WO2007018169A1 - Method for detection of inosine-containing site in rna - Google Patents

Abstract

Disclosed is a method for detection of an inosine-containing site in RNA, which can distinguish the inosine-containing site from a noise or SNP readily, can be performed only using RNA as a sample to be analyzed, can analyze even on a small quantity of a sample, can reduce the detection background at a low level, and can detect the presence of inosine at a high sensitivity. The method comprises the step of treating RNA with a compound having an α,β-unsaturated bond and an electron-attracting group to chemically modify an inosine-containing site in the RNA.

Description

 Specification

 Method for detecting the inosine site in RNA

 Technical field

 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.

 Background art

 [0002] Modification by deamination (deamination) is called RNA editing. 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. There are three types of mammals, ADAR1, ADAR2, and ADAR3. 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. It is known that 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. In addition to GluR-B, AD to A from I through serototone receptor (5-HT2c), potassium channel (Kvl.l), etc. The editing is in sight. Interestingly, 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. The

[0003] It is estimated that brain mRNA in which ADAR is highly expressed contains inosine at a frequency of 17000 bases, and human mRNA is predicted to contain a large amount of unknown editing sites. It had been. Recently, analyzes using bioinformatics methods have reported a large number of editing candidates from A to I in the repetitive sequence of the Alu factor in the UTR of human mRNA. Only a few of these have been experimentally proven 1 The estimate that inosine exists in more than 12000 sites in 600 genes suggests that editing from A to I plays a major role in the control of the gene's global expression. Only primates have the Alu factor, and ADAR is highly expressed in the brain, and considering the increase in transcriptome complexity due to editing from A to I is highly evolved. This may be related to the acquisition of complex neural circuitry in the brain. Diseases resulting from abnormal RNA editing have also been reported. In malignant glioma and amyotrophic lateral sclerosis, editing of glutamate receptor subunit protein (GluR2) from A to I is markedly reduced. Thus, inosine in mRNA gives a qualitative change to RNA, and is highly likely to be closely related to higher life phenomena.

 [0004] The matched-tissue method has been the most common method for detecting the RNA editing site from A to I (where 1 is present) (Fig. 1). In this method, RNA (amplified as cDNA) derived from the same tissue of the same individual is compared with the sequence of the corresponding region in the genome. Since 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. However, in this method, nonspecific amplification of PCR, genomic contamination, SNP between sequence allele 'noise' alleles (A1 lele SNP) genes It is difficult to determine the target inosine-derived G mixture. In addition, in the case of exon boundary regions or regions where pseudogenes exist, it is difficult to determine the genomic region corresponding to RNA, so this method itself cannot be used. Furthermore, since the inosine site identified by this method is detected as G on the sequence, it does not strictly prove the existence of inosine. In addition, since it is necessary to prepare both genome and RNA from the same individual, there is a difficulty in restricting the samples that can be analyzed.

[0005] In addition, there have been two reports on chemical modifications of inosine in the field of molecular biology. The first is the inosine-specific cleavage method for the identification of inosine sites in RNA (Morse, DP (2004). Iaentincation of substrates for adenosine deaminas es that act on RNA.Methods Mol Biol 265 , 199-218; Morse, DP, and Bass, BL (1997). Detection of inosine in messenger RNA by inosine- specific cleavage. Bioche mistry 36, 8429-8434; and Morse, DP, and Bass, BL (1999). Long RNA hairpin s that contain inosine are present in Caenorhabditis elegans poly (A) + RNA. Proc Natl Acad Sci USA 96, 6048-6053) (Figure 2). This method consists of the following three steps.

[0006] (1) Base modification of guanosine (G) and inosine (I) in an RNA strand is performed using dalyoxal as a chemical modifying reagent. In the case of G, darioxal is attached to the 1st and N'2 positions to form cis-diol, which is further compounded with boric acid (G * in the figure). On the other hand, inosine has no amino group at the 2-position, so darioxal is added only at the 1-position. Furthermore, since the added structure is unstable, the reverse reaction is easily caused to return to the original inosine.

 (2) Cut RNA after treatment with dalyoxal / boric acid with RNase Tl specific for G and inosine. At this time, since G is modified to G *, cleavage does not occur at the G site. On the other hand, inosine is modified, and it will be cut back at the inosine site. As a result, site-specific cleavage of inosine can be achieved.

 (3) Remove the force factor from the G * in the RNA after cleavage, attach an anchor sequence to the RNA fragment containing inosine, and reverse-transcribe the PCR with this site force and analyze the sequence.

[0007] By this method, Morse et al. Identified several inosine sites in mRNA! /, Ruga (Morse, DP, and Bass, BL (1999). Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly (A) + RNA. Proc Natl Acad Sci USA 96, 6048-6053). The key to the success of this analysis is the RNase Tl-insensitive modification of G by treatment with dalyoxal / boric acid. It is difficult to modify all G that occupies 1/4 of the bases of RNA, and it is inevitable that unmodified G remains. Furthermore, there are only about 0 to 30 bases of inosine in a single mRNA of several thousand to tens of thousands of bases, and the background due to cleavage of the unmodified G site is the target inosinization site. This makes it very difficult to detect cuts. Therefore, even if candidate sites are obtained, confirmation by the matched tissue method is necessary.

[0008] Inosine-specific chemical modification has also been reported by Yoshida et al.!, Ru (Yoshida, M., Fur uichi, Y., Ukita, Τ ·, ana aziro, Y. (1967). The effect of cyanoethylation on codon r ecognition of yeast tRNA containing inosine. Biochim Biophys Acta 149, 308-310). In this analysis method, in order to elucidate the function of inosine present in the anticodon site of transfer RNA (transfer RNA; tRNA) (which corresponds to the codon on mRNA that defines the genetic code), position 1 of inosine is acrylonitrile. Is used to inhibit codon-anticodon pair formation and analyze its effects. Although the reaction efficiency is low, Shiuduridine (mouth) is also cyanoethylated (Yoshida, M., and Ukita, T. (1968). Modification of nu cleosides and nucleotides. 8. The reaction rates of pseudouridine residues with acryl nitrile and its relation to the secondary structure of transfer ribonucleic acid. Bioc him Biophys Acta 157, 466-475), Mengeh Jorgensen et al. analyzed the change in molecular weight of pseudouridine by cyanoethylation by mass spectrometry, and identified the position on tRNA A method has been reported (MengehJorgensen, J., and Kirpekar, F. (2002). Detection of ps eudouridine and other modifications in tRNA by cyanoethylation and MALDI mass s pectrometry. Nucleic Acids Res 30, el35). These methods are intended to analyze relatively abundant tRNAs and analyze the effect of anticodon inactivation or the change in mass caused by cyanoethyl.

 Disclosure of the invention

 Problems to be solved by the invention

[0009] 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.

[0010] As a result of diligent investigations to solve the above-mentioned problems, the present inventors have chemically modified the inosine site by treating RNA with a compound having an α, 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.

[0011] That is, according to the present invention, RNA has an α, β unsaturated bond and an electron-withdrawing group. There is provided a method for detecting an inosine site, comprising the step of chemically modifying the inosine site by treatment with a compound.

 [0012] Preferably, 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.

 Preferably, 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.

[0013] Preferably, the compound having an ex, j8-unsaturated bond and an electron-withdrawing group is

 Formula: C d ^ I ^^ C d ^ E

(Wherein 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, and 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. A full group having a group or an electron withdrawing group, and E represents an electron withdrawing group.

 It is a compound represented by these.

 Preferably, the electron withdrawing group is CN, NO, SO H, CONH, COCH, COOCH,

 2 3 2 3 3

COOC H, COCH, COC H, or COC H.

 2 5 3 2 5 6 5

 [0014] Preferably, the chemically-modified RNA is subjected to a reverse transcription reaction to synthesize cDNA, and then a cDNA amplification reaction is performed.

 Preferably, as a control, 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.

 Preferably, the RNA is chemically modified by treating the RNA with an oc, β-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.

 Preferably, chemically modified inosine is detected by detecting the length of cDNA derived from the chemically modified RNA.

[0015] Preferably, RNA is treated with a compound having an a, β 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.

 Preferably, 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.

 Preferably, 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.

 [0016] According to another aspect of the present invention, there is provided an inosine moiety modifying agent in RNA comprising a compound having a, | 8-unsaturated bond and electron withdrawing group.

 According to still another aspect of the present invention, there is provided a reagent kit for performing the above-described method of the present invention, comprising the above-described modifying agent of the present invention.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in more detail.

 The method for detecting an inosine site according to the present invention includes a step of chemically modifying an inosine site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group. And In the present invention, the type of RNA is not particularly limited and may be any of mRNA, rRNA, tRNA or non-coding (nc) RNA! /.

[0018] In 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. In addition, since the inosine-specific modification is performed in the method of the present invention, 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. Further, 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. Furthermore, 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).

 Hereinafter, the method of the present invention will be specifically described.

 [0020] (1) Inosine-specific chemical modification

 In the method according to the present invention, first, inosine-specific chemical modification is applied to RNA. As a reagent, a, j8-unsaturated electron withdrawing group compound can be used.

 [0021] The reaction mechanism basically proceeds by a mechanism called Michael addition (Fig. 3A). First, the β-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. Depending on the selection of the modifying reagent, it is also possible to isolate an RNA molecule containing inosine specifically for the added functional group. The reaction can be carried out by selecting an appropriate solvent according to the modifying reagent. In the examples described below, acrylonitrile (CH = CHCN) is used.

 2

 is doing. Similarly, in the case of acrylonitrile, it is known that pseudouridine also reacts. By shortening the reaction time, it is possible to make reaction conditions almost specific for inosine.

 [0022] Examples of the compound having an a, β unsaturated bond and an electron-withdrawing group include:

 Formula: C d ^ I ^^ C d ^ E

(Wherein 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, and 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. A full group having a group or an electron withdrawing group, and E represents an electron withdrawing group.

The compound represented by these can be mentioned. Examples of functional groups in the above compound and specific examples of the above compound are shown in FIGS. 3B and 3C, respectively. [0023] Specific examples of the above compounds include CH = CHCN, CH CH = CHCN, CH CH = C

 2 6 5 3

HCOCH, CH CH = CHCOC H, CH CH = C (CH) COCH, (CH3) C = CH

 3 3 6 5 3 3 3 2

COCH, CH CH = C (CH) COC H, CH = C (CH) COC H, CH = C (CH C

3 3 3 6 5 2 3 6 5 2 2

H) COC H, CH = CHCOOCH, CH = C (CH) COOCH, CH OC H CH =

3 6 5 2 3 2 3 3 3 6 5

CHCOOCH CH, CH OC H CH = CHCOOCH, CH = COOCH CH, CH

 2 3 3 6 5 3 2 2 3 2

= COOCH, CH = CONH, CH = C (OCOCH) CN, and

 3 2 2 2 3

[0024] [Chemical 1]

 [0025] and the like.

 [0026] (2) Reverse transcription reaction (amplification as necessary)

 In the method of the present invention, reverse transcription can be performed on RNA that has been chemically modified specifically for inosine. When not chemically modified, cytidine (C) is incorporated into the inosine site by reverse transcriptase. On the other hand, when chemical modification is applied, the incorporation of C is inhibited by this chemical modification, and the elongation of the reverse transcribed strand stops before that.

 [0027] The reverse transcription reaction can be performed by a conventional method using a primer, chemically modified RNA (铸 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.

 [0028] In the present invention, it is preferable that a chemically amplified RNA is subjected to a reverse transcription reaction to synthesize cDNA and then a cDNA amplification reaction is performed. In this case, reverse transcription and amplification can be performed by a series of operations using the RT-PCR method. Alternatively, it can also be performed by transcription amplification using reverse cDNA-cRNA as a cage.

 [0029] Further, in the reverse transcription reaction, the 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.

 [0030] (3) Detection

In detection, 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. Thus, the inosine site in RNA may be detected. In this case, the detection target information includes its length, base sequence, or amount.

 [0031] In the first embodiment of detection, the inosine site is chemically modified by treating RNA with a compound having an α, β unsaturated bond and an electron-withdrawing group, and then chemically modified. By subjecting the RNA to mass spectrometry, chemically modified inosine sites can be detected. For mass analysis, in order to analyze the presence and total amount of inosine, 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. For example, NucleasePl can be used. The phosphatase is not particularly limited, and the species of origin such as nocteria is not limited. For example, Bacterial Alkaline Phospatase derived from E. coli can be used.

 [0032] For example, RNA is treated with acrylonitrile as a modifier for the inosine site, and then degraded to nucleosides. Compare this sample with 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) Alternatively, the inosine wrinkle site can be detected by the increase.

 [0033] In addition, the region containing inosine may be identified by using the chemically modified RNA as a sample. Preferably, 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.

 [0034] For example, 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). The region containing inosine can be identified by the appearance or increase of 53 molecular weight increase calories per site).

[0035] According to this method, it is possible to detect the presence or absence of an inosine sputum site, quantify the amount of inosine, and identify a region containing Z or inosine. [0036] In the second mode of detection, α, β unsaturated bonds and electron-withdrawing groups are detected by detecting the length of cDNA or the amplification product of the reverse transcript of chemically modified RNA. It is possible to detect inosine chemically modified by a compound having The length of cDNA can be detected by conventional methods known to those skilled in the art, such as electrophoresis. As described above, chemical modification (for example, Cyanethyl 匕 etc.) 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.

 [0037] Further, as a third aspect of detection, a compound having an α, β 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.

[0038] Furthermore, as a fourth embodiment of detection, cDNA derived from chemically modified RNA is detected by hybridization using a probe containing a sequence only upstream of the inosinization site of RNA. By doing so, inosine chemically modified by a compound having an a, j8-unsaturated bond and an electron-withdrawing group can be detected. For example, if the 3 'end force is extended using a poly-T primer after modification of the mRNA, 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. As a control, when cDNA derived from non-chemically modified RNA is 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. Thus, the site of inosine can be detected by the presence or absence of a signal. Also, depending on the probe design As a result, it is possible to identify the region including the inosin wrinkle site and the position thereof. This method may be performed in combination with a microarray. As another example, after the mRNA is chemically modified, 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. As a control, when cDNA derived from non-chemically modified RNA is 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. As 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.

 As described above, 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.

[0039] In a fifth embodiment of detection, 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. On the other hand, except for the inosine site, 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.

[0040] From the thus obtained cDNA population, a 3'-OH group was added to the 3 'end terminated at the inosine site. Select only cDNAs possessed.

 [0041] As an example of the method, first, 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. At this time, 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. Continue! Double-stranded the cDNA using primers complementary to the adapter sequence. Thirdly, either poly dA, dT, dC or dG sequence (poly dN) is added to the 3 ′ end of cDNA by using an enzyme having terminal deocynucleotidyl transferase activity. At this time, 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.

 [0042] In some cases, the double-stranded cDNA is amplified and the sequence thereof is analyzed. For example, 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. Alternatively, a protruding end can be created at the end in a double-stranded DNA amplification step using PCR. Thus, the sequence analysis can be carried out by incorporating double-stranded DNA with protruding ends into a cloning vector such as a plasmid. In this case, for efficiency, 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.

[0043] As described above, 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. [0044] 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.

 [0045] 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.

 [0046] A reagent kit for detecting the inosine bud site can be designed according to the detection method.

 For example, in the case of the reagent kit for the first detection method, Rnase or phosphatase can be combined in addition to the inosine modifying agent. As a reagent kit for the second detection method, in addition to the above-mentioned inosine modifier, a reverse transcriptase for generating a reverse transcript or a buffer for reverse transcription as necessary may be combined. it can. In the case of a reagent kit for the third detection technique, a set of reagents for base sequence determination can be combined with the inosine modifier. As a set of reagents for determining the base sequence, for example, polymerase, dNTP, ddNTP, reaction buffer and the like can be mentioned as examples. As a reagent for the fourth detection method, a probe (probe set) 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.

 [0047] The ability to more specifically describe the present invention by the following examples The present invention is not limited by the examples.

 Example

 [0048] Example 1: Inosine-specific chemical modification (1-cyanoethylation) reaction

 10 μg of the RNA fraction to be analyzed is dissolved in 30 μ 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 In addition, the sample was incubated at 70 ° C for 15 or 30 minutes in light shielding, and a sample incubated without acrylonitrile as a control (CE-) was also prepared.After the reaction, the sample was rapidly cooled on ice, and RNeasy MinElute Cleanup After purification with kit (QIAGEN), the RNA in the eluate was ethanol precipitated and rinsed with 70% ethanol, and then lyophilized.

[0049] Example 2: Inosin that was specifically cyanoylated was confirmed by liquid chromatography / mass spectrometry (LC / MS method). For the sample, total RNA extracted from yeast was used and reacted basically according to the method described in Example 1. However, the reaction time should be 70 ° C 0 min, 15 min, 30 min, or 60 min, and the RNA after the reaction should be rinsed with ethanol without RNeasy MinElute Cleanup kit, rinsed with 70% ethanol, and then lyophilized. It was. Subsequently, RNA after drying is used in Sakurai, M., Ontsuki, T., buzuki, T., and Watanabe, K. (2005) .Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum.FEB S Lett LC / MS analysis was performed after degradation to nucleosides using Nuclease PI and bacterial alkaline phosphatase according to 579, 2767- 2772.

[0050] As a result, the inosine peak (m / z 269) observed with CE- (£ yano _£ thylation, Fig. 4A) decreased with the reaction time of cyanobacteria, but was not detected with CE- instead. A strong cyanoethylated inosine (CE-I) (m / z 322) peak was observed (Figure 4B, C, D, E). The peak of cyanoethylated pseudouridine (CE-Ψ) (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.

 [0051] 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.

 2

.3), Incubated in 50 μC for 60 minutes in 10 μl hot metal containing 100 U Superscript III reverse transcriptase (Invitrogen). [0052] After the reaction, add 2 μLoading solution (7M urea, 0.05% bromophenol blue, 0.05% xylene cyanol, lx TBE) 10 μ1, 100% formamide 20 μ1 and boil at 95 ° C for 5 minutes. Was used for electrophoresis. Electrophoresis was performed on a 15% polyacrylamide, 7M urea, ΙχΤΒΕ gel.

 [0053] In the case of untreated (CE-) total RNA, C was incorporated into the reverse transcribed strand with respect to the inosine site, and the extension proceeded without delay. [For confirmation, ddATP (Dideoxy ATP) stops the elongation. On the other hand, in the case of cyanoethylation (CE +), as shown in Fig. 5, the 1-cyanoethyl group of cyanoethylinosine (CE-I) inhibits C uptake, so that the reverse transcription chain elongation is Stopped one base before. From this result, it can be said that the elongation of the reverse transcribed strand can be specifically stopped at the inosynthetic site by means of Cyanochrydo and the inosine site can be identified by the extended length.

 [0054] 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. On the other hand, 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 μ1. 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. Subsequently, Big Dye Terminator vl.l was used, and ycle sequencing kit (Applied BiosystemsJ was used to analyze ~ Jense anti-Jj and analyzed with ABI PRISM 3700 DNA Analyzer (Applied Biosystems). Design primers, perform genomic PCR, I did.

 [0055] The principle of this method is as follows. First, in the case of untreated (CE-) total RNA, the reverse transcribed strand extended from the reverse primer incorporates C into the inosine site and reaches the forward primer complementary site. There is a G that reflects inosine at the site of inosine corresponding to the sense strand of the PCR amplification product sense strand of this 1st strand cDNA (Fig. 6A). However, 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. Therefore, when inosine is partially mixed, G reflecting inosine disappears from the corresponding portion of the PCR product sense strand (Fig. 6B). In addition, when complete inosine replacement occurs, the entire PCR amplification is not observed (Figure 6C).

 [0056] In the example (Fig. 7), comparing the genomic sequence of mouse brain serotonin receptor 2c mRNA with the cDNA NA-sequence, G reflected I in 5 A-to-I RNA editing sites. Mixed! / However, the G! Peak was disappeared specifically as the cyanoethylation reaction progressed, and became the peak of A alone. Therefore, it is possible to detect inosine in trace amounts of RNA by comparing the CE- and CE + sequences. A technique for specifically eliminating inosine-derived G by this chemical modification is called Inosine Chemical Erasing (ICE method).

 [0057] 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

 twenty four

The reverse transcription reaction using DNA primer (Ambion)] 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.

[0058] 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. First, in the case of untreated (CE-) total RNA, the reverse transcribed strand extended from the reverse primer incorporates C into the inosine site and extends. Then double stranded c DNAa 匕 · 7 RNA amplification with RNA polymerase 'labeling and hybridization with the probe on the array, and the labeling intensity (signal intensity) of the aRNA on this array is quantified (Figure 8A). On the other hand, in the case of CE +, the extension of the reverse transcription chain to mRNA containing inosine stops before the site of inosine, so a short reverse transcription chain is synthesized. The length of the synthesized reverse transcribed strand is directly reflected in the length of aRNA via transcription amplification (Fig. 8B). Therefore, a short aRNA resulting from a reverse transcription strand that is specifically stopped inosine does not contain a region complementary to the probe on the array and cannot hybridize with the probe. As a result, the signal strength of the aRNA on the probe is thought to decrease, reflecting the amount of reverse transcription chain arrested before the inosine site. In other words, when the signal intensity of CE + decreases compared to the signal intensity of CE−, it can be said that inosine exists in the region between the probe and the reverse transcription primer on the array.

 In the examples, Hitachi Software's Ace Gene was used due to the restriction of the probe design position on the array. Based on the probe design position and the information of the A-to-IRNA editing site confirmed by the present inventors by the ICE method, an inosine site exists between the probe position on the array and the mRNA end. Species were extracted (Fig. 9, X marked black line). The signal intensity ratio of CE + to CE— [Log2 (CE + / CE-)] for these 68 species and all genes on the array (Figure 9, lip-gray line), negative in the horizontal axis in the graph As the analysis progressed, it means that the CE + signal decreased. As a result, the distribution of 68 types of mRNA undergoing RNA editing was biased in the negative direction, and the average value was -0.42, which is the average value for all genes in the array. -Significant signal reduction compared to 0.12. Therefore, it was shown that the presence of inosine in mRNA can be detected by this method.

 [0060] 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. In 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 μg and dNTP (20 μl final concentration 2 μΜ) A reaction solution 131 containing 1 was prepared. For CE +, a reaction solution containing 500 ng of RNA and 250 g of random primer and a reaction solution containing dNTP (final concentration 500 μΜ on a 20 μ1 scale) 13 μ 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 μ \, 5x First strand buffer (Invitrogen, 250 mM Tris-HCl ( pH 8.3), 375 mM KC1, 15 mM MgCl) 4 μ1, 200 U / μ 1 SuperScriptlll RT (

 2

 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 µl 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.

 [0061] Subsequently, 1st strand cDNA was purified using S-300 HR column (GE Healthcare) and QIA quick nucleotide removal kit (QIA GEN) in this order.

 [0062] Next, a poly dC chain was attached to the 3 'end of the cDNA. The obtained 1st strand cDNA was prepared to 11 μ1, boiled at 95 ° C for 2 minutes, rapidly cooled, and then 5x TdT buffer (1 M potassium cacodylate, 125 mM Tris—HCl, 1.25 mg / ml) BSA (pH 6.6) 4 1, 10 mM CoCl 2 ^ 1, 400 ^ M

 2

 A reaction solution 201 containing dCTP, 400 U / μ 1 terminal deoxynucleotidyl transferase (Roche) 1 μ 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.

 Next, the first PCR amplification was performed using the obtained cDNA solution as a saddle shape. PCR was performed using 2 μ1 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

 Four

 bo), lx KOD-Plus reaction buffer containing 50 μ 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.

[0064] Further, a second PCR amplification was performed using the PCR product as a saddle. PCR was performed using 1 μ 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 μ μ dNTPs, 1U KOD- Plus (Toyobo ), 50 μ 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.

 [0065] Finally, in each case of CE- and CE +, quantitative analysis of cDNA by real-time PCR was carried out using the first and second PCR products as saddles. For analysis, primers designed to have an amplification length of approximately 50 base pairs with a spacing of 50 bases from the entire region of mouse glutamate receptor B mRNA and serotonin receptor 2c mRNA. The amount of cDNA in each region was quantified.

 [0066] The reaction was analyzed using SYBR Premix Ex Taq (Takara) and LightCycler 480 (Roche). The amplification was performed on a 20 μ1 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. The reaction conditions are Denature; 95 ° C for 10 seconds, PCR (45 cycles); 95 ° C for 3 seconds 4.8 ° C / second, 57 ° C for 10 seconds 2.5 ° C / second, 72 ° C for 6 seconds 4.8 ° C / Seconds acquisition = single, Melting; 95 ° C 3 seconds 4.8 ° C / second, 65 ° C 15 seconds 2.5 ° C / second, 95 ° C acquisition = 20point / ° C, Cooling; 37 ° C 30 minutes 2.0 ° C Per second protocol. 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)

 MGluRBQRf-700 (TGTTACAAGTCAAGGGCCGAG) (SEQ ID NO: 11)

 MGluRBQRr-550 (CAATTTGTCCAACAGGCCTTG) (SEQ ID NO: 12)

 MGluRBQRf-500 (TAACCTCGCAGTACTAAAACTG) (SEQ ID NO: 13)

 MGluRBQRr-450 (AATGAGGATCCTTTAGGTGTGG) (SEQ ID NO: 14)

 MGluRBQRf-400 (AACCTGGATTCCAAAGGCTAC) (SEQ ID NO: 15)

 MGluRBQRr-400 (ATGGTGTCGCAAGGCTTCCT) (SEQ ID NO: 16)

 MGluRBQRf-350 (CTGGAGTCCACAATGAATGAG) (SEQ ID NO: 17)

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 (TCACTACTTTGTGTTTCTCTTCC) (SEQ ID NO: 28) MGluRBQRf + 110 (AGTGGCACACTGAGGAATTTG) (SEQ ID NO: 29) MGluRBQRr + 150 (CCCAATGTAGGCAAACACAATG) (SEQ ID NO: 30) MGluRBQRf + 200 (TCCTTTAGCCTATGAGATCTGG) TAGlu 31 (SEQ ID NO: 32) MGluRBQRf + 300 (AGAGGTGATTGACTTCTCGAAG) (SEQ ID NO: 33) MGluRBQRr + 350 (TTCCCATATACAAGTTCTCCAAC) (SEQ ID NO: 34) MGluRBQRf + 400 (TGCAGACACCAAAATTTGGAATG) (SEQ ID NO: 35) MGluRBQRATCTC C) (SEQ ID NO: 36) MGluRBQRf + 500 (TGAAGGGAATGAGCGTTATGAG) (SEQ ID NO: 37) MGluRBQRr + 1000 (GGGCCGAAGTATACTTAATTGTC) (SEQ ID NO: 38) MGluRBQRf + 1050 (GAAAAGAATACCCTGGAGCAC) (SEQ ID NO: 39) MGluRBQRGACGTC ) MGluRBQRf + 1550 (TTTCGCAGTCACCAATGCTTTC) (SEQ ID NO: 41) M5HT2cRr-1550 (TACCACTATGTTTGGGGACAGA) (SEQ ID NO: 42) M5HT2cRf-1500 (TGTTGTGTCGGTATTTTGCTGC) (SEQ ID NO: 43) M5HT2cRTGTGTAGTAG ) (SEQ ID NO: 45) M5HT2cRr-500 (GCACCACATGATCAGAAACACA) (SEQ ID NO: 46) M5HT2cRf-450 (CTTCCAAAGTCCTTGGCATTG) (SEQ ID NO: 47)

M5HT2cRr-400 (TTTCTTCTTTCGACGTGGCTTC) (SEQ ID NO: 48)

M5HT2cRf-350 (AGAACGCTCCCAACCCCAAT) (SEQ ID NO: 49)

M5HT2cRr-300 (TGATATTACGCAGTTCCTCCTC) (SEQ ID NO: 50)

M5HT2cRf-250 (CGTCAAACCCTGATGTTACTTC) (SEQ ID NO: 51)

M5HT2cRr-200 (CAACGGGATGAAGAATGCCAC) (SEQ ID NO: 52)

M5HT2cRf-150 (GACCCGAACTTCGTTCTCATC) (SEQ ID NO: 53)

M5HT2cRr-100 (CTCCTATTGATATTGCCCAAACG) (SEQ ID NO: 54)

M5HT2cRf-50 (GGACTAAGGCCATCATGAAGAT) (SEQ ID NO: 55)

M5HT2cRr-25 (GAATTGAACCGGCTATGCTCAA) (SEQ ID NO: 56)

M5HT2cRf + 25 (TATCGCTGGACCGGTATGTAG) (SEQ ID NO: 57)

M5HT2cRr + l (CATACCGGTCCAGCGATATG) (SEQ ID NO: 58)

M5HT2cRf + 50 (TTTTCAACTGCGTCCATCATGC) (SEQ ID NO: 59)

M5HT2cRr + 50 (TGGACGCAGTTGAAAATAGCAC) (SEQ ID NO: 60)

M5HT2cRf + 100 (TTTGTGCCCCGTCTGGATTTC) (SEQ ID NO: 61)

M5HT2cRr + 150 (TGACAAGTAGTCCCACCAGC) (SEQ ID NO: 62)

M5HT2cRf + 200 (TCTTAATGTCCCTAGCCATTGC) (SEQ ID NO: 63)

M5HT2cRr + 250 (AGAATGTTGCCCCCTATTGTC) (SEQ ID NO: 64)

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. Set the amplification region on mRNA with the Q to R site of GluR-B and the most upstream editing site of 5HT2cR, each containing +1, and the primer name Shown at the end. The more negative it is, the closer to the 3 'end of mRNA. The amplification region including the length of the primer was approximately 50 bp.

 [0068] The principle of this method is as follows.

 First, reverse transcription reaction is performed using a random primer with an adapter sequence added to the 5 'end using RNA as a saddle. In this case, in the case of untreated (CE-) RNA, 1st strand cDNA is generated equally over the entire region of RNA. On the other hand, when chemically modified (CE +), 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. Subsequently, 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. As a result, 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!

 [0069] In Examples, mouse glutamate receptor B mRNA and the amount of cDNA in each region were comparatively quantified with CE- and CE + (Figs. 10 and 11).

 [0070] With respect to the signal intensity obtained by real-time PCR, 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.

[0071] In glutamate receptor B, about 40% of the inosine site known as the R to G site between the -400-450 and -500 # -550 regions and the Q to R site at the +1 position There are almost 100% inosine sites that can be found. In the graph, the signal intensity ratio decreases in the -400 # 450 region in the upstream region closest to the R to G site and the + 25 # -25 region including the +1 position. The result was obtained. The degree of decrease gradually decreased toward 5 'upstream. In addition, the magnitude of the decrease decreased to about -1 at the R to G site (40%) and to about -2.8 at the Q to R site (100%) in proportion to the efficiency of the respective inosine sites.

[0072] On the other hand, in serotonin receptor 2C, there are 10 to 80% inosine sites at positions +1, +3, +7, +8, and +13. In the graph, a peak of decrease in the signal intensity ratio was obtained in the + 25 # -25 region including these sites. From these results, it was shown that the presence of the inosine site can be detected in the region where the signal intensity ratio is significantly reduced or in the vicinity of the 3 'end by this method. This technique can be applied to tiling arrays. Industrial applicability

[0073] 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.

 Brief Description of Drawings

[0074] FIG. 1 shows detection of inosine by the matched-tissue method.

 [Fig. 2] Fig. 2 shows the identification of inosine 匕 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] 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] Figure 6 shows the principle of the ICE method.

 [FIG. 7] 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] 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] 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.

Claims

The scope of the claims  [1] A method for detecting an inosine site, comprising a step of chemically modifying the inosine site by treating RNA with a compound having an oc, β unsaturated bond and an electron-withdrawing group.  [2] The method according to claim 1, further comprising 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. Method.  [3] The method according to claim 1 or 2, wherein the ability to detect an inosine site is detection of the presence or absence of an inosine site, quantification of the amount of inosine, and identification of a region containing Z or inosine or an inosine moth site.  [4] a, | 8—a compound having an unsaturated bond and an electron-withdrawing group is  Formula: cd ^ i ^^ cd ^ E (Wherein 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, and 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. A full group having a group or an electron withdrawing group, and E represents an electron withdrawing group.  The method according to any one of claims 1 to 3, wherein the compound is represented by the formula: [5] Electron withdrawing groups are CN, NO, SO H, CONH, COCH, COOCH, COOC H,  2 3 2 3 3 2 5 5. The method of claim 4, wherein the method is COCH, COC H, or COC H.  3 2 5 6 5  [6] The method according to any one of [1] to [5], wherein the chemically-modified RNA is subjected to reverse transcription to synthesize cDNA, and then the cDNA is amplified. [7] As a control, detect the inosine site by comparing RNA synthesized in reverse transcription reaction with cDNA synthesized from chemically modified RNA. Item 7. The method according to any one of Items 2 to 6. [8] After chemically modifying the inosine site by treating RNA with a compound having an oc, β unsaturated bond and an electron withdrawing group, the chemically modified RNA is subjected to mass spectrometry. 6. The method according to any one of claims 1, 3, 4 and 5, wherein inosine chemically modified by is detected. [9] The method according to any one of [2] to [7], wherein the chemically modified inosine is detected by detecting the length of cDNA derived from the chemically modified RNA. [10] After chemically modifying the inosine site by treating RNA with a compound having an a, β unsaturated bond and an electron withdrawing group, the chemically modified RNA or c derived therefrom The method according to any one of claims 1 to 7, wherein chemically modified inosine is detected by determining a base sequence of DNA. [11] The chemically modified inosine is detected by detecting cDNA derived from the chemically modified RNA using a probe containing a sequence only upstream of the inosine site of RNA. 8. The method according to any one of 7. [12] The chemically modified inosine is detected by selectively extracting the cDNA derived from the chemically modified RNA at the inosine site and analyzing its sequence. The method in any one of. [13] A, β Inosine site-modifying agent in RNA comprising a compound having an unsaturated bond and an electron-withdrawing group.  [14] A reagent kit for performing the method according to any one of claims 1 to 12, comprising the modifying agent according to claim 13.