JP2008259425A - Method for detecting inosine-containing site in rna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting an inosine-containing site in RNA, which can easily distinguish from noises and SNP, analyze only with RNA as an analysis sample, analyze even a small amount of the sample, control a back ground low, and highly sensitively detect the presence of inosine. <P>SOLUTION: This method for detecting the inosine-forming site in RNA includes a process for treating the RNA with a compound having an α, β-unsaturated bond and an electron attractive group to chemically modify the inosine-containing site. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting an inosynthetic site in RNA. More specifically, the present invention relates to a method for detecting an inosynthetic site by chemically modifying the inosineated site in RNA.

  Modification by deamination is called RNA editing. RNA editing causes a change from A to I, and changes the codons on the mRNA, causing changes in the amino acid sequence. Double-stranded RNA adenosine deaminating action (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 for ADAR2, in which adenosine in exon 11 is edited to inosine, and the amino acid sequence changes from glutamine to arginine. Since ADAD2 knockout mice do not produce this editing, it is known that the calcium permeability of glutamate receptors cannot be controlled, 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, editing from A to I is performed using serotonin receptor (5-HT2c), potassium channel (Kv1.1), etc. Has been found. Interestingly, ADAR2 is known to induce variable splicing and feedback control the expression level of ADAR2 by editing its mRNA intron site from A to I.

  There is an estimate that ADAR is highly expressed in brain mRNA at a frequency of 17,000 bases, and human mRNA was predicted to have a large amount of unknown editing sites. . Recently, analyzes using bioinformatics techniques have reported a large number of A to I editing candidate sites in the repetitive sequence of the Alu factor in the UTR of human mRNA. Although only a few examples have been experimentally verified, the estimation that inosine exists at more than 12,000 sites in the 1600 gene suggests that editing from A to I greatly affects the global expression control of the gene. Suggests that it plays a role. Considering that only primates have Alu factors, and ADAR is highly expressed in the brain, the increased complexity of the transcriptome by editing from A to I is highly evolved. It may be related to the acquisition of the complexity of the neural circuit of the brain. In addition, diseases caused by abnormal RNA editing have 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 likely to be deeply related to higher life phenomena.

  The matched-tissue method is the most common method for detecting an RNA editing site from A to I (= site where I exists) (FIG. 1). In this method, RNAs (amplified as cDNA) derived from the same tissue of the same individual are compared with sequences of corresponding regions in the genome. Since I forms a base pair with C, G is incorporated into the I-corresponding site in the cDNA after reverse transcription and PCR amplification from mRNA. Therefore, although the base on the genome is A at the editing site, it is a mixture of G or A / G on the cDNA. However, in this method, non-specific amplification of PCR, genome contamination, sequence error, noise, SNP between alleles (Allele SNP), pseudogene, gene copy, splicing variant, etc. It is difficult to determine the mixture of G derived from inosine. In addition, in the case of an exon boundary region or a region where a pseudogene exists, it is difficult to discriminate a 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 presence of inosine. In addition, since it is necessary to prepare both genome and RNA derived from the same individual, there is a problem that the samples that can be analyzed are limited.

  In addition, there have been two reports on chemical modifications of inosine in the field of molecular biology. One is an inosine-specific cleavage method aimed at identifying inosine sites in RNA in vivo (Non-patent Documents 1, 2 and 3) (FIG. 2). This method consists of the following three steps.

(1) Using glyoxal as a chemical modifying reagent, base modification of guanosine (G) and inosine (I) in the RNA strand is performed. In the case of G, glyoxal is added to the 1-position and N′2-position to form a 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 glyoxal 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) The RNA after glyoxal / boric acid treatment is cleaved with RNase T1 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, since inosine has not been modified (returned to the original), cleavage at the inosine site occurs. As a result, inosine site-specific cleavage becomes possible.
(3) Remove the adduct from G * in the RNA after cleavage, attach an anchor sequence to the RNA fragment containing inosine, perform reverse transcription / PCR from this site, and analyze the sequence.

  In this method, Morse et al. Have identified inosine sites in several mRNAs (Non-Patent Document 3), but there has been no report. The key to the success of this analysis method is the RNase T1-insensitive modification of G by glyoxal / boric acid treatment. It is difficult to modify all G occupying 1/4 of the bases constituting RNA, and it is inevitable that unmodified G remains. Furthermore, the amount of inosine in a single mRNA consisting of thousands to tens of thousands of bases is so small that only about 0 to 30 bases are present, and the background due to cleavage of the unmodified G site detects the cleavage at the target inosine site. It is very difficult. Therefore, even if candidate sites are obtained, confirmation by the matched tissue method is necessary.

  Inosine-specific chemical modification has been reported by Yoshida et al. (Non-patent Document 4). The purpose of this analysis is to clarify the function of inosine present in the anticodon site of transfer RNA (tRNA) in yeast (which corresponds to the codon on the mRNA that defines the genetic code). Using cyanoethylation to inhibit codon-anticodon pair formation and analyzing its effect. Although the reaction efficiency is low, pseudouridine (□) is also cyanoethylated (Non-patent Document 5), and Mengel-Jorgensen et al. Analyzed the change in molecular weight of pseudouridine by cyanoethylation by mass spectrometry and determined the position on tRNA. A method for identifying is reported (Non-Patent Document 6). These methods are intended to analyze relatively abundant tRNAs, and analyze the influence of inactivation of anticodons or the change in mass due to cyanoethylation.

Morse, D. P. (2004) .Identification of substrates for adenosine deaminases that act on RNA.Methods Mol Biol 265, 199-218. Morse, D. P., and Bass, B. L. (1997). Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36, 8429-8434. Morse, D. P., and Bass, B. L. (1999) .Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly (A) + RNA.Proc Natl Acad Sci U S A 96, 6048-6053. Yoshida, M., Furuichi, Y., Ukita, T., and Kaziro, Y. (1967) .The effect of cyanoethylation on codon recognition of yeast tRNA containing inosine.Biochim Biophys Acta 149, 308-310. Yoshida, M., and Ukita, T. (1968) .Modification of nucleosides and nucleotides. 8. The reaction rates of pseudouridine residues with acrylonitrile and its relation to the secondary structure of transfer ribonucleic acid. . Mengel-Jorgensen, J., and Kirpekar, F. (2002) .Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry.Nucleic Acids Res 30, e135.

  The present invention can be easily distinguished from noise and SNP, can be analyzed only with RNA as an analysis sample, can be analyzed with a small amount of sample, and can further reduce the background. Therefore, an object of the present invention is to provide a method for detecting an inosine site in RNA, which can detect the presence of inosine with high sensitivity.

  As a result of diligent studies to solve the above-mentioned problems, the present inventors have chemically modified the inosynthetic site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group. As a result, it was found that an inosinization site in RNA can be detected, and the present invention has been completed.

  That is, according to the present invention, there is provided a method for detecting an inosynthetic site comprising a step of chemically modifying an inosynthetic site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group. Is done.

Preferably, the method of the present invention further comprises a step of subjecting the chemically modified RNA to a reverse transcription reaction to synthesize cDNA, and a step of detecting an inosynthetic site based on the synthesized cDNA.
Preferably, the detection of an inosine site is detection of the presence or absence of an inosine site, quantification of the amount of inosine, and / or identification of a region or inosine site containing inosine.

Preferably, the compound having an α, β-unsaturated bond and an electron-withdrawing group is
Formula: C (R ¹ ) R ² = C (R ³ ) E
(Wherein, R ¹ and R ² each independently represents a hydrogen atom, a phenyl group having an alkyl group, a phenyl group, or an alkoxyl group having 1 to 6 carbon atoms from 1 to 6 carbon atoms, and R ¹ and R ² Any one of these may be bonded to E to form a ring, R ³ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a phenyl group, a phenyl group having an alkoxyl group having 1 to 6 carbon atoms, Alternatively, it represents 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 ₂ , SO ₃ H, CONH ₂ , COCH ₃ , COOCH ₃ , COOC ₂ H ₅ , COCH ₃ , COC ₂ H ₅ , or COC ₆ H ₅ .

Preferably, the chemically modified RNA is subjected to a reverse transcription reaction to synthesize cDNA, and then the cDNA amplification reaction is performed.
Preferably, as a control, an inosynthetic site is detected by comparing cDNA synthesized by subjecting unmodified RNA to reverse transcription and cDNA synthesized from the chemically modified RNA.
Preferably, the RNA is chemically modified by treating the RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group, and then chemically modifying the inosine-modified site, and then subjecting the chemically modified RNA to mass spectrometry. Detect inosine.
Preferably, chemically modified inosine is detected by detecting the length of the cDNA derived from the chemically modified RNA.

Preferably, after the inosylation site is chemically modified by treating RNA with a compound having an α, β-unsaturated bond and an electron withdrawing group, the base sequence of the chemically modified RNA or cDNA derived therefrom is determined. By doing so, chemically modified inosine is detected.
Preferably, 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.

According to another aspect of the present invention, an inosynthetic site modifier in RNA comprising a compound having an α, β-unsaturated bond and an electron withdrawing group is provided.
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.

  At present, most of the inosine sites, which are said to be 12,000 or more, have not been identified. Given the large amount of inosine present in brain mRNA, it is thought to be associated with abnormalities in mental disorders (schizophrenia, panic syndrome, bipolar disorder, autism, etc.). If inosinating 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 pathogenesis of the diseases and considering therapeutic methods. In addition, it is possible to estimate the risk of developing lifestyle-related diseases and various diseases such as SNP by analyzing the variation of the inosine site for each individual.

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. In the present invention, the type of RNA is not particularly limited, and may be any of mRNA, rRNA, or tRNA.

  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 easy to distinguish from noise and SNP. Furthermore, since no genome is required as an analysis sample, analysis can be performed using RNA alone, so even a small amount of valuable sample can be analyzed. For this reason, it is possible to specify an inosine site even when 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 background can be kept very low, and the presence of inosine can be detected and proved with high sensitivity. Furthermore, by performing detection in the method of the present invention using a microarray, inosine sites in RNA in vivo can be comprehensively specified. Furthermore, the method of the present invention targets all RNA species and utilizes the conventionally reported inosine-specific chemical modification (Yoshida, M., et al.) 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 cyanoethylation 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.

(1) Inosine-specific chemical modification In the method according to the present invention, first, inosine-specific chemical modification is applied to RNA. As the reagent, an α, β-unsaturated-electron withdrawing group compound can be used.

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 modifying reagent. This carbon atom undergoes electrophilic addition to the nitrogen atom at the 1-position, which is the active amine of inosine, whereby the addition modification to inosine proceeds. 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 ₂ ═CHCN) is used. In the case of acrylonitrile, it is known that pseudouridine also reacts in the same manner, but this can be made almost inosine-specific reaction conditions by shortening the reaction time.

Examples of the compound having an α, β-unsaturated bond and an electron-withdrawing group include:
Formula: C (R ¹ ) R ² = C (R ³ ) E
(Wherein, R ¹ and R ² each independently represents a hydrogen atom, a phenyl group having an alkyl group, a phenyl group, or an alkoxyl group having 1 to 6 carbon atoms from 1 to 6 carbon atoms, and R ¹ and R ² Any one of these may be bonded to E to form a ring, R ³ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a phenyl group, a phenyl group having an alkoxyl group having 1 to 6 carbon atoms, Alternatively, it represents 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 compound and specific examples of the compound are shown in FIGS. 3B and 3C, respectively.

Specific examples of the _{compounds, CH 2 = CHCN, C 6} H 5 CH = CHCN, CH 3 CH = CHCOCH 3, CH 3 CH = CHCOC 6 H 5, CH 3 CH = C (CH 3) COCH 3, ( _{CH3) 2 C = CHCOCH 3,} CH 3 CH = C (CH 3) COC 6 H 5, CH 2 = C (CH 3) COC 6 H 5, CH 2 = C (CH 2 CH 3) COC 6 H 5, _{CH 2 = CHCOOCH 3, CH 2} = C (CH 3) COOCH 3, CH 3 OC 6 H 5 CH = CHCOOCH 2 CH 3, CH 3 OC 6 H 5 CH = CHCOOCH 3, CH 2 = COOCH 2 CH 3, CH ₂ = COOCH ₃ , CH ₂ = CONH ₂ , CH ₂ = C (OCOCH ₃ ) CN, and

And so on.

(2) Reverse transcription reaction (amplification as necessary)
In the method of the present invention, reverse transcription reaction 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, C chemical uptake is inhibited by this chemical modification, and the elongation of the reverse transcribed strand stops before that.

  The reverse transcription reaction can be performed by a conventional method using a primer, chemically modified RNA (template), 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 predetermined time.

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

  In the reverse transcription reaction, RNA that has not been chemically modified is subjected to a reverse transcription reaction to synthesize cDNA, which can be used as a control in the following detection step.

(3) Detection In the detection, the presence or absence of chemical modification by inosine may be detected by mass spectrometry, or the reverse transcribed strand that has been inhibited by extension is detected and compared in the presence or absence of inosine chemical modification. The inosine site may be detected. In this case, the detection target information includes its length, base sequence, or amount.

  As a first mode of detection, after chemically inosinating a site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group, the chemically modified RNA is subjected to mass spectrometry. By applying, a chemically modified inosinization site can be detected. When mass spectrometry is performed, in order to analyze the presence and the total amount of inosine, RNA after chemical modification may be used as a sample. However, preferably, the chemically modified RNA is decomposed to nucleoside with RNase and phosphatase. The RNase used here is not particularly limited as long as it can be decomposed to a nucleoside. For example, Nuclease P1 can be used. There is no particular limitation on phosphatase, and there is no limitation on the species of organisms such as bacteria. For example, the bacterium Alkaline Phospatase can be used.

  For example, RNA is treated with acrylonitrile as a modifier of the inosine site and then degraded to nucleosides. This sample is compared to a nucleotide sample from non-chemically modified RNA to reduce inosine peak (m / z 269) or cyanoethylated inosine peak (m / z) in liquid chromatography / mass spectrometry (LC / MS) The inosinylated site can be detected by the appearance of 322) or its increase.

  In addition, for identification of a region containing inosine, RNA after chemical modification may be used as a sample, but preferably after 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. For example, G-specific RNase T1 can be used.

  For example, RNA is treated with acrylonitrile as a modifier of the inosine site and then broken down into RNA fragments. Compare this sample with an RNA fragmented sample from non-chemically modified RNA to reduce the peak of inosine-containing fragments in liquid chromatography / mass spectrometry (LC / MS) or the peak of fragments containing cyanoethylated inosine (inosine The region containing inosine can be identified by the appearance or increase of 53 molecular weight increase per site).

  According to this method, it is possible to detect the presence or absence of an inosine site, quantify the amount of inosine, and / or identify a region containing inosine.

  As a second mode of detection, a compound having an α, β-unsaturated bond and an electron-withdrawing group is detected by detecting the length of cDNA or its amplified product, which is a reverse transcription product of chemically modified RNA. Chemically modified inosine can be detected. The length of cDNA can be detected by conventional methods known to those skilled in the art, such as electrophoresis. As described above, the extension of the reverse transcribed strand is specifically stopped at the inosine site by chemical modification (for example, cyanoethylation), and the length extended from the 3 ′ end of the primer used for reverse transcription is determined as the mobility of electrophoresis. It is possible to detect the inosine site from the above. According to this method, it is possible to detect the presence or absence of an inosine site, quantify the amount of inosine, and / or identify a region containing inosine or an inosine site.

  In addition, as a third aspect of detection, chemical modification is performed with 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. Inosine can be detected. In the presence of chemically modified inosine as described above, the reverse transcription reaction stops before the inosine site, and a short cDNA is generated. By determining the base sequence of this cDNA, it is possible to detect an 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, quantify the amount of inosine, and / or identify a region containing inosine or an inosine site.

  Furthermore, as a fourth aspect of the detection, cDNA derived from chemically modified RNA is detected by hybridization using a probe containing a sequence only upstream of the inosine site of RNA, whereby α, β- Inosine chemically modified by a compound having an unsaturated bond and an electron-withdrawing group can be detected. For example, when mRNA is chemically modified and then extended from the 3 'end using a poly-T primer, the reverse transcription product stops at the inosine site, and a short cDNA lacking a sequence complementary to the upstream sequence of the mRNA. Is generated. 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 a probe containing a sequence only upstream of the inosine site of RNA. In the derived cDNA, a signal reacted with the probe can be detected. Thus, the inosine site can be detected by the presence or absence of a signal. In addition, depending on the design of the probe, it is possible to identify the region including the inosine site and also the position. This method may be performed in combination with a microarray. As described above, according to this method, it is possible to detect the presence or absence of an inosine site, quantify the amount of inosine, and / or identify a region containing inosine.

  The present invention relates to a modifying agent that modifies inosine in RNA, and further to a reagent kit for detecting an inosine site comprising the same.

  The modifying agent that modifies inosine in RNA contains a compound having an α, β-unsaturated bond and an electron-withdrawing group. Since the “compound having an α, β-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 an inosine 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, in addition to the inosine modifying agent, a set of reagents for base sequence determination can be combined. Examples of the set of reagents for determining the base sequence include, for example, polymerase, dNTP, ddNTP, reaction buffer, and the like. As a reagent for the fourth detection technique, in addition to the inosine modifying agent, a probe (probe set) based on the upstream sequence of RNA to be detected, a hybridization buffer solution, a washing solution, and the like can be combined. The probe set may be provided by being immobilized on a microarray.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Inosine-specific chemical modification (1-cyanoethylation) reaction After dissolving 10 μg of RNA fraction to be analyzed in 30 μl of CE buffer (41% ethanol, 1.1 M TEA-acetic acid (pH 8.6)) Then, 4 μl of 15.2M acrylonitrile (Tokyo Kasei) was added, and the mixture was allowed to react at 70 ° C. for 15 minutes or 30 minutes in the dark. As a control (CE-), a sample incubated with acrylonitrile not added was also prepared. The sample after the reaction was rapidly cooled on ice and purified by RNeasy MinElute Cleanup kit (QIAGEN). Furthermore, RNA in the eluate was ethanol precipitated and rinsed with 70% ethanol, and then lyophilized.

Example 2:
Specific cyanoethylated inosine was confirmed by liquid chromatography / mass spectrometry (LC / MS method). The sample was total RNA extracted from yeast, and basically reacted according to the method described in Example 1. However, the reaction time was 70 ° C. for 0 minutes, 15 minutes, 30 minutes, or 60 minutes, and the RNA after the reaction was directly rinsed with ethanol precipitation / 70% ethanol without using the RNeasy MinElute Cleanup kit, and then lyophilized. Subsequently, RNA after drying was used in Sakurai, M., Ohtsuki, T., Suzuki, T., and Watanabe, K. (2005) .Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum.FEBS Lett 579, LC / MS analysis was performed after degrading to nucleoside using Nuclease P1 and bacterial alkaline phosphatase according to 2767-2772.

As a result, CE- (C yano e thyl reduction, Fig. 4A) cyanoethylation observed inosine peak (m / z 269) is decreased with reaction time of cyanoethylated, which were not detected in CE- instead in A peak of inosine (CE-I) (m / z 322) was observed (FIGS. 4B, C, D, E). In addition, the peak of cyanoethylated pseudouridine (CE-Ψ) (m / z 298) increased with the cyanoethylation time, but the reaction rate was slow (Fig. 4B, C, D, F), reaction for 60 minutes. Later, about 60% of unreacted pseudouridine (m / z 245) was detected. Since no change was detected for other bases, this cyanoethylation reaction can be said to be inosine specific.

Example 3 Detection of Inosine by Primer Extension Method Inosine-specific chemical modification reaction was performed on mouse brain total RNA by the reaction procedure described in Example 1. 25 μg of the RNA after the reaction and 0.4 pmol of DNA primer labeled with ³² P at the 5 ′ end were dissolved in 5 μl, 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). And its base sequence is 5'-GATCTTGGCGAAATATCGCATC-3 '(SEQ ID NO: 1). 10 μl containing 150 μM dGTP, dCTP, dTTP at the same concentration as ddATP, 0.5 mM DTT, 3 mM MgCl ₂ , 175 mM KCl, 50 mM Tris-HCl (pH 8.3), 100 U SuperScript III reverse transcriptase (Invitrogen) Incubated in solution at 50 ° C. for 60 minutes.

  After the reaction, 10 μl of 2 × Loading solution (7M urea, 0.05% bromophenol blue, 0.05% xylene cyanol, 1 × TBE) and 20 μl of 100% formamide were added, boiled at 95 ° C. for 5 minutes, and then 10 μl was used for electrophoresis. Electrophoresis was performed on a 15% polyacrylamide, 7M urea, 1xTBE gel.

  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, the first U appeared site was extended by ddATP (dideoxy ATP). Has stopped]. On the other hand, in the case of cyanoethylation (CE +), as shown in FIG. 5, C uptake is inhibited by the 1-cyanoethyl group of cyanoethylinosine (CE-I). Stopped at. From this result, it can be said that the elongation of the reverse transcription chain is specifically stopped by inosine site by cyanoethylation, and the inosine site can be identified by the extended length.

Example 4: Direct sequencing inosine detection by (I nosine C hemical E rasing 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 a region 250 nt centering on 5 A-to-I RNA editing sites in mouse serotonin receptor 2c (5HT2cR) mRNA. The base sequence of the DNA primer used here is forward primer; 5'-ATGGAGAAGAAACTGCACAATG-3 '(SEQ ID NO: 2), 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 amount of total RNA added here is respectively CE- (control under the condition 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 μl. Reaction was reverse transcription; 55 ° C. for 30 minutes, heat denaturation; 94 ° C. for 2 minutes, followed by 38 cycles of 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 30 seconds. The template was purified by treating 5 μl of the reaction solution with ExoSAP-IT (Usb). Subsequently, a sequencing reaction was performed using Big Dye Terminator v1.1 Cycle sequencing kit (Applied Biosystems) and analyzed by ABI PRISM 3700 DNA Analyzer (Applied Biosystems). For comparison, primers for the genome were designed, followed by genomic PCR and sequencing in the same manner.

  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. G reflecting inosine is present at the site corresponding to inosine in the sense strand of the PCR amplification product of the 1st strand cDNA (FIG. 6A). However, when cyanoethylation is performed, extension of the reverse transcribed strand for mRNA containing inosine 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 site of the PCR product sense strand (FIG. 6B). In addition, when the substitution with inosine completely occurs, the entire amplification by PCR is not observed (FIG. 6C).

In the example (FIG. 7), comparing the genomic sequence of mouse brain serotonin receptor 2c mRNA with the cDNA CE-sequence, G reflecting I was mixed in five A-to-I RNA editing sites. However, this mixed G peak disappeared specifically with the progress of the cyanoethylation reaction and became a peak of A alone. Therefore, it is possible to detect inosine in a small amount of RNA by comparing the CE- and CE + sequences. Specifically approach to eliminate G-derived inosine This chemical modification, referred to as I nosine C hemical E rasing (ICE method).

Example 5: Detection of inosine by microarray (ICE-microarray method)
Microarray analysis was performed using Human brain total RNA (CE−) and (CE + 15 min) prepared in Example 1. From the reverse transcription reaction using oligo dT primer with T7 promoter sequence [T7 (dT) ₂₄ cDNA primer (Ambion)], we asked Hitachi Soft DNA Chip Research Laboratories for the AceGene Human Olig Chip 30K 1Chip version. It was performed using. The company uses the Amino Allyl Message Amp aRNA kit (Ambion) for reverse transcription, 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. 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. Thereafter, it is hybridized with a probe on the array through double-stranded cDNA, aRNA transcription amplification with T7 RNA polymerase, and labeling, and the labeling intensity (signal intensity) of the aRNA on this array is quantified (FIG. 8A). On the other hand, in the case of CE +, the elongation of the reverse transcription chain for mRNA containing inosine stops before the inosine site, so that a short reverse transcription chain is synthesized. The length of the synthesized reverse transcribed strand is directly reflected in the length of aRNA through transcription amplification (FIG. 8B). Therefore, a short aRNA resulting from a reverse transcription strand that is specifically stopped inosine does not contain a complementary region to the probe on the array and cannot hybridize with the probe. As a result, it is considered that the signal intensity of aRNA on the probe decreases to reflect the amount of reverse transcription chain stopped before the inosine site. That is, when the signal intensity of CE + decreases compared to the signal intensity of CE−, it can be said that inosine is present in the region between the probe and the reverse transcription primer on the array.

  In the examples, Hitachi Soft's Ace Gene was used due to the restriction of the probe design position on the array. 68 types of mRNA with an inosine site between the probe position on the array and the mRNA end are extracted from the probe design position and the information of the A-to-I RNA editing site confirmed by the inventors using the ICE method. (Fig. 9, x mark-black line). The signal intensity ratio of CE + to CE− for these 68 species and all genes on the array (Figure 9, □ -gray line) This means that the CE + signal decreased], and the distribution of 68 mRNA species undergoing RNA editing was negatively biased, with an average value of -0.42 and an average value of -0.12 for all genes in the array A significant signal reduction. Therefore, it was shown that the presence of inosine in mRNA can be detected by this method.

FIG. 1 shows the detection of inosine by the matched-tissue method. FIG. 2 shows the identification of inosine sites by the inosine specific cleavage method. FIG. 3 shows an inosine specific chemical modification reagent. FIG. 4 shows the detection of inosine after the cyanoethylation reaction by mass spectrometry. FIG. 5 shows the detection of inosine by the primer extension method. FIG. 6 shows the principle of the ICE method. FIG. 7 shows the detection of inosine by the ICE method. FIG. 8 shows the detection of inosine by microarray. FIG. 9 shows the detection results of mRNA containing inosine by microarray.

SEQUENCE LISTING
<110> The University of Tokyo
<120> A method for detecting inosine site in RNA
<130> A51628A
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<212> DNA
<213> Artificial Sequence
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<223> Description of Artificial Sequence: Synthetic DNA
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<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 2
atggagaaga aactgcacaa tg 22
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
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Claims (13)

A method for detecting an inosynthetic site, comprising a step of chemically modifying an inosinylated site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group. The method according to claim 1, further comprising a step of subjecting the chemically modified RNA to a reverse transcription reaction to synthesize cDNA, and a step of detecting an inosine site based on the synthesized cDNA. The method according to claim 1 or 2, wherein the detection of an inosine site is detection of the presence or absence of an inosine site, quantification of the amount of inosine, and / or identification of a region or inosine site containing inosine. A compound having an α, β-unsaturated bond and an electron-withdrawing group is
Formula: C (R ¹ ) R ² = C (R ³ ) E
(Wherein, R ¹ and R ² each independently represents a hydrogen atom, a phenyl group having an alkyl group, a phenyl group, or an alkoxyl group having 1 to 6 carbon atoms from 1 to 6 carbon atoms, and R ¹ and R ² Any one of these may be bonded to E to form a ring, R ³ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a phenyl group, a phenyl group having an alkoxyl group having 1 to 6 carbon atoms, Alternatively, it represents an electron-withdrawing group, and E represents an electron-withdrawing group.
The method in any one of Claim 1 to 3 which is a compound represented by these. An electron-withdrawing _{group, CN, NO 2, SO 3} H, CONH 2, COCH 3, COOCH 3, COOC 2 H 5, COCH 3, COC 2 H 5, or COC ₆ H _5, Claim 4 the method of. 6. The method according to claim 1, wherein the chemically amplified RNA is subjected to a reverse transcription reaction to synthesize cDNA, and then the cDNA amplification reaction is performed. The inosinization site is detected by comparing cDNA synthesized by subjecting RNA not chemically modified to reverse transcription reaction as a control and cDNA synthesized from chemically modified RNA. The method in any one. The inosine-modified 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 inosine is subjected to mass spectrometry. The method according to claim 1, wherein the method is detected. The method according to any one of claims 2 to 7, wherein the chemically modified inosine is detected by detecting the length of cDNA derived from the chemically modified RNA. By chemically modifying the inosynthetic site by treating RNA with a compound having an α, β-unsaturated bond and an electron-withdrawing group, and then determining the base sequence of the chemically modified RNA or cDNA derived therefrom The method according to claim 1, wherein chemically modified inosine is detected. 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. The method described. An inosine-modified site modifier in RNA comprising a compound having an α, β-unsaturated bond and an electron-withdrawing group. The reagent kit for performing the method in any one of Claim 1 to 11 containing the modifier of Claim 12.

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