WO2006078042A1 - Procede de detection des petits arn et reactif de detection des petits arn - Google Patents

Procede de detection des petits arn et reactif de detection des petits arn Download PDF

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WO2006078042A1
WO2006078042A1 PCT/JP2006/301074 JP2006301074W WO2006078042A1 WO 2006078042 A1 WO2006078042 A1 WO 2006078042A1 JP 2006301074 W JP2006301074 W JP 2006301074W WO 2006078042 A1 WO2006078042 A1 WO 2006078042A1
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rna
small molecule
gene
double
nucleotide sequence
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PCT/JP2006/301074
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English (en)
Japanese (ja)
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Kojiro Ishii
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Kurume University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a method for easily detecting small molecule RNA in a sample, a reagent for detecting small molecule RNA, and the like.
  • RNA interference RNA i nt er f rn c e
  • Dicer an enzyme responsible for the RNA-induced silencing cAMP lex .
  • RNA i method si RNA method
  • MiRNA is a regulatory RNA consisting of approximately 22 bases.
  • miRNAs such as Drosophila play an important role in gene regulation in the differentiation process. It attracts attention because it specifically suppresses post-transcriptional translation by forming base pairs.
  • Northern blotting is a well-known method for identifying RNA by hybridizing RNA transcribed from isolated DNA with a complementary probe.
  • detection of small RNAs consisting of 20 to 30 bases, such as siRNA and miRNA has not been easy.
  • RNA probe is fragmented by acid treatment, but it is an extremely advanced treatment method and has not been widely adopted in related technical fields. Oligo RNA probes are also used, but the search sequence is limited, resulting in failure in sequence selection. Disclosure of the invention
  • An object of the present invention is to provide a method for easily detecting small molecule RNA, such as siRNA and miRNA, which has been increasing in importance in recent years, and a reagent for detecting small molecule RNA.
  • the present inventor synthesized double-stranded RNA containing the full length or a part of the nucleotide sequence of the target gene, and converted the double-stranded RNA into RNa se.
  • the present invention was completed by finding that small molecule RNA can be easily detected by using the obtained digested fragment as a probe, and detecting a reagent for detecting the small molecule RNA.
  • the present invention provides the following:
  • a method for detecting small molecule RNA derived from a target gene comprising the following steps:
  • Reagent for detecting small molecule RNA including RNase digestion product of double-stranded RNA containing the full length or a part of the nucleotide sequence of the gene of interest,
  • a method for preparing a probe for detecting small molecule RNA derived from a target gene comprising the following steps:
  • a reagent containing an RNase digestion product of double-stranded RNA containing the full length or a part of the nucleotide sequence of the gene of interest, and the reagent can or should be used for detection of small molecule RNA A commercial package containing a statement stating that there is. Brief Description of Drawings
  • FIG. 1 is a diagram showing an example of detection of siRNA in a cell extract by performing a Northern plot using a centrisomal sequence known to produce siRNA in a clonal analysis of fission yeast as a probe.
  • Lane 1 is the ago 1 gene disruption strain
  • Lane 2 is the rdpi gene disruption strain
  • Lane 3 is the dcrl gene disruption strain
  • Lane 4 is the wild strain (h-)
  • Lane 5 is the wild strain (h 90 ) Show.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for efficiently detecting small molecule RNA.
  • double-stranded RNA containing the full length or a part of the nucleotide sequence of the target gene is subjected to RNase digestion, and small molecule RNA is detected using the obtained digested product.
  • the present invention also provides a method for preparing a probe containing the digested product.
  • the small RNA detected in the present invention is an RNA having a size of about 10-50 bases, preferably about 20-30 bases.
  • the RNA may be single-stranded or double-stranded. When the RNA is a double strand, the double strand may have an overhang.
  • the duplex may have a stem loop structure, a hairpin structure, or the like. When the duplex has a stem loop structure, a hairpin structure, or the like, the “RNA size” is a loop. It is the length of the part that forms a complementary pair that does not include. Specifically, for example, siRNA, miRNA, and stRNA (smal lte emporary RNA tered RNA), which have been suggested to be involved in RNAi, can be mentioned.
  • the target gene means a gene containing a desired nucleotide sequence region (exploration region) to be examined whether or not it can have a small molecule RNA as a product.
  • “gene” includes “nucleic acid”.
  • Nucleotide sequence of target gene refers to the nucleotide sequence of chromosomal DNA or mRNA (cDNA) encoding the target gene.
  • the size of the “part of the nucleotide sequence of the target gene” is not particularly limited, but may be a size sufficient to have a small molecule RNA as a product, for example, 100 bases or more.
  • the “part of the nucleotide sequence of the target gene” includes the above exploration region.
  • “Small molecule RNA derived from the target gene” may be a small molecule RNA directly derived from the target gene or an indirect small molecule RNA, but is preferably derived directly.
  • Small molecule RNA directly derived means the transfer of the gene. If the transcript is a small molecule RNA itself, or the transcript itself is not a small molecule RNA (eg, tRNA, mRNA, rRNA, etc.), but is digested by an endogenous degrading enzyme (RNase, etc.) This means that the digested product contains small molecule RNA.
  • “indirectly derived” is a small molecule RNA derived from a gene other than the target gene, and the small molecule RNA has the same sequence as a digested product derived from the target gene described later, or, for example, a stringent described later. And those having high homology (70% or more, preferably 80% or more, and more preferably 90% or more) that can be hyper-predated under various conditions.
  • the origin of the target gene is not particularly limited, and any organism (eg, animal, plant, fungus, etc.) can be appropriately used depending on the purpose.
  • Double-stranded RNA containing the full length or a part of the nucleotide sequence of the gene of interest used in the present invention can be prepared using any method. For example, it can be prepared by RNA transcription of both the normal and reverse strands of double-stranded DNA containing the full-length nucleotide sequence of the gene of interest or a partial nucleotide sequence thereof, and mixing both products. .
  • the double-stranded DNA is prepared by a known method, for example, a method in which the full length or a part of the nucleotide sequence of the target gene is inserted into a vector, and the like.
  • the nucleotide sequence is preferably amplified by, for example, PCR.
  • a method for amplifying the full length or a part of the nucleotide sequence of the gene of interest by PCR is, for example, as follows. Cycle reaction (dissociation of a double-stranded template into a single strand, primer and primer) using a primer that is complementary to both ends of the nucleotide sequence and a heat-resistant DNA polymerase, using the full length or a part of the nucleotide sequence of the target gene as a template. By annealing the single-stranded template, forming a new double-stranded template by synthesizing the template complementary strand following the primer, and repeating the three steps of Nucleotides are added according to the base sequence, and the normal and reverse strands extend. This reaction By repeating, the full length or a part of the nucleotide sequence of the target gene is amplified.
  • the cloning vector is a plasmid derived from E. coli (eg, pBR322, pBR325, pAT153, pUC8, pUC12, pUC13, pGEM); derived from Bacillus subtilis Plasmids (eg, pUB 110, pTP 5, p C 1 94); yeast-derived plasmids (eg, p SH 19, p SH 15); bacteriophages such as ⁇ phage; retrovirus, vaccinia Animal viruses such as virus 4 and baculovirus can be used.
  • E. coli eg, pBR322, pBR325, pAT153, pUC8, pUC12, pUC13, pGEM
  • Bacillus subtilis Plasmids eg, pUB 110, pTP 5, p C 1 94
  • yeast-derived plasmids eg, p SH 19, p SH 15
  • bacteriophages such as ⁇
  • a method used in general cloning can be used as appropriate.
  • the full length of the nucleotide sequence of the target gene or its For example, a method may be used in which a part is cleaved, removed, and cloned by ligation with a vector. Initiation of transcription of RNA polymerases ( ⁇ ⁇ 7 RNA polymerase, SP 6 RNA polymerase, etc.) that can be contained in the cloned Jung vector, the full-length nucleotide sequence of the gene of interest is located downstream and forward of the DNA sequence. Alternatively, a part of the sequence is preferably inserted.
  • RNA transcription is performed by a method known per se.
  • the RNA transcription may be performed in vivo or in vitro, but is preferably performed in vitro.
  • In vitro RNA transcription is performed by a method known per se. Specifically, for example, the above-mentioned cloned plasmid is used as a saddle and the substrate (ribonucleotide (ATP, GTP, UTP, CTP, etc.)) and RNA polymerase are mixed in an appropriate buffer solution, RNA is transcribed by reacting at a temperature (37 ° C, etc.) for an appropriate time (about 1 to 5 hours).
  • substrate ribonucleotide (ATP, GTP, UTP, CTP, etc.)
  • RNA polymerase RNA polymerase
  • the reaction is preferably performed in the presence of a ribonuclease inhibitor in order to avoid degradation of RNA.
  • the plasmid is preferably linearized with an appropriate restriction enzyme in advance.
  • the restriction enzyme cleaves the full length of the nucleotide sequence of the target gene incorporated into the plasmid or a partial sequence thereof. Appropriate ones can be chosen so that they do not. Examples of the restriction enzyme include BamHI, Xbal, Sail, Accl, HincII, Pstl, Sphl, Hindlll. NotI, and the like.
  • RNA polymerase examples include T 7 RNA polymerase, SP 6 RNA polymerase and the like, and those corresponding to the transcription initiation DNA sequence possessed by the cloned vector are used.
  • the DNA in a cage shape is removed after RNA transcription in vitro.
  • the removal is preferably carried out, for example, by digestion with a DNase such as DNaseI.
  • RNA forward and reverse strands can also be prepared, for example, by chemical synthesis.
  • the chemical synthesis method include a phosphoramidite method, a phosphoric acid triester method, and an H-phosphonate method.
  • the normal and reverse strands are prepared by RNA transcription in the form of a non-limiting force DN A.
  • RNA transcript is preferably purified.
  • purification method include PAGE purification, purification using a simple column, phenol / chloroform extraction, ethanol precipitation, and the like.
  • RNA double-stranded nucleic acid
  • RNA normal- and reverse-stranded nucleic acid
  • the conditions for the high temperature heating are not particularly limited, but conditions of 70 ° C. to 100 ° C. for 3 minutes to 5 minutes are preferable.
  • the heat treatment temperature can be set as appropriate depending on other reaction conditions.For example, in the purification of RNA transcripts, when water is used for RNA elution from the column, heat treatment is performed in water that does not contain salts.
  • the heat treatment can be performed at about 75 ° C. Moreover, it is preferable to return to room temperature after heating.
  • the formation of double-stranded RNA of labeled strand and unlabeled strand is It is preferred that the unlabeled chain does not become an excess mole.
  • the RNase used in the present invention is not particularly limited, but an RNase I I I family protein such as Dicer having double-strand RNA cleavage activity (Dicer) is preferred.
  • RNase III family protein include dicers derived from mammals such as humans, RNase Ill I derived from Escherichia coli, psychrophilic bacteria, and the like.
  • the treatment conditions for RNase are not particularly limited because they vary depending on the RNase used. For example, when RNaseIl I derived from E. coli is used, it may be about 30 minutes to 1 hour at about 37 ° C.
  • the size of the double-stranded RNA fragment obtained by RNase treatment is not particularly limited, but is preferably 10-30 bases, more preferably 12-30 bases. Further, it may have a 2-3 base overhang at the 3 ′ end, and preferably has a 2 base overhang.
  • one or both strands of the digested product may be labeled in order to increase detection / quantitative sensitivity.
  • the label include, but are not limited to, fluorescence (FITC, rhodamine, Texas red, 6-carboxyfluorescein (FAM), tetrachrome mouth 6-carboxyfluorescein (TET), 2 , 7-Dimethoxy-1,4,5-Dichloro-6-Carboxyfluorescein (JOE), Hexachlorine 6-Carboxyfluorescein (HEX), 6_Carboxytetramethylrhodamine (TAMRA) Etc.) Labels, piotin labels, digoxigenin labels, radioisotope ( 32 P, 3 H, etc.) labels, etc.
  • the timing for labeling the digested product is not particularly limited, and it may be during RNA transcription or after RNase digestion.
  • labeling during RNA transcription for example, when RNA is transcribed in a test tube, a radiolabeled ribonucleotide (eg, [a- 32 P] UTP) is mixed in the test tube as a substrate. In the transfer reaction, those radiolabeled ribonucleotides are incorporated into RNA and labeled. RNA can be obtained. In addition, in double-stranded RNA, only one strand may be labeled, or both strands may be labeled.
  • a radiolabeled ribonucleotide eg, [a- 32 P] UTP
  • those radiolabeled ribonucleotides are incorporated into RNA and labeled.
  • RNA can be obtained.
  • double-stranded RNA only one strand may be labeled, or both strands may be labeled.
  • the digested product can be used in a method for detecting a small molecule RNA.
  • the digested product can be used as a probe for detecting a small molecule RNA.
  • the method for detecting a small molecule RNA using the digested product is not particularly limited.
  • an in situ hybridization, Northern plot method, etc. can be used, and the Northern blot method is particularly preferably used.
  • the Northern plot method can be carried out by a method known per se. Specifically, for example, RNA (including small molecule RNA) in a sample is subjected to agarose gel electrophoresis or polyacrylamide gel electrophoresis. Next, it is transferred to an appropriate membrane such as a nitrocellulose membrane, nylon membrane, PVDF membrane, etc., and solidified.
  • RNA and the probe comprising the digested fragment according to the present invention are hybridized under stringent conditions, and the presence / absence / intensity of the hybridization detects the presence / absence of the small molecule RNA derived from the target gene. Or quantify.
  • “Stringent conditions” refers to conditions under which a probe, primer or oligonucleotide hybridizes to its target sequence but does not hybridize to other sequences. That is, in the present invention, “stringent conditions” are conditions that enable specific and detectable hybridization between a small molecule RNA derived from a target gene and a probe comprising the digested fragment.
  • “Stringent conditions” refers to the ratio of guanine (G) and cytosine (C) (GC content), size, and hybridization composition (salt concentration, formaldehyde) in the nucleotide sequence that can be hybridized. Concentration), temperature, and other known conditions. That is, the higher the GC content of the nucleotide that can be hybridized and the larger the size, the more stable the hybridization. Therefore, the salt concentration in the composition of the hybridization buffer is decreased, the organic solvent concentration is increased, or the hybridization is increased. Increase stringency by raising the temperature of the dispersion. In the present invention, the hybridization is carried out under low stringency conditions.
  • Hybridization can be detected by a method known per se, and can be detected by, for example, autoradiography, although it depends on the type of label of the probe comprising the deleted fragment.
  • the specific band When a specific band is detected by the detection method, the specific band indicates the presence of a small molecule RNA. Since it has been suggested that small molecule RNA is involved in RNA i, the appearance of the specific band is also related to the regulation mechanism of expression of RNA i and the like by small molecule RNA in vivo. Indicates the possibility of being.
  • RNAI RNA-binding protein
  • the mutant species of a gene involved in RNA i eg, ago 1 gene mutant strain, rdpi gene mutant strain, dcrl gene mutant strain, etc.
  • a probe prepared by labeling only one strand of double-stranded RNA identify whether the small molecule RN A derived from the forward or reverse strand of the nucleotide sequence of the target gene exists. It is also possible. Specifically, a forward-labeled probe Alternatively, if a reverse band labeled probe is used and a specific band is detected only on one side and not on the other side, it can be said that there is a small molecule RN A derived from a strand complementary to the labeled strand.
  • the present invention also provides a reagent for detecting a small RNA, comprising as a probe a double-stranded RNA RNase digestion product containing the full length or a part of the nucleotide sequence of the gene of interest.
  • the reagent may contain a buffer solution, a standard solution, and the like.
  • the detection reagent it is not particularly limited.
  • small molecule RNA can be easily detected by the above detection method.
  • the present invention can also provide a small molecule RNA detection kit derived from the target gene, including an RNase digestion product of double-stranded RNA containing the full length or a part of the nucleotide sequence of the target gene.
  • the kit may contain a reagent for detecting a small molecule RNA, a reagent for preparing an RNase digestion product, an instruction describing a preparation protocol, and the like. These elements can be mixed in advance if necessary.
  • detection of small molecule RNA becomes easy. Since small molecule RNA is involved in RNA i, the kit is very useful for confirming and experimenting with medical techniques using the RNA i method. It is also possible to make a Northern plot kit containing the digested product.
  • double-stranded RNA containing the full length or a part of the nucleotide sequence of the gene of interest is subjected to RNase digestion, and the resulting digested product is used as a probe. Difficult techniques are not included, and there are no region-specific requirements or sequence selection failures like oligo RNA probes.
  • RNA probe labeled with only one strand since it is possible to prepare an RNA probe labeled with only one strand, a small molecule RNA derived from the full-length nucleotide sequence of the target gene or a part of its normal or reverse sequence can be obtained. It is easy to tell if it exists. From the above, the method of the present invention is extremely useful because it has very high experimental accuracy and detection sensitivity and is easy to operate.
  • PCR fragment of the centromeric sequence of the fission yeast gene see Nakaseko et al., EM BO Journal, Vol. 5, p. 1 0 1 1— 1 02 1, (1 986)
  • pGEM— T P ro me ga In both directions.
  • These two types of plasmids were linearized at the transcription termination site by treatment with Not I restriction enzyme, etc., and then transferred in vitro with 7 RNA polymerase in the presence of [a- 32 P] UTP. (MAX I script T7 kit, Amb ion). After DNase I treatment, the transcript was purified by Wizard SVG eland PCR Cien-up System (Promega).
  • Equal molar amounts of each purified product and RNase III buffer (Amb ion) were mixed, treated at 75 ° C for 5 minutes, and then slowly returned to room temperature to promote double-stranded RNA formation.
  • RNase III derived from E. coli; Am bion
  • RNA small molecule of fission yeast was purified and concentrated using mirVanami RNA I solation Kit (Amb ion) and separated by 15% polyacrylamide amide TBE gel electrophoresis containing 8M urea.
  • the gel was electrically transferred to a nylon membrane (Hy bond— XL, Amersham) and the RNA molecules were immobilized by UV cross-linking. After visualizing markers and tRNA molecules by methylene blue staining, the membrane region of interest was cut out and hybridization was performed with U 1 trahyb -0 1 igo (Amb ion). Hybridization with the RNA probe was carried out by reacting at 42 ° C for 16 hours or longer. 2 After rinsing twice with XSSC 0.5% SDS at 42 ° C for 30 minutes, the sigma on the membrane was washed with Typhoon 9200 (Amersham). Null was detected.
  • Figure 1 shows an example of detection of siRNA in a cell extract by performing a Northern plot using a centrisomal sequence known to produce siRNA in fission yeast clonal analysis as a probe.
  • the signals around 25 nucleotides seen in lanes 4 and 5 were lost due to mutations in the RNA i mechanism (lanes 1, 2, and 3), which means that the si RNA signal depends on the RNA i mechanism.
  • RNA i involving small molecule RNA becomes simple, and it is very useful as a method for confirming and experimenting with medical techniques using the RNA i method.

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Abstract

Ce procédé consiste à détecter facilement des petits ARN, et concerne un réactif destiné à être utilisé dans cette détection. Plus précisément, ce procédé consiste à détecter des petits ARN dérivés d’un gène cible en utilisant un produit de digestion obtenu par la digestion par une ribonucléase pour l’ARN double brin comprenant la longueur totale ou une partie de la séquence nucléotidique du gène cible ; et concerne un réactif destiné à être utilisé dans la détection des petits ARN qui comprend le produit de digestion.
PCT/JP2006/301074 2005-01-20 2006-01-18 Procede de detection des petits arn et reactif de detection des petits arn WO2006078042A1 (fr)

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WO2016130811A1 (fr) * 2015-02-11 2016-08-18 Biogen Ma Inc. Méthodes et compositions pour la détection d'acides nucléiques cibles

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WO2004057017A2 (fr) * 2002-12-18 2004-07-08 Third Wave Technologies, Inc. Detection de petits acides nucleiques

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WO2004057017A2 (fr) * 2002-12-18 2004-07-08 Third Wave Technologies, Inc. Detection de petits acides nucleiques

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LAGOS-QUINTANA M. ET AL.: "Identification of Novel Genes Coding for Small Expressed RNAs", SCIENCE, vol. 294, 2001, pages 853 - 858, XP002971837 *
LEE Y. ET AL.: "The nuclear RNase III Drosha initiates microRNA processing", NATURE, vol. 425, 2003, pages 415 - 419, XP002384567 *
YANG D. ET AL.: "Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells", PROC. NATL. ACAD. SCI. USA, vol. 99, no. 15, 2002, pages 9942 - 9947, XP002277296 *

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