the average difference length of the generated RNA degradation products correlates with the appearance frequency of the base sequence recognized by the polypeptide. That is, the present invention provides an RNA degradation product having a certain chain length distribution. Furthermore, it is possible to excise a specific region in RNA using its sequence specificity. [0033] Furthermore, single-stranded RNA can be selectively degraded by the polypeptide of the present invention.
  • protein synthesis systems for example, cell-free translation systems and mRNAs in transformants can be degraded with the polypeptide of the present invention to inhibit protein synthesis.
  • the mRNA encoding the desired protein which is artificially prepared so as not to contain the base sequence recognized by the polypeptide of the present invention, is allowed to exist in the above system, so that only the mRNA is degraded.
  • the desired protein is specifically produced in the system. This embodiment is particularly useful for producing highly pure protein.
  • Example 1 Isolation of NMB2038 homologue derived from Neisseria meningitides ATCC No. 13090 strain and construction of expression plasmid
  • pemK—related protein / locus-tag NMB2038 of Neisseria meningitides MC58 strain the amino acid sequence and nucleotide sequence of the polypeptide encoded therein were obtained from the NCBI database (accession No. NP—275029 and NC—003112) . Based on the nucleotide sequence information of NMB2038, primer NMB2038-F (SEQ ID NO: 1) and primer NMB2038-R (SEQ ID NO: 2) were synthesized so that DNA in the region encoding the entire polypeptide could be amplified by PCR.
  • Genomic DNA was prepared from Neisseria meningitides (ATCC No. 13090) by the SDS-protenaseK method. PCR was performed using TaKaRa LA-PCR Kit ver. 2.1 (manufactured by Takara Bio Co., Ltd.) using 50 ng of genomic DNA and primers NMB2038-F and NMB2038-R to obtain an amplified DNA fragment of 344 bp. This amplified fragment was digested with restriction enzymes Ndel and Xhol and subjected to agarose electrophoresis, and a 323 bp DNA fragment was recovered from the gel. PET21a vector digested with restriction enzymes Ndel and Xhol ( E.
  • coli strain JM109 was transformed with the recombinant plasmid obtained by ligating this 323 bp DNA fragment to Novagen.
  • a plasmid was prepared from the mouth of the transformant thus obtained, its nucleotide sequence was confirmed, and this was named the expression vector pET-NMB2038Hlg.
  • the base sequence of the NMB meningitides-derived NMB2038 homolog polypeptide inserted into the expression vector pET-NMB2038Hlg is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4, respectively.
  • a polypeptide in which a histidine monotag having 8 amino acid residues and including 6 histidine residues is added to the C-terminal of the polypeptide having the amino acid sequence of SEQ ID NO: 4 Is expressed.
  • Escherichia coli BL21 (DE3) strain (manufactured by Novagen) was transformed with the expression vector PET-NMB2038 Hlg obtained in Example 1 to obtain E. coli pET-NMB2038 Hlg / BL21 (DE3) for expression.
  • pET—NMB2038 Hlg / BL21 (DE3) 37 in 5 ml LB medium containing 100 ⁇ g / ml ampicillin.
  • IPTG manufactured by Takara Bio Inc.
  • IPTG manufactured by Takara Bio Inc.
  • the suspension was centrifuged and the supernatant was collected. The same elution procedure was repeated two more times to obtain a total of 60 1 samples containing NMB2038 homolog polypeptide. A part of this sample was subjected to SDS-PAGE to confirm that it contained a polypeptide of the expected size.
  • the protein concentration in the sample was about 400 ngZ ⁇ 1.
  • Example 3 Preparation of Substrate RNA
  • Substrate RNA was prepared by in vitro transcription using lambda phage DNA as a cage.
  • RNA of the sequence corresponding to the region corresponding to 28411 to 29074 of lambda phage DNA (Genbank accession No. J02459)
  • primer LAS28951F (SEQ ID NO: 5)
  • primer LAS2895 IF (SEQ ID NO: 6) were respectively synthesized.
  • Primer 1 The base sequence of T7 promoter was inserted into LAS28951F.
  • the obtained reaction solution was treated with DNase 1 (manufactured by Takarabio Co., Ltd.) at a final concentration of 0.5 U / ⁇ 1 for 30 minutes at 37 ° C, and then gel filtered using Centrisep (manufactured by Princeton Separation Inc.). The filtrate was subjected to phenol / black mouth form treatment, black mouth form treatment, and isopropanol precipitation to purify RNA. The RNA precipitate was dissolved in sterile distilled water. By this operation, 667 bases of LAS28951RNA (SEQ ID NO: 7 shows the base sequence of LAS28951RNA) was obtained.
  • LAS28951PEROX a primer having the base sequence shown in SEQ ID NO: 8 and having a ROX label added to the 5 ′ end, was synthesized.
  • a 51 reaction solution consisting of LAS28951RNA 25ngZ 1 obtained in Example 3 and NMB2038 homolog polypeptide 4ngZ 1 obtained in Example 2 and 10 mM Tris-HCl (pH 7.5) was prepared at 37 ° C, Incubated for 30 minutes.
  • reverse transcription reaction was performed as follows. Using the total amount of the above reaction mixture, 2.5 / ⁇ 1 MMLV reverse transcriptase (Takara Bio), 0.5mM dNTPs, 1U / ⁇ l RNase Inhibitor (Takara Bio), 0.5pmol / ⁇ 1 LAS28951PEROX A total of 101 RT reaction solution containing primers was prepared and incubated at 42 ° C for 60 minutes. After completion of the reaction, 3 times the amount of sample buffer (95% formamide, 20 mM EDTA) was added.
  • sample buffer 95% formamide, 20 mM EDTA
  • the cleavage base number is indicated by the base number on the 3 ′ side of the cleavage site (G e n b a n k a c c e ss! O n N o.
  • oligoribonucleotides were synthesized and cleaved with NMB2038 homolog polypeptide.
  • oligoribonucleotides MRI001 to MRI016 were synthesized as substrates.
  • 10 ⁇ oligoribonucleotide, ⁇ 2038 homolog protein obtained in Example 2 4 ngZ ⁇ 1, 5 / z 1 reaction solution consisting of 10 mM Tris-HCl (pH 7.5) was incubated at 37 ° C for 30 minutes .
  • the reaction product was subjected to 20% denaturing acrylamide gel (20% acrylamide, 7M urea, 0.5 X TBE buffer) electrophoresis, stained with SYBR GREEN II (Takara Bio), and then fluorescence image analyzer FMBIOII Multiview (Takara). Fluorescence images were analyzed using Biotechnology. Table 2 shows the state of cleavage of each oligoribonucleotide.
  • Each oligoribonucleotide has three sequences that can be cleaved by the NMB2038 homolog polypeptide.
  • Table 2 shows 5 ′ side force cutting sites 1, 2, and 3. Also, the cutting situation is complete cutting +++, partial cutting +++, very weak cutting +, Complete undegradation is indicated by one. Furthermore, based on the presence or absence of the cleavage of each oligoribonucleotide and the strength of the cleavage, the base sequence around the cleavage site was compared to evaluate the specificity of the sequence.
  • NMB2038 homolog polypeptide preferentially recognizes the sequence of 5, -GA / ACU-3 '(/ indicates the cleavage site) and specifically cleaves RNA. It was easy. It was also found that 5'-GA / AUU-3 ', 5'-GAZACC (or A) -3', 5'-A AZACU-3 'and 5'-GUZACU can also be cleaved.
  • Cut site The cut site is cut between -1 and +1.
  • Cut site The cut site is cut between -1 and +1.
  • Industrial applicability [0053]
  • the present invention provides a novel sequence-specific endoribonuclease. Since the enzyme can recognize and cleave a specific sequence in RNA, analysis of RNA molecules, preparation of RNA fragments, control of cells through RNA cleavage in cells (for example, inhibition of protein production), etc. Useful to
  • SEQ ID NO: l PCR primer NMB2038— F to amplify a DNA fragment encoding NMB2 038 protein.
  • SEQ ID NO: 2 PCR primer NMB2038-R to amplify a DNA fragment encoding NMB2 038 protein.
  • SEQ ID NO 7 SEQ ID NO 7; LAS28951RNA transcribed from a portion of lambda DNA.

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Abstract

L’invention concerne un nouveau polypeptide ayant une activité d’endoribonucléase ; un acide nucléique codant ce polypeptide ; un ADN recombiné contenant cet acide nucléique ; un transformant ayant été transformé par l'ADN recombiné ; une procédé de production du polypeptide décrit ci-dessus caractérisé en ce qu’il comprend l'étape de culture du transformant et l'étape de collecte du polypeptide à partir de la culture ; un procédé de production d’un produit de digestion d'ARN simple brin caractérisé en ce qu’il comprend l'étape de traitement d’un ARN simple brin avec le polypeptide décrit ci-dessus ; et un procédé de digestion d’un ARN simple brin.
PCT/JP2006/308981 2005-05-16 2006-04-28 Nouvelle endoribonuclease WO2006123537A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133137A1 (fr) 2007-04-20 2008-11-06 Takara Bio Inc. Vecteur pour une thérapie génique
JP2013528372A (ja) * 2010-05-10 2013-07-11 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア エンドリボヌクレアーゼ組成物およびその使用方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077183A2 (fr) * 2001-03-21 2002-10-03 Elitra Pharmaceuticals, Inc. Identification de genes essentiels dans des microorganismes
WO2004113498A2 (fr) * 2003-06-13 2004-12-29 University Of Medicine And Dentistry Of New Jersey Arn interferases et leurs procedes d'utilisation
WO2005074986A2 (fr) * 2004-02-10 2005-08-18 Genobiotix Aps Especes bioactive capables d'interferer avec un complexe toxine-antitoxine microbien et procedes d'evaluation et d'utilisation de ladite espece bioactive

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077183A2 (fr) * 2001-03-21 2002-10-03 Elitra Pharmaceuticals, Inc. Identification de genes essentiels dans des microorganismes
WO2004113498A2 (fr) * 2003-06-13 2004-12-29 University Of Medicine And Dentistry Of New Jersey Arn interferases et leurs procedes d'utilisation
WO2005074986A2 (fr) * 2004-02-10 2005-08-18 Genobiotix Aps Especes bioactive capables d'interferer avec un complexe toxine-antitoxine microbien et procedes d'evaluation et d'utilisation de ladite espece bioactive

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENPEPT NCBI; 22 May 2006 (2006-05-22), PARKHILL J.: "Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491", XP000918875 *
DATABASE GENPEPT NCBI; 22 May 2006 (2006-05-22), TETTELIN H. ET AL.: "Complete genome sequence of Neisseria meningitidis serogroup B strain MC58", XP000914963 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133137A1 (fr) 2007-04-20 2008-11-06 Takara Bio Inc. Vecteur pour une thérapie génique
JP2013528372A (ja) * 2010-05-10 2013-07-11 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア エンドリボヌクレアーゼ組成物およびその使用方法
EP2569425A4 (fr) * 2010-05-10 2014-03-12 Univ California Compositions d'endoribonucléases et leurs procédés d'utilisation
US9115348B2 (en) 2010-05-10 2015-08-25 The Regents Of The University Of California Endoribonuclease compositions and methods of use thereof
US9605246B2 (en) 2010-05-10 2017-03-28 The Regents Of The University Of California Endoribonuclease compositions and methods of use thereof
US9708646B2 (en) 2010-05-10 2017-07-18 The Regents Of The University Of California Endoribonuclease compositions and methods of use thereof

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