WO2007018169A1 - Procédé de détection d’un site contenant de l’inosine dans l’arn - Google Patents

Procédé de détection d’un site contenant de l’inosine dans l’arn Download PDF

Info

Publication number
WO2007018169A1
WO2007018169A1 PCT/JP2006/315581 JP2006315581W WO2007018169A1 WO 2007018169 A1 WO2007018169 A1 WO 2007018169A1 JP 2006315581 W JP2006315581 W JP 2006315581W WO 2007018169 A1 WO2007018169 A1 WO 2007018169A1
Authority
WO
WIPO (PCT)
Prior art keywords
inosine
rna
site
cdna
chemically modified
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2006/315581
Other languages
English (en)
Japanese (ja)
Inventor
Tsutomu Suzuki
Masayuki Sakurai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Tokyo NUC
Original Assignee
University of Tokyo NUC
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 University of Tokyo NUC filed Critical University of Tokyo NUC
Publication of WO2007018169A1 publication Critical patent/WO2007018169A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to a method for detecting an inosine site in RNA. More specifically, the present invention relates to a method for detecting an inosine site by chemically modifying the inosine site in RNA.
  • RNA editing causes a change from A to I and changes the amino acid sequence by changing the codons on the mRNA.
  • Double-stranded RNA adenosine deaminase acting on RNA (ADAR) is an enzyme that specifically recognizes the double-stranded part of RNA and converts adenosine residues into inosine.
  • ADAR Double-stranded RNA adenosine deaminase acting on RNA
  • the glutamate receptor subunit GluR-B mRNA is a substrate of ADAR2, adenosine in exon 11 is edited to inosine, and the amino acid sequence changes from glutamine to arginine.
  • ADAR2 knockout mice do not undergo this editing and therefore cannot control the calcium permeability of glutamate receptors, causing epilepsy symptoms and premature death.
  • ADAR is known to show a wide range of substrate recognition ability for double-stranded RNA.
  • the editing is in sight.
  • ADAR2 is known to be able to induce variable splicing by editing the intron position of its mRNA from A to I, and to feedback control the expression level of ADAR2.
  • RNA editing may be related to the acquisition of complex neural circuitry in the brain.
  • Diseases resulting from abnormal RNA editing have also been reported.
  • GluR2 glutamate receptor subunit protein
  • RNA amplified as cDNA
  • I forms a base pair with C
  • mRNA is also reverse transcribed.
  • G is incorporated into the I-corresponding site in the cDNA after PCR amplification. Therefore, the editing site is a mixture of G or A / G on the cDNA, despite its basic strength on the genome.
  • the present invention can be easily distinguished from noise and SNP, can be analyzed using RNA alone as an analysis sample, can be analyzed even with a small amount of sample, and the background is kept low. It is possible to detect the presence of inosine with high sensitivity, and to provide a method for detecting an inosine site in RNA.
  • the present inventors have chemically modified the inosine site by treating RNA with a compound having an ⁇ , j8-unsaturated bond and an electron-withdrawing group. As a result, it was found that an inosine site in RNA can be detected, and the present invention has been completed.
  • RNA has an ⁇ , ⁇ unsaturated bond and an electron-withdrawing group.
  • a method for detecting an inosine site comprising the step of chemically modifying the inosine site by treatment with a compound.
  • the method of the present invention further comprises a step of synthesizing cDNA by subjecting the chemically modified RNA to a reverse transcription reaction, and a step of detecting an inosine site based on the synthesized cDNA. including.
  • the detection of the inosine site is the detection of the presence or absence of an inosine site, the quantification of the amount of inosine, and the identification of the region or inosine site containing Z or inosine.
  • the compound having an ex, j8-unsaturated bond and an electron-withdrawing group is
  • R 1 and R 2 each independently represent a hydrogen atom, a alkyl group having 1 to 6 carbon atoms, a phenol group, or a phenyl group having 1 to 6 carbon atoms
  • R Either 1 or R 2 may combine with E to form a ring
  • R 3 is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a full group, or an alkoxyl group having 1 to 6 carbon atoms.
  • the electron withdrawing group is CN, NO, SO H, CONH, COCH, COOCH,
  • COOC H COCH, COC H, or COC H.
  • the chemically-modified RNA is subjected to a reverse transcription reaction to synthesize cDNA, and then a cDNA amplification reaction is performed.
  • the inosinization site is detected by comparing a cDNA synthesized by subjecting RNA that has not been chemically modified to reverse transcription to a cDNA that has been chemically modified and synthesized by RNA.
  • the RNA is chemically modified by treating the RNA with an oc, ⁇ -unsaturated bond and an electron-withdrawing group, followed by chemical modification of the inosynthetic site, and then subjecting the chemically modified RNA to mass spectrometry. Detected inosine.
  • chemically modified inosine is detected by detecting the length of cDNA derived from the chemically modified RNA.
  • RNA is treated with a compound having an a, ⁇ unsaturated bond and an electron-withdrawing group. Then, after chemically modifying the inosinization site, the chemically modified inosine is detected by determining the base sequence of the chemically modified RNA or cDNA derived therefrom.
  • chemically modified inosine is detected by detecting cDNA derived from chemically modified RNA using a probe containing a sequence only upstream of the inosine synthesizing site of RNA.
  • the chemically modified inosine is detected by selectively extracting cDNA derived from chemically modified RNA and analyzing the sequence of the cDNA that has been stopped at the inosinization site.
  • an inosine moiety modifying agent in RNA comprising a compound having a,
  • a reagent kit for performing the above-described method of the present invention comprising the above-described modifying agent of the present invention.
  • the method for detecting an inosine site includes a step of chemically modifying an inosine site by treating RNA with a compound having an ⁇ , ⁇ -unsaturated bond and an electron-withdrawing group.
  • the type of RNA is not particularly limited and may be any of mRNA, rRNA, tRNA or non-coding (nc) RNA! /.
  • the method of the present invention it is possible to prove the presence of inosine in cDNA by detecting inosine-specific modification / inhibition of reverse transcription elongation, and it is also easy to distinguish between noise and SNP. is there. Furthermore, genomes are not required as analysis samples, and analysis can be performed with RNA alone, so even a small amount of valuable samples can be analyzed. For this reason, it is possible to identify an inosine site even if the gene structure such as a splice site or the coding region on the genome is unknown. In these respects, the method of the present invention is superior to the conventional matched-tissu method.
  • the knock ground can be kept very low, and there is an advantage that the presence of inosine can be detected and proved with high sensitivity.
  • detection in the method of the present invention is performed by a microarray. This makes it possible to comprehensively identify inosine sites in RNA in vivo.
  • the method of the present invention targets all RNA species and uses the inosine-specific chemical modification (Yoshida, M., et al.), which has been reported in the past, in that it uses the inhibition of reverse transcription strand elongation by inosine-specific chemical modification. Furuichi, Y "Ukita, T” and Kaziro, Y. (1967). The effect of cyan oetnylation on codon recognition of yeast tRNA containing inosine. Biochim Biophys Acta 149, 308-310).
  • RNA inosine-specific chemical modification
  • a reagent a, j8-unsaturated electron withdrawing group compound can be used.
  • the reaction mechanism basically proceeds by a mechanism called Michael addition (Fig. 3A).
  • the ⁇ -position carbon atom is positively charged due to the electron-withdrawing group of the modified reagent.
  • This carbon atom undergoes addition modification to inosine by electrophilic addition to the 1st nitrogen atom, which is the active amine of inosine.
  • the modifying reagent it is also possible to isolate an RNA molecule containing inosine specifically for the added functional group.
  • Examples of the compound having an a, ⁇ unsaturated bond and an electron-withdrawing group include:
  • R 1 and R 2 each independently represent a hydrogen atom, a alkyl group having 1 to 6 carbon atoms, a phenol group, or a phenyl group having 1 to 6 carbon atoms
  • R Either 1 or R 2 may combine with E to form a ring
  • R 3 is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a full group, or an alkoxyl group having 1 to 6 carbon atoms.
  • reverse transcription can be performed on RNA that has been chemically modified specifically for inosine.
  • C cytidine
  • reverse transcriptase reverse transcriptase
  • the reverse transcription reaction can be performed by a conventional method using a primer, chemically modified RNA ( ⁇ type), four kinds of dNTPs, reverse transcriptase and the like. That is, the reverse transcription reaction can be performed by mixing the above reagents in an appropriate reaction solution (for example, a buffer solution containing an appropriate salt) and incubating at a predetermined temperature for a fixed time.
  • an appropriate reaction solution for example, a buffer solution containing an appropriate salt
  • RNA is subjected to a reverse transcription reaction to synthesize cDNA and then a cDNA amplification reaction is performed.
  • reverse transcription and amplification can be performed by a series of operations using the RT-PCR method.
  • it can also be performed by transcription amplification using reverse cDNA-cRNA as a cage.
  • RNA is chemically modified, and RNA is subjected to the reverse transcription reaction to synthesize cDNA, which can be used for the control in the following detection step.
  • the presence or absence of chemical modification by inosine may be detected by mass spectrometry, or the reverse transcribed strand that is inhibited from elongation is detected and compared with the presence or absence of inosine chemical modification.
  • the inosine site in RNA may be detected.
  • the detection target information includes its length, base sequence, or amount.
  • the inosine site is chemically modified by treating RNA with a compound having an ⁇ , ⁇ unsaturated bond and an electron-withdrawing group, and then chemically modified.
  • chemically modified inosine sites can be detected.
  • the chemically modified RNA can be used as a sample! /, but preferably the chemically modified RNA is degraded to nucleosides with RNase and phosphatase. After that.
  • the RNase used here is not particularly limited as long as it can be decomposed to nucleoside.
  • NucleasePl can be used.
  • the phosphatase is not particularly limited, and the species of origin such as nocteria is not limited.
  • Bacterial Alkaline Phospatase derived from E. coli can be used.
  • RNA is treated with acrylonitrile as a modifier for the inosine site, and then degraded to nucleosides.
  • a nucleotide sample derived from non-chemically modified RNA and reduce the inosine peak (mZz 269) or the appearance of the cyanoethylated inosine peak (mZz 322) in liquid chromatography Z mass spectrometry (LCZMS)
  • LCZMS liquid chromatography Z mass spectrometry
  • the region containing inosine may be identified by using the chemically modified RNA as a sample.
  • the chemically modified RNA is fragmented with an appropriate RNase.
  • the RNase used here is not particularly limited as long as RNA can be fragmented to a length of several bases to about 100 bases, such as G-specific RNase T1.
  • RNA is treated with acrylonitrile as a modifier of the inosine site, and then decomposed into RNA fragments. Compare this sample with an RNA fragmentation sample derived from non-chemically modified RNA and reduce the peak of the fragment containing inosine or the peak of the fragment containing cyanoethylated inosine (LC) in liquid chromatography Z mass spectrometry (LCZMS).
  • LCZMS liquid chromatography Z mass spectrometry
  • chemical modification stops the reverse transcription strand specifically at the inosine site, and the 3 ′ end force of the primer used for reverse transcription is extended. It is possible to detect the inosinylated site from the degree or the like. According to this method, it is possible to detect the presence or absence of an inosine site, to quantify the amount of inosine, and to identify a region containing Z or inosine or an inosine sputum site.
  • a compound having an ⁇ , ⁇ unsaturated bond and an electron-withdrawing group by determining the base sequence of chemically modified RNA or cDNA derived therefrom. Can detect chemically modified inosine. In the presence of chemically modified inosine as described above, the reverse transcription reaction stops before the site of inosine and a short cDNA is generated. By determining the base sequence of this cDNA, it is possible to detect the inosine site where the extension of the cDNA has stopped. That is, according to this method, it is possible to detect the presence or absence of an inosine site, to quantify the amount of inosine, and to identify a region or inosine site containing Z or inosine.
  • cDNA derived from chemically modified RNA is detected by hybridization using a probe containing a sequence only upstream of the inosinization site of RNA.
  • inosine chemically modified by a compound having an a, j8-unsaturated bond and an electron-withdrawing group can be detected.
  • the reverse transcription product will stop at the site of elongation reactivity S inosine and complement the upstream sequence of the mRNA.
  • a short cDNA is generated that lacks.
  • cDNA derived from non-chemically modified RNA when used, cDNA derived from chemically modified RNA containing inosine does not react with probes containing sequences only upstream of the inosine site of RNA.
  • the cDNA reacted with the probe can be detected.
  • the site of inosine can be detected by the presence or absence of a signal.
  • This method may be performed in combination with a microarray.
  • the whole region force of the mRNA is also extended using a random primer.
  • This reverse transcription product stops at the site of elongation reaction inosinization and produces a short cDNA lacking a sequence complementary to the upstream sequence of mRNA.
  • cDNA derived from non-chemically modified RNA when used, cDNA derived from chemically modified RNA containing inosine does not react with probes containing sequences only upstream of the inosine site of RNA. Signals reacted with the probe can be detected from cDNA derived from. Thus, the inosine wrinkle site can be detected by the presence or absence of the signal or the decrease.
  • This cDNA may be amplified in some cases.
  • the probe it is possible to use a tiling array that covers the entire genome or a complementary sequence for a transcription product, or a primer set for quantitative PCR.
  • a, j8-unsaturated bond and electron-withdrawing property are obtained by detecting only the cDNA whose elongation is stopped at the inosine site, using chemically modified RNA as a saddle type. Inosine chemically modified by a compound having a group can be detected. For example, after chemical modification of mRNA, the random region is used to extend the whole region force of mRNA. At the beginning of this extension reaction, a reaction solution containing dNTPs is used. In reverse transcripts, the elongation reaction stops at the inosinization site, and a short cDNA is generated lacking a sequence complementary to the upstream sequence of mRNA.
  • the reverse transcript extension reaction proceeds smoothly and a long cDNA is generated. Then, in the middle of the extension reaction, replace dNTP with Didiox (dd) NTP, or add an excessive amount of ddNTP to dNTP. By this operation, ddNTP is incorporated into the 3 ′ end of the reverse transcript that was extended outside the inosine site, and the extension was stopped. In the resulting cDNA, the 3 'end of the cDNA stopped at the inosine site has a 3'-OH group. The 3' end of the cDNA extended outside the inosine site becomes a 3'-H group.
  • dd Didiox
  • a ddNTP derivative is used during the extension reaction. As a result, this derivative is present only in cDNA with a 3'-H group at the 3 'end. Subsequently, by using a carrier that specifically binds to this derivative, it is possible to remove the extended cDNA other than the inosine site from the cDNA population. Next, the cDNA stopped at the inosinization site remaining in the system is converted into double-stranded DNA. Second, the adapter DNA or RNA is ligated to the 3 'end of the cDNA using ligase.
  • the adapter was not ligated to the extended cDNA other than the inosine site having 3'-H group at the 3 'end, and as a result, at the inosinization site having 3'-OH group at the 3' end.
  • the adapter is only ligated to the stopped cDNA.
  • Double-stranded the cDNA using primers complementary to the adapter sequence.
  • the cDNA extended at a site other than the inosine site with 3'-H group at the 3 'end does not have a poly dN sequence added to the cDNA, and as a result, an inosine with a 3'-OH group at the 3' end.
  • Poly dN sequences are added only to cDNAs that are stopped at the anchor site.
  • the cDNA is then double-stranded using primers complementary to the poly dN sequence.
  • the double-stranded cDNA is amplified and the sequence thereof is analyzed.
  • this double-stranded cDNA can be controlled by adding a restriction enzyme recognition sequence to the 5 'end of the random primer during reverse transcription and the 5' end of the primer during double-stranded DNA synthesis.
  • Protruding ends can be created by degradation with a restriction enzyme.
  • a protruding end can be created at the end in a double-stranded DNA amplification step using PCR.
  • the sequence analysis can be carried out by incorporating double-stranded DNA with protruding ends into a cloning vector such as a plasmid.
  • a plurality of sequence analyzes can be performed at a time by linking double-stranded DNAs having protruding ends and incorporating them into a force-cloning vector.
  • An inosine site exists immediately upstream of the RNA corresponding to the finally detected sequence.
  • the present invention relates to a modifying agent that modifies inosine in RNA, and also to a reagent kit for detecting an inosine moiety containing the modifying agent.
  • the modifying agent that modifies inosine in RNA contains a compound having an a, j8-unsaturated bond and an electron-withdrawing group. Since “ ⁇ ,) 8-compound having an unsaturated bond and an electron-withdrawing group” is the same as the compound that can be used in the above method, the description thereof is omitted.
  • a reagent kit for detecting the inosine bud site can be designed according to the detection method.
  • Rnase or phosphatase can be combined in addition to the inosine modifying agent.
  • a reverse transcriptase for generating a reverse transcript or a buffer for reverse transcription as necessary may be combined. it can.
  • a set of reagents for base sequence determination can be combined with the inosine modifier.
  • a set of reagents for determining the base sequence for example, polymerase, dNTP, ddNTP, reaction buffer and the like can be mentioned as examples.
  • a probe based on the upstream sequence of RNA to be detected, a hybridization buffer, a washing solution, and the like can be combined in addition to the inosine modifier.
  • the probe set may be provided fixed to the microarray.
  • RNA fraction to be analyzed 10 ⁇ g is dissolved in 30 ⁇ 1 of CE buffer (41% ethanol, 1.1 M TE A-acetic acid (pH 8.6)), and then 15.2 M acrylonitrile (Tokyo Kasei) 4 1
  • CE buffer 41% ethanol, 1.1 M TE A-acetic acid (pH 8.6)
  • Tokyo Kasei 15.2 M acrylonitrile
  • CE- acrylonitrile
  • Example 2 Inosin that was specifically cyanoylated was confirmed by liquid chromatography / mass spectrometry (LC / MS method).
  • LC / MS method liquid chromatography / mass spectrometry
  • the inosine peak (m / z 269) observed with CE- decreased with the reaction time of cyanobacteria, but was not detected with CE- instead.
  • the peak of cyanoethylated pseudouridine (CE- ⁇ ) (m / z 298) also increased with the time of cyanoethylation, but the reaction rate was slow (Fig. 4B, C, D, F). About 60% of unreacted pseudouridine (m / z 245) was detected. Since no change was detected for other bases, this cyanoethyl reaction is inosine-specific.
  • Example 3 Detection of inosine by the primer extension method
  • Inosine-specific chemical modification reaction was performed on mouse brain total RNA according to the reaction procedure described in Example 1. 25 g of RNA after the reaction and 0.4 pmol of DNA primer labeled with 32 P at the 5 ′ end were dissolved in 51, incubated at 65 ° C. for 3 minutes, and then cooled to room temperature.
  • the DNA primer used here was designed downstream of the Q / R A-to-I RNA editing site in mouse glutamate receptor B mRNA (99% of A has been edited into inosine).
  • the nucleotide sequence is 5′-GATCTTGGCGAAATATCGCA TC-3 ′ (SEQ ID NO: 1).
  • Samples cooled to room temperature were ddATP, 0.5 mM DTT, 3 mM MgCl, 175 mM KCl, 50 mM Tris—HCl (pH 8) at the same concentration as 150 M dGTP, dCTP, dT TP.
  • Example 4 Detection of inosine by direct sequencing (Inosine Chemical Erasing method; ICE method)
  • Inosine-specific chemical modification reaction was performed on mouse brain total RNA according to the reaction procedure described in Example 1.
  • a forward primer and a reverse primer were designed to amplify 250 nt of the region centering on five A-to-I RNA editing sites in mouse serotonin receptor 2c (5HT2cR) mRNA.
  • the base sequence of the DNA primer used here is a forward primer; 5′-ATGGAGAAGAAACTGCACAATG-3 ′ (SEQ ID NO: 2), a reverse primer; 5-ATGATGGCCTTAGTCCGCGAAT-3 ′ (SEQ ID NO: 3). Both primers were mixed with 2.5 pmol each and mouse brain total RNA after reaction.
  • the total RNA amounts obtained here are respectively CE- (control without acrylonitrile) condition; 10 ng, CE + 15 min condition; 50 ng, CE + 30 min; 50 ng.
  • the mixed sample was subjected to RT-PCR using a Superscript III One-Step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) at a scale of 12.5 ⁇ 1. Reaction was reverse transcription; 55 ° C for 30 minutes, heat denaturation; after 94 ° C for 2 minutes, 38 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 68 ° C for 30 seconds were performed.
  • the reaction solution 51 was treated with ExoSAP-IT (Usb) to purify the mold.
  • the reverse transcribed strand extended from the reverse primer incorporates C into the inosine site and reaches the forward primer complementary site.
  • cyanobyl when cyanobyl is applied, the reverse transcription chain extension to the inosine-containing mRNA stops before the inosine site, does not reach the forward primer complementary site, and is not amplified by subsequent PCR.
  • Example 5 Detection of inosine by microarray (ICE-microarray method)
  • Microarray analysis was performed using human brain total RNA (CE ⁇ ) and (CE + 15min) prepared in Example 1. Oligo dT primer with T7 promoter sequence [T7 (dT) c
  • the reverse transcription reaction using DNA primer was requested from Hitachi Soft's DNA Chip Laboratory, and the company's AceGene Human Olig Chip 30K IChip version was used.
  • the company uses Amino Allyl Message Amp aRNA kit (Ambion) for reverse transcription to cDNA synthesis and aminoallyl-labeled aRNA (amplified RNA) transcription amplification.
  • the principle of this method is as follows.
  • the setting is made when a primer for reverse transcription is located downstream (3 ') of the inosine site and a probe on the array is designed upstream (5') of the inosine site.
  • the reverse transcribed strand extended from the reverse primer incorporates C into the inosine site and extends.
  • Example 6 Detection of inosine by real-time PCR
  • the reverse transcription reaction was performed using the random primer with the mouse brain total RNA (CE ⁇ ) and (CE + 15min) prepared in Example 1 as a saddle type.
  • CE- 500 ng of RNA, N9 random primer (5 and AGCAGAGGATTGACGACTACAGNNNNNNN NN-3 ') (SEQ ID NO: 4) with adapter sequence and reaction solution containing 250 ⁇ g and dNTP (20 ⁇ l final concentration 2 ⁇ )
  • a reaction solution 131 containing 1 was prepared.
  • a reaction solution containing 500 ng of RNA and 250 g of random primer and a reaction solution containing dNTP (final concentration 500 ⁇ on a 20 ⁇ 1 scale) 13 ⁇ 1 was prepared.
  • the reaction solution was heat denatured at 65 ° C for 2 minutes, and 0.1 M DTT 1 1, 40 U / ⁇ 1 RNaseOUT (Invitrogen 1 ⁇ ⁇ , 5x First strand buffer (Invitrogen, 250 mM Tris-HCl ( pH 8.3), 375 mM KC1, 15 mM MgCl) 4 ⁇ 1, 200 U / ⁇ 1 SuperScriptlll RT (
  • Invitrogen 1 1 was added and incubated at 25 ° C for 5 minutes, followed by an extension reaction at 50 ° C for 30 minutes. After the reaction, the enzyme was inactivated by incubation at 70 ° C for 15 minutes. To this reaction solution, 2 ⁇ l of IN NaOH was added and heated at 70 ° C for 10 minutes to degrade RNA, and then IN HC1 2 1, 1M Tris-HCl (pH 7.6) was added and neutralized.
  • 1st strand cDNA was purified using S-300 HR column (GE Healthcare) and QIA quick nucleotide removal kit (QIA GEN) in this order.
  • a reaction solution 201 containing dCTP, 400 U / ⁇ 1 terminal deoxynucleotidyl transferase (Roche) 1 ⁇ 1 was prepared. The reaction was carried out at 37 ° C for 15 minutes and incubated at 65 ° C for 10 minutes to inactivate the enzyme.
  • PCR was performed using 2 ⁇ 1 of the cDNA solution after the above reaction.
  • Primer complementary to dC-tail (5 and AGCAGAGGATTGACGACTACAGGGGGGGGGGGGGGHN-3,) (SEQ ID NO: 5) 2.5 pmol for amplification.
  • Primer (5'-AGCAGAGGATTGACGACTACAG-3,) (SEQ ID NO: 6) 2.5 pmol, 1 mM MgSO, 200 ⁇ M dNTPs, 1 U KOD-Plus (Toyo
  • lx KOD-Plus reaction buffer containing 50 ⁇ 1 was prepared. First, 94 ° C for 2 minutes 15 seconds, 50 ° C for 2 minutes, 68 ° C for 2 minutes, dsDNA synthesis, 94 ° C for 15 seconds, 60 ° C for 30 seconds, 68 ° C for 90 seconds This cycle was performed 16 to 24 cycles. After confirming this amplified product by electrophoretic movement, the stage 1 or 2 cycles before the amplification reached the plateau was determined as the optimal cycle. The PCR product in this cycle was purified using the QIA quick PCR purification kit.
  • PCR was performed using 1 ⁇ 1 of the PCR reaction solution from the first round.
  • Primer for amplification (5'-AGCAGAGGATTGACGACTAC AG-3,) (SEQ ID NO: 7) 3 pmol, 1 mM MgSO, 200 ⁇ ⁇ dNTPs, 1U KOD- Plus (Toyobo ), 50 ⁇ 1 of reaction solution containing lx KOD-Plus reaction buffer was prepared.
  • the reaction was performed by heat denaturation at 94 ° C for 2 minutes, followed by 4 to 12 cycles of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 68 ° C for 90 seconds. After confirming this amplification product by electrophoresis, the stage before the cycle at which amplification reached a plateau was determined as the optimal cycle.
  • the PCR product in this cycle was purified using the QIA quick PCR purification kit.
  • the reaction was analyzed using SYBR Premix Ex Taq (Takara) and LightCycler 480 (Roche). The amplification was performed on a 20 ⁇ 1 scale using 4 pmol each of the forward primer and reverse primer of each primer set and 5 ng of the amplified and purified PCR product dsDNA.
  • Each primer sequence used is as follows.
  • MGluRBQRr-1050 (CAGTCACACTGACATTCATTCC) (SEQ ID NO: 8)
  • MGluRBQRf-1000 (ATGCTGTCCCTTACGTGAGTC) (SEQ ID NO: 9)
  • MGluRBQRr-750 GCATTCTTTGCCACCTTCATTC (SEQ ID NO: 10)
  • MGluRBQRr-300 CCGTAGTCCTCACAAACACAG
  • SEQ ID NO: 18 MGluRBQRf-250 (TGTGGACTTATATGAGGAGTGC)
  • SEQ ID NO: 19 MGluRBQRr-200 (GAGCCAGAGTCTAATGTTCCAT)
  • SEQ ID NO: 20 MGluRBQRf-150 (GAGGATCTGTCTAAGCAAACAG)
  • SEQ ID NO: 21) MGluRBQRr-100 GTCCTGTGTAGGATCGTGTGATGTGGGTGTC
  • SEQ ID NO: 23 MGluRBQRr-25 (GATCTTGGCGAAATATCGCATC)
  • SEQ ID NO: 24 MGluRBQRf + 25 (GTTTTCCTTGGGTGCCTTTATG)
  • SEQ ID NO: 25 MGluRBQRr + 5 (CATAAAGGCACCCAAGGAAAAC)
  • SEQ ID NO: 26 MGluRBQRTGATGARATAT +60 (TCACTACTTTGTGTTTC
  • M5HT2cRr-400 (TTTCTTCTTTCGACGTGGCTTC) (SEQ ID NO: 48)
  • M5HT2cRr-200 (CAACGGGATGAAGAATGCCAC) (SEQ ID NO: 52)
  • M5HT2cRf + 25 (TATCGCTGGACCGGTATGTAG) (SEQ ID NO: 57)
  • M5HT2cRr + l (CATACCGGTCCAGCGATATG) (SEQ ID NO: 58)
  • M5HT2cRf + 50 (TTTTCAACTGCGTCCATCATGC) (SEQ ID NO: 59)
  • M5HT2cRr + 150 (TGACAAGTAGTCCCACCAGC) (SEQ ID NO: 62)
  • M5HT2cRf + 200 (TCTTAATGTCCCTAGCCATTGC) (SEQ ID NO: 63)
  • M5HT2cRf + 300 (CCAGCACTTTCAATAGTCGTG) (SEQ ID NO: 65)
  • M5HT2cRr + 350 (CCACCATCGGAGGAATTAAAAG) (SEQ ID NO: 66)
  • M5HT2cRf + 400 (GTCCAGTAGCAGCTATAGTAAC) (SEQ ID NO: 67)
  • M5HT2cRr + 450 (CCAGGTTCACCATTATTGCTTC) (SEQ ID NO: 68)
  • M5HT2cRf + 500 (CCCAATTCTCAGTTTGAAACTGG) (SEQ ID NO: 69)
  • M5HT2cRr + 1000 (TTCTTCTTTGAAGGCCCCTAAC) (SEQ ID NO: 70)
  • M5HT2cRf + 1050 (TCTTCGTCCGCTTAGAATAGTG) (SEQ ID NO: 71)
  • T for the forward primer and "r" for the reverse primer are shown after the gene name.
  • the amplification region including the length of the primer was approximately 50 bp.
  • reverse transcription reaction is performed using a random primer with an adapter sequence added to the 5 'end using RNA as a saddle.
  • RNA untreated (CE-) RNA
  • 1st strand cDNA is generated equally over the entire region of RNA.
  • CE + chemically modified
  • the extension of cDNA stops just before the inosine site, so the amount of complementary cDNA just upstream of the inosin site is the same as the stopped cDNA amount. descend.
  • terminal deocynucleotidyl transferase (Roche) was used to attach a poly dC chain to the end of the cDNA, and this cDNA was doubled with a primer complementary to the adapter sequence and an oligo dG primer with the adapter sequence added. Perform chaining and PCR amplification. For the final product, the amount of cDNA in each region of RNA is compared between CE- and CE + using a tiling array probe or a primer set for real-time PCR that mimics a tiling array.
  • the signal intensity ratio of CE + / CE- decreases in the region containing the inosynthetic site or immediately upstream (5 'side), reflecting the amount of 1st strand cDNA in the CE + RNA stopped. Conceivable. In other words, when the signal intensity of CE + decreases compared to the signal intensity of CE-, it can be said that inosine exists near the quantified region!
  • mouse glutamate receptor B mRNA and the amount of cDNA in each region were comparatively quantified with CE- and CE + (Figs. 10 and 11).
  • the intensity ratio of CE + / CE- was taken, and a log with 2 as the base was calculated. Furthermore, correction was performed by subtracting the average of log values on the same mRNA from the log value in each region. The log value is shown on the vertical axis and the position of each region is shown on the horizontal axis. The graph also shows the values when the first PCR product was in a saddle shape and when the second PCR product was in a saddle shape.
  • inosine sites At present, most of the inosine sites, said to be more than 12000, have not been identified. Given that inosine is abundant in brain mRNA, it is thought to be associated with abnormalities in mental disorders (schizophrenia, panic syndrome, bipolar disorder, autism, etc.). If inosin fistula sites clearly associated with these diseases can be identified by the method of the present invention, very important knowledge can be obtained in elucidating the onset mechanism of the disease and considering a therapeutic method. In addition, it is possible to estimate the risk of developing lifestyle-related diseases and various diseases such as SNP by analyzing the fluctuations of the inosin cocoon site for each individual.
  • FIG. 1 shows detection of inosine by the matched-tissue method.
  • FIG. 2 shows the identification of inosine ⁇ site by inosine-specific cleavage (Morse, D. P., and Bass, BL (1997). Detection of inosine in messenger RNA by inosine-specin c cleavage. (Quoted from Biochemistry 36, 8429-8434).
  • FIG. 3 shows an inosine-specific chemical modification reagent.
  • FIG. 4 shows the detection of inosine after the cyanoethyl ester reaction by mass spectrometry.
  • FIG. 5 shows inosine detection by the primer extension method.
  • Figure 6 shows the principle of the ICE method.
  • FIG. 7 shows the detection of inosine by the ICE method.
  • FIG. 8 shows inosine detection by microarray.
  • FIG. 9 shows the detection results of mRNA containing inosine by microarray.
  • FIG. 10 shows the reverse transcription reaction using a random primer and the detection of inosine using a tiling array.
  • the gray area indicates the probe complementary area on the tiling array, and the amount of cDNA containing this area is measured by each probe.
  • the hatched triangle indicates the amount of probe that disappears due to chemical modification of inosine.
  • FIG. 11 shows the quantitative results of real-time PCR of cDNA after reverse transcription reaction with random primers. After reverse transcription using random primers, poly dC chain is added to cDNA, cDNA is double-stranded, mouse glutamate receptor B and serotonin receptor -2C in the product of the first PCR and second PCR Compared.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention concerne un procédé de détection d’un site contenant de l’inosine dans l’ARN, susceptible de distinguer facilement entre le site contenant de l’inosine et un bruit ou du SNP, que l’on peut réaliser uniquement en utilisant de l’ARN comme échantillon à analyser, capable d’analyser même sur une petite quantité d’échantillon, de réduire le fond de détection à un faible niveau, et de détecter la présence d’inosine avec une grande sensibilité. Le procédé comprend la phase de traitement d’ARN avec un composé ayant une liaison α,β insaturée et un groupe attracteur d’électrons pour modifier chimiquement un site contenant de l’inosine dans l’ARN.
PCT/JP2006/315581 2005-08-08 2006-08-07 Procédé de détection d’un site contenant de l’inosine dans l’arn Ceased WO2007018169A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005229335A JP2008259425A (ja) 2005-08-08 2005-08-08 Ｒｎａ中のイノシン化部位の検出方法
JP2005-229335 2005-08-08

Publications (1)

Publication Number Publication Date
WO2007018169A1 true WO2007018169A1 (fr) 2007-02-15

Family

ID=37727352

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/315581 Ceased WO2007018169A1 (fr) 2005-08-08 2006-08-07 Procédé de détection d’un site contenant de l’inosine dans l’arn

Country Status (2)

Country Link
JP (1) JP2008259425A (fr)
WO (1) WO2007018169A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014136870A1 (fr) * 2013-03-08 2014-09-12 国立大学法人熊本大学 Procédé de détection simple pour la modification d'arn et procédé de détection du diabète de type ii à l'aide dudit procédé de détection
CN115125292A (zh) * 2022-06-02 2022-09-30 武汉大学 一种内切酶辅助的rna中肌苷修饰的单碱基分辨率定位分析方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MENGEL-JORGENSEN J. ET AL.: "Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry", NUCLEIC ACIDS RES., vol. 30, no. 23, 1 December 2002 (2002-12-01), pages E135, XP003008728 *
MORSE D.P. ET AL.: "Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA", PROC. NATL. ACAD. SCI. U S A, vol. 96, no. 11, 25 May 1999 (1999-05-25), pages 6048 - 6053, XP002336889 *
SUZUKI T.: "RNA Shushoku no Sekai-Misugosarete iru RNA no Shitsuteki na Joho", SEIKAGAKU, vol. 77, no. 12, 25 December 2005 (2005-12-25), pages 1481 - 1496, XP003008730 *
YOSHIDA M. ET AL.: "Selective Modifications of Inosine and phi-Uridine with Acrylonitrile out of the Other Ribonucleosides", J. BIOCHEM. (TOKYO), vol. 57, no. 6, 1965, pages 818 - 821, XP003008727 *
YOSHIDA M. ET AL.: "The effect of cyanoethylation on codon recognition of yeast tRNA containing inosine", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - NUCLEIC ACIDS AND PROTEIN SYNTHESIS, vol. 149, no. 1, 1967, pages 308 - 310, XP003008729 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014136870A1 (fr) * 2013-03-08 2014-09-12 国立大学法人熊本大学 Procédé de détection simple pour la modification d'arn et procédé de détection du diabète de type ii à l'aide dudit procédé de détection
JPWO2014136870A1 (ja) * 2013-03-08 2017-02-16 国立大学法人 熊本大学 Ｒｎａ修飾の簡易検出法、及び該検出法を用いた２型糖尿病の検査方法
US10526654B2 (en) 2013-03-08 2020-01-07 National University Corporation Kumamoto University Simple detection method for RNA modification, and method for detecting type-II diabetes using said detection method
CN115125292A (zh) * 2022-06-02 2022-09-30 武汉大学 一种内切酶辅助的rna中肌苷修饰的单碱基分辨率定位分析方法

Also Published As

Publication number Publication date
JP2008259425A (ja) 2008-10-30

Similar Documents

Publication Publication Date Title
KR100527265B1 (ko) 폴리뉴클레오티드 서열의 선형 등온 증폭을 위한 방법 및조성물
JP5619702B2 (ja) 二重特異性オリゴヌクレオチドを使用した方法
JP6382189B2 (ja) Ｒｎａテンプレートから開始する等温ｄｎａ増幅用のキット
JP2004537257A (ja) 核酸配列の検出および／または定量のための、方法およびプローブ
CN113748216B (zh) 一种基于自发光的单通道测序方法
EA035092B1 (ru) Синтез двухцепочечных нуклеиновых кислот
US20240076633A1 (en) Pcr buffer composition for increasing activity of dna polymerase with increased gene mutation specificity
CN114286867B (zh) 一种基于发光标记物光信号动力学及二次发光信号对多核苷酸进行测序的方法
NZ533221A (en) Oligonucleotides having dual functions for generating specific hybridisation and verifying hybridisation results quantitatively
US20090023151A1 (en) Method For The Labeling And Detection Of Small Polynucleotides
US20240368686A1 (en) Methylation sequencing methods and compositions
WO2007018169A1 (fr) Procédé de détection d’un site contenant de l’inosine dans l’arn
CN114008063A (zh) 用于dna和rna聚合和测序的修饰的核苷酸和方法
JP5692954B2 (ja) Ｈｉｖ−１を検出しそして定量するためのアッセイ
US20080050728A1 (en) MITIGATION OF Cot-1 DNA DISTORTION IN NUCLEIC ACID HYBRIDIZATION
US10036053B2 (en) Determination of variants produced upon replication or transcription of nucleic acid sequences
EP1688493B1 (fr) Procede de detection de polymorphisme genetique
KR102079963B1 (ko) 소거 프로브 기반 염색체 수적이상 검출방법 및 염색체 수적이상 검출용핵산의 조성물
WO2006070666A1 (fr) Procede de detection simultanee de polymorphismes genetiques
JP4453910B2 (ja) 遺伝子多型の検出方法
JP2005287447A (ja) プローブ及びプライマーおよびこれらを用いた核酸断片相補鎖合成法
HK40055932A (en) Single-channel sequencing method based on self-luminescence
WO2009029875A1 (fr) Procédé pour le marquage et la détection de petits polynucléotides

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06782423

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP