WO2025079347A1 - Method for labeling inosine base, method for detecting inosine base, method for determining sequence of nucleic acid, method for concentrating nucleic acid containing inosine base, inosine base labeling agent, and kit - Google Patents

Abstract

This method for labeling an inosine base in a nucleic acid includes bringing a nucleic acid and an inosine base labeling agent into contact with each other. The inosine base labeling agent contains: (A) an inosine base labeling site that contains an annular structure containing a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and electron-withdrawing groups respectively bonded to the two carbon atoms adjacent to the carbon-carbon double bond; and (B) an additional chemical structure directly or indirectly bonded to the nitrogen atom of the inosine base labeling site.

Description

Method for labeling inosine bases, method for detecting inosine bases, method for determining the sequence of nucleic acid, method for concentrating nucleic acid containing inosine bases, inosine base labeling agent, and kit

The present disclosure relates to a method for labeling inosine bases, a method for detecting inosine bases, a method for determining the sequence of a nucleic acid, a method for concentrating a nucleic acid containing an inosine base, an inosine base labeling agent, and a kit.

Many cells have a mechanism for modifying the chemical structure of bases in DNA and RNA. One known mechanism for modifying bases is the conversion of adenosine (A) to inosine (I) through deamination (known as "A-to-I editing"). Inosine is a nucleoside formed by binding hypoxanthine (in this disclosure, the base contained in inosine is also referred to as the "inosine base") to ribose. In RNA, adenosine is converted to inosine by deamination of its base adenine into hypoxanthine (inosine base). ADAR (adenosine deaminase acting on RNA), an adenosine deaminase enzyme, is responsible for A-to-I editing. For example, in the transcriptome of the human brain, adenosine is edited to inosine at 30,000 to 50,000 sites. Inosine forms base pairs with cytidine (C) just like guanosine (G), so converting adenosine to inosine has the same effect as converting adenosine to guanosine, and is thought to regulate the information and function of nucleic acids. This A-to-I editing can be a cause of various diseases, so elucidating its function is of great significance.

Technologies for detecting inosine in nucleic acids with the aim of elucidating the nucleic acid editing mechanism have been reported. For example, Patent Document 1 describes a method for detecting an inosine site, which includes a step of chemically modifying the inosine site by treating RNA with a compound having an α,β-unsaturated bond and an electron-withdrawing group. Non-Patent Document 1 proposes a method for detecting an inosine site in RNA by cyanoethylation of inosine using acrylonitrile. Non-Patent Document 2 proposes a method for labeling and affinity capturing inosine in RNA using acrylamide. Non-Patent Document 3 proposes a method for chemical profiling of inosine sites in RNA using phenylacrylamide.

　Conventionally, A-to-I editing was thought to occur only in double-stranded RNA due to the properties of the enzyme ADAR. However, in recent years, it has been discovered that RNA:DNA hybrid strands can also serve as substrates, and that deoxyadenosine in not only RNA but also DNA can be converted to deoxyinosine. This suggests that an active base editing mechanism by A-to-I editing is inherent in mammalian genomic DNA. As evidence of this, the function of A-to-I editing has been elucidated in telomere elongase-positive cultured human cancer cells (Non-Patent Document 4).

International Publication No. 2007/018169

Masayuki Sakurai et al., Nature Chemical Biology, vol.6, 733-740 (2010). Steve D. Knutson et al., Bioconjugate Chem, 29, 9, 2899-2903 (2018). Steve D. Knutson et al., Chemistry. 2020 Aug 6;26(44):9874-9878. Yusuke Shiromoto et al., Nature Communications volume 12, 1654 (2021).

　Although methods for modifying and detecting inosine on RNA have been reported, there is a need to develop a method for efficiently detecting inosine or deoxyinosine in RNA and in DNA that are present in trace amounts. In light of this situation, the present disclosure relates to a method for labeling inosine bases that can efficiently label inosine bases in nucleic acids; a method for detecting inosine bases, a method for determining the sequence of nucleic acids, and a method for concentrating nucleic acids that contain inosine bases that utilize this method; and an inosine base labeling agent and kit that can be used for these.

Means for solving the above problems include the following aspects.
<1> A method comprising contacting a nucleic acid with an inosine base labeling agent,
The inosine base labeling agent is
(A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
A method for labeling an inosine base in a nucleic acid, comprising:
<2> The method for labeling an inosine base according to <1>, wherein the additional chemical structure is selected from the group consisting of a detection label, an identification label, a purification label, a reaction label, and a functional group capable of binding these labels.
<3> The method for labeling an inosine base according to <1> or <2>, wherein the additional chemical structure is a detection label selected from the group consisting of a fluorescent dye, a visible light dye, and a luminescent substrate; or a purification label which is an affinity tag.
<4> The method for labeling an inosine base according to any one of <1> to <3>, wherein the cyclic structure of the inosine base labeling site is a five-membered cyclic structure.
<5> The method for labeling an inosine base according to any one of <1> to <4>, wherein the cyclic structure of the inosine base labeling moiety contains a maleimide ring.
<6> The method for labeling an inosine base according to any one of <1> to <5>, wherein the contact is carried out in a solution in which triethylamine is dissolved in ethanol, or in a mixed solvent of a phosphate buffer, NaCl, and an aprotic polar solvent.
<7> Labeling an inosine base in a nucleic acid by the labeling method according to any one of <1> to <6>;
detecting the labeled inosine base; and
A method for detecting an inosine base in a nucleic acid, comprising:
<8> The method for detecting an inosine base according to <7>, further comprising quantifying the labeled inosine base.
<9> The method for detecting an inosine base according to <7> or <8>, further comprising identifying a position of the labeled inosine base.
<10> The method for detecting an inosine base according to any one of <7> to <9>, wherein detecting the labeled inosine base comprises carrying out a complementary strand extension reaction using a nucleic acid containing the labeled inosine base as a template.
<11> A method for determining a sequence of a nucleic acid, comprising detecting an inosine base by the detection method according to any one of <7> to <10>.
<12> Labeling an inosine base in a nucleic acid by the labeling method according to any one of <1> to <6>;
concentrating nucleic acids containing labeled inosine bases;
A method for concentrating a nucleic acid containing an inosine base, comprising:
<13> (A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
13. An inosine base labeling agent comprising:
<14> The inosine base labeling agent according to <13>, wherein the additional chemical structure is selected from the group consisting of a detection label, an identification label, a purification label, a reaction label, and a functional group capable of binding these.
<15> The inosine base labeling agent according to <13> or <14>, wherein the additional chemical structure is a detection label selected from the group consisting of a fluorescent dye, a visible light dye, and a luminescent substrate; or a purification label which is an affinity tag.
<16> The inosine base labeling agent according to any one of <13> to <15>, wherein the cyclic structure of the inosine base labeling moiety is a five-membered ring.
<17> The inosine base labeling agent according to any one of <13> to <16>, wherein the cyclic structure of the inosine base labeling moiety contains a maleimide ring.
<18> A kit comprising the inosine base labeling agent according to any one of <13> to <17>, which is stored in a container.

The present disclosure provides a method for labeling inosine bases that can efficiently label inosine bases in nucleic acids; a method for detecting inosine bases, a method for determining the sequence of a nucleic acid, and a method for concentrating nucleic acids that contain inosine bases that utilize the method; and an inosine base labeling agent and kit that can be used for these.

The outline of the mechanism of inosinization is shown below. A-to-I RNA editing from adenosine (A) to inosine (I) is catalyzed by adenosine deaminase (ADAR) acting on RNA. Since I is not U/T but has a structure similar to G, as a result of editing, I forms a G=C-like base pair, stabilizing the I=C base pair. Therefore, in the central dogma, A-to-I editing can have an effect synonymous with editing the genetic code from A to G. A-to-I editing has traditionally been thought to only use double-stranded RNA (dsRNA) structures as substrates. In addition, inosine (or its base structure, hypoxanthine) in DNA chains was previously recognized as a base change phenomenon due to spontaneous deamination, and its existence was only recognized as a factor in the mutation phenomenon with an unknown cause and inevitable. However, recent research has revealed that ADAR has the ability to edit DNA chains in RNA/DNA hybrids. A comparison of the ICE method and the ICLAMP method is shown. (a) In the ICE method, acrylonitrile is used as an inosine base labeling agent, and 1-cyanoethylinosine is produced by a Michael addition reaction mechanism with the 1-position of inosine. Of acrylonitrile and the cyanoethyl group, which is the added structure after labeling, CN (cyano group) is extremely chemically stable. Therefore, it is not possible to add a further chemical structure to the cyano group before or after the labeling reaction, and the applicability as a detection technique for inosine is limited. On the other hand, maleimide can cause a Michael addition reaction with inosine like acrylonitrile, and further, by replacing the hydrogen atom of the NH group with another added chemical structure, fluorescent labeling or biotin labeling of inosine is possible. A new labeling technique for rare inosines in DNA and RNA strands is outlined. FITC-maleimide is used as a fluorescent label for inosine, allowing visual detection and quantification. Biotin-maleimide can be used to introduce a biotin tag to the inosine base of nucleic acids. Inosine-containing nucleic acids can be further enriched by affinity purification using streptavidin-conjugated magnetic beads. The acid dissociation constant pKa values of each base are compared. The reactivity of FITC-maleimide with DNA and RNA oligonucleotides was compared by denaturing PAGE analysis. The reaction rate increased over time and the trend was almost the same. The fluorescent signal of the band using 10 mM FITC-maleimide became brighter and more distinct. The reactivity of inosine-specific fluorescent labeling was evaluated by denaturing PAGE analysis. The reaction between DNA oligonucleotides and FITC-maleimide peaked at 1 hour. The efficiency of labeling DNA oligonucleotides with 10 mM FITC-maleimide within 1 h was examined by denaturing PAGE analysis. A maximum labeling rate of 40% was derived from band densitometry, and the I/A ratio indicated that the most specific reaction time was 15 min. Values represent the mean ± standard deviation (n = 3). A comparison of FITC-maleimide labeling efficiency at 2 mM, 10 mM, and 50 mM concentrations is shown, with minimal non-specific labeling as judged by the I/A ratio at 10 mM for 15 min, while the labeling efficiency of I was 80% at 50 mM for 30 min. The efficiency of labeling DNA oligonucleotides with 50 mM FITC-maleimide within 1 h was examined by denaturing PAGE analysis. A maximum labeling rate of 80% was derived from band densitometry, and the I/A ratio indicated that the most specific reaction time was 15 min. Values represent the mean ± standard deviation (n = 3). The reaction specificity of maleimide with inosine and other bases is shown in the comparative quantification results by densitometry of PAGE analysis of nucleic acids after the labeling reaction. Maleimide specifically labels inosine. Values represent the mean ± standard deviation (n = 3). Optimization of the inosine-specific biotin labeling method and streptavidin-based enrichment and purification is shown. The sequences of four oligonucleotides of different lengths are listed on the left. Biotin-maleimide labeling increased the band distribution position in the gel. PAGE analysis and densitometric quantification of pulled-down DNA oligonucleotides showed that biotin-maleimide-labeled inosine was significantly separated and purified, while the contamination of inosine-free FAM-30nt-dU and FAM-24nt-dA was negligible. An outline of the procedure for confirming biotinylation enrichment and purification using an endogenous RNA editing site as a model by the primer extension method is shown. The outline and results of the confirmation procedure of biotinylation enrichment purification using the endogenous RNA editing site as a model by the primer extension method are shown. Primer extension was performed from several bases downstream of the I site for mouse glutamate receptor mRNA. Detection was performed by autoradiography using a DNA primer with a 32P-labeled 5' end. Under non-reactive conditions, the primer is extended and cDNA incorporating a C base is synthesized at the site complementary to the I base on the template mRNA, terminating at the end of the RNA. Under CE+ and MI+ conditions, transcription ends immediately adjacent to the labeled I residue, as shown in the PAGE analysis on the right. The results of biotinylation enrichment and purification using the primer extension method as a model of endogenous RNA editing sites are shown. Primer extension begins several bases downstream of the I site of mouse glutamate receptor mRNA, and cDNA is synthesized by replacing dCTP with α-32P dCTP. Under non-reactive conditions, the primer is extended to synthesize cDNA incorporating a C base at the site complementary to the I base on the template mRNA, and terminates at the end of the RNA. The extended cDNA is radioactively labeled with ^32P at all C bases. Under CE+ and MI+ conditions, transcription terminates immediately adjacent to the labeled I residue, and cDNA containing two C residues labeled with ^32P is generated as shown in the left figure. The enrichment and purification steps can significantly reduce nonspecific background interference. 1 shows a standard curve prepared in the verification of the quantification of inosine using an inosine-containing oligonucleotide of known concentration and FITC-maleimide. The horizontal axis shows the substrate concentration, and the vertical axis shows the fluorescence intensity of FITC. 1 shows the results of PAGE analysis in the study of aqueous reaction solvents. This shows the fluorescence intensity ratio of FITC to Cy5 (FITC/Cy5) for each sample in the study of aqueous reaction solvents. The horizontal axis represents the sample number. 1 shows the fluorescence intensity ratio (I/A) of a deoxyinosine-containing oligonucleotide to a deoxyadenine-containing oligonucleotide in each buffer in an investigation of aqueous reaction solvents. The outline and results of the comparison of reaction characteristics with acrylonitrile and verification of the mechanism are shown. Workflow based on the reactivity of acrylonitrile and inosine (top). As for the conditions for comparison, first, inosine was labeled singly with FITC-maleimide. Second, inosine was reacted with acrylonitrile first, and then the reaction product was incubated with FITC-maleimide for labeling. Third, acrylonitrile and FITC-maleimide were added simultaneously to the reaction system to perform a competitive reaction with inosine by both labeling agents. The labeling efficiency by FITC-maleimide after carrying out these reaction conditions was compared by quantifying the fluorescence intensity detection of FITC by PAGE analysis of the reaction product (bottom). As a result of the analysis, the fluorescent signal of the label was attenuated under the condition where the product was labeled with maleimide after the reaction with acrylonitrile (ce ¹ I → MI) compared to the condition where the product was labeled with maleimide only (MI). This suggests that the Michael addition reaction of maleimide to inosine occurs at the N1 position of inosine, similar to the Michael addition reaction of acrylonitrile to inosine, and therefore the labeling with maleimide was inhibited by the reaction with acrylonitrile. On the other hand, the fluorescence intensity was slightly decreased in CE+MI, in which acrylonitrile and maleimide were simultaneously applied to inosine. This indicated that maleimide reacts with inosine faster than acrylonitrile. The results of the verification of the superiority of maleimide derivatives in inosine labeling reactions compared with acrylamide derivatives are shown below. Acrylamide fluorescein labeling reactions were performed in TEAA buffer containing 250 mM FITC-acrylamide and 10 pmol/μL DNA oligonucleotide, as described in the literature. Meanwhile, FITC-maleimide labeling reactions were performed in TEAA buffer containing 10 mM FITC-maleimide and 1 pmol/μL DNA oligonucleotide. Reaction times were between 15 min and 24 h, and the labeling efficiency of inosine was compared by PAGE analysis and densitometry quantification using green fluorescence. In the case of acrylamide fluorescein, inosine labeling was not detected until the reaction time was 8 hours or more, whereas the labeling efficiency of FITC-maleimide was higher after 15 min to 1 h reaction than after 24 h reaction with acrylamide fluorescein. The results of the verification of the superiority of maleimide derivatives in inosine labeling reactions in comparison with acrylamide derivatives are shown. The results of the verification of the superiority of maleimide derivatives in comparison with acrylamide derivatives are shown. Inosine labeling was performed according to the FITC-maleimide method, with acrylamide fluorescein and FITC-maleimide at the same concentration of 10 mM, in TEAA buffer, and 1 pmol/μL DNA oligonucleotide, at 70°C for 15 minutes to 24 hours. The labeling efficiency of inosine was compared by PAGE analysis and densitometry quantification using green fluorescence. In the case of acrylamide fluorescein at a concentration of 10 mM, only slight inosine labeling was detected after 24 hours of reaction, while in the case of FITC-maleimide, a higher labeling efficiency was observed in the reaction from 15 minutes to 1 hour than in the reaction with acrylamide fluorescein after 24 hours. In addition, the FITC-maleimide labeling reaction reached a plateau after 1 hour of reaction, and no improvement in the labeling rate was observed by extending the reaction time beyond that.

Below, the form for carrying out the embodiment of the present disclosure will be described in detail. However, the embodiment of the present disclosure is not limited to the following embodiment. In the following embodiment, the components (including element steps, etc.) are not essential unless specifically stated. The same applies to the numerical values and their ranges, and they do not limit the embodiment of the present disclosure.

In the present disclosure, the term "step" includes not only a step that is independent of other steps, but also a step that cannot be clearly distinguished from other steps as long as the purpose of the step is achieved.
In the present disclosure, the numerical range indicated using "to" includes the numerical values before and after "to" as the minimum and maximum values, respectively.
In the numerical ranges described in the present disclosure in stages, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit value of the numerical range may be replaced with a value shown in the examples.
In the present disclosure, the hypoxanthine moiety as a base contained in a nucleoside or nucleotide may be referred to as an "inosine base,""inosine," or "I." Note that in the present disclosure, "inosine base,""inosine," or "I" as a base all encompass hypoxanthine as a base. For example, the term "inosine base" includes not only the hypoxanthine portion of inosine (i.e., a nucleoside in which hypoxanthine and ribose are bonded), but also, for example, the hypoxanthine portion of deoxyinosine (i.e., a nucleoside in which hypoxanthine and deoxyribose are bonded); the hypoxanthine portion of a nucleotide in which these nucleosides are bonded to a phosphate group; the hypoxanthine portion of a DNA or RNA containing these nucleosides or nucleotides; the hypoxanthine portion obtained by converting adenosine in adenosine monophosphate (AMP), adenosine diphosphate (ADP), or adenosine triphosphate (ATP) to hypoxanthine, etc. In the art, the hypoxanthine portion contained in the above molecule is generally referred to as "inosine" or "I", and therefore, in accordance with commonly understood terms, the term "inosine base", "inosine" or "I" is used in the present disclosure for the above hypoxanthine portion.

<Method for labeling inosine base>
In one aspect of the present disclosure, a method for labeling an inosine base in a nucleic acid includes contacting a nucleic acid with an inosine base labeling agent, the inosine base labeling agent comprising:
(A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
In the present disclosure, "labeling" a base in a nucleic acid, or a nucleoside or nucleotide containing the same means chemically labeling the base, or the nucleoside or nucleotide containing the same.

First, we will explain the mechanism by which adenosine in nucleic acids is converted to inosine. As an example, the mechanism by which adenosine on RNA is converted to inosine is shown in the diagram below. The conversion of adenosine to inosine occurs when adenine is deaminated and converted to an inosine base, as shown below.

Since inosine has a similar structure to guanosine, the inosinization of adenosine can essentially be regarded as the guanosinization of adenosine. Therefore, the inosinization of adenosine can also be regarded as a mechanism for introducing mutations from adenosine to guanosine, and elucidating this genetic mechanism is of great significance. Although techniques for detecting inosinized sites on RNA have been reported to date, the present inventors have devised a method that can be applied to both RNA and DNA and that efficiently labels inosine bases specifically. This labeling can be applied to various applications, such as the detection of inosine bases, the detection of nucleic acids containing inosine bases, sequencing, concentration, and purification, in RNA, DNA, and various other nucleic acids.

The labeling mechanism of inosine bases by the inosine base labeling agent used in the labeling method disclosed herein is shown in the diagram below. In the diagram below, the inosine base labeling agent is shown as a hook-shaped inosine base labeling site (A) to which an additional chemical structure (B) shown as a circle is linked.

The reactivity of Michael addition at the N1 position of inosine, the relationship between pH and pK, and the equilibrium state have been verified and reported for inosine on transfer RNA in M Yoshida, T Ukita, J Biochem. 1965 Jun;57(6):818-21. The same document also shows that acrylonitrile is added to inosine. Based on this feature, Masayuki Sakurai et al., Nature Chemical Biology, vol.6, 733-740 (2010) reported the verification results using acrylonitrile as an electrophilic labeling agent.
On the other hand, acrylonitrile is difficult to label with any desired label and is therefore less convenient. However, according to the labeling method of the present disclosure, a specific inosine base labeling agent is used, which improves the convenience of labeling.

Among A, G, C, T, and I that constitute the nucleic acid bases in DNA and RNA, I has an acid dissociation constant pKa value closest to 7. Since the reaction mode of the labeling agent in the present disclosure is an electrophilic addition reaction, I, which has a pKa value closest to the pH and whose equilibrium is tilted toward a negative charge state, has the highest reactivity among the nucleic acid bases that have a negative charge in a neutral to weakly alkaline solution.
On the other hand, the inosine base labeling agent used in the labeling method of the present disclosure has the inosine base labeling site (A) and the addition chemical structure (B). Due to the influence of the electron-withdrawing group at the inosine base labeling site (A) of the inosine base labeling agent, the carbon atom forming the carbon-carbon double bond is positively charged. This carbon atom undergoes electrophilic addition to the nitrogen atom at position 1, which is the active amine of the inosine base, and the addition modification to the inosine base proceeds. Since the inosine base labeling site (A) has a cyclic structure, it is considered that there is little steric hindrance and that this contributes to the high reaction rate with the inosine base. Therefore, it is considered that the inosine base labeling agent has high reactivity with the inosine base and that the binding proceeds quickly. In addition, the nitrogen atom at the inosine base labeling site (A) facilitates binding to the addition chemical structure.

As an example, the reaction mechanism of maleimide with inosine base when maleimide is used as the inosine base labeling site and an alcohol-containing solvent is used as the solvent is shown in the figure below. In the figure, R-OH represents the alcohol in the solvent (e.g., ethanol), and R represents any organic group.

The inosine base has an α,β-unsaturated carbonyl structure containing a nitrogen atom, and the electron at the N1 position is in a conjugated state with the oxygen at the 1st position or the nitrogen at the 3rd position via a double bond. Therefore, the hydrogen at the N1 position is in a state that is easily liberated as a proton, and the free inosine base and the non-free inosine base exist in equilibrium. In a weakly alkaline pH environment, the free inosine base is most abundant at the N1 position.
On the other hand, in maleimide, the electron-withdrawing property of the oxygen atom generates a positive charge at the carbon atom at position 4 in the figure through the unsaturated electron pair and double bond. Since maleimide has linear symmetry, a positive charge can also be generated at the carbon atom at position 3. An electrophilic addition reaction (Michael addition reaction) occurs from the positively charged carbon atom of maleimide to the negatively charged nitrogen atom at position N1 of the inosine base. This causes the inosine base to be labeled with maleimide.
Based on the above-mentioned mechanism of action, it is believed that the inosine base labeling agent used in the labeling method of the present disclosure can efficiently label inosine bases in nucleic acids.

(Nucleic Acid)
The nucleic acid to be used in the labeling method of the present disclosure may be any nucleic acid (DNA, RNA, a mixture thereof, an analog thereof, a natural product, an artificial product, etc.), or a nucleic acid to which a low molecular weight compound, a group, a molecule other than nucleic acid, a structure, etc. is linked. The DNA may be any DNA, such as genomic DNA, non-genomic DNA, viral DNA, etc. The RNA may be any RNA, such as mRNA, rRNA, tRNA, non-coding (nc) RNA, viral DNA, etc.

The origin of the nucleic acid that is the subject of the labeling method of the present disclosure is not particularly limited, and may include organisms such as animals (e.g., mammals (e.g., humans and non-human mammals), birds, amphibians, reptiles, fish, chordates, arthropods, etc.), plants, fungi, bacteria, etc.; viruses, etc.

The labeling method disclosed herein labels the inosine base, and therefore can label both inosine, a nucleoside in which the inosine base is bound to ribose, and deoxyinosine, a nucleoside in which the inosine base is bound to deoxyribose. In addition, the labeling method disclosed herein can label any nucleoside that contains the inosine base as a base.

(Inosine base labeling agent)
Inosine base labeling agent is
(A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
Includes.

(A) Inosine base labeling site The inosine base labeling site is a site of an inosine base labeling agent that targets and binds to an inosine base, and is typically a site that chemically bonds to an inosine base. The inosine base labeling site includes a carbon-carbon double bond, a cyclic structure including two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and electron-withdrawing groups that are bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond.

In the present disclosure, the "cyclic structure" refers to a single ring structure. The cyclic structure of the inosine base labeling site is preferably a cyclic structure having an odd number of atoms (5 or more) on the ring, preferably a 5-membered ring structure or a 7-membered ring structure, and more preferably a 5-membered ring structure. When the cyclic structure has an odd number of atoms on the ring, the molecular linear symmetry is high and positive charges are likely to be generated on each of the two carbon atoms adjacent to the carbon-carbon double bond, which is thought to enhance reactivity with inosine bases. When the cyclic structure is a 5-membered ring structure, the molecular structure has a large strain, which is thought to enhance reactivity with inosine bases and enable more efficient labeling of inosine bases.

The number of carbon-carbon double bonds in the cyclic structure may be one or two or more, and from the viewpoint of ease of obtaining or producing the compound, one to three are preferred, one or two are more preferred, and one is even more preferred.

Each carbon atom in the carbon-carbon double bond of the cyclic structure has an adjacent carbon atom. In other words, the "two carbon atoms adjacent to the carbon-carbon double bond" means both carbon atoms adjacent to and bonded to each carbon atom in the carbon-carbon double bond. However, the number of carbon atoms adjacent to and bonded to each carbon atom in the carbon-carbon double bond does not have to be one, and may be two or more.

The nitrogen atom in the cyclic structure functions as a site for linking the additional chemical structure (i.e., the site to which the additional chemical structure is to be added). The number of nitrogen atoms in the cyclic structure may be one or more than one, and from the viewpoint of ease of obtaining or producing the compound, one to three is preferred, one or two is more preferred, and one is even more preferred.

The cyclic structure may have additional atoms in addition to the carbon-carbon double bond, the two carbon atoms adjacent to the carbon-carbon double bond, and the nitrogen atom. Additional atoms include carbon atoms and heteroatoms such as oxygen atoms and sulfur atoms.

In one embodiment, the cyclic structure of the inosine base labeling site preferably contains a maleimide ring.

Examples of the electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond include -CN, _-NO2 , _-SO3H , -CONH2, _-COCH3 , _-COOCH3 , _-COOC2H5 , _-COCH3 , _{-COC2H5 , -COC6H5 , =} O, and _the like.

In one embodiment, the inosine base labeling site preferably has a structure represented by the following formula (M1). The structure represented by formula (M1) is a five-membered ring and has high linear symmetry, and is therefore considered to have high reactivity with inosine bases and to enable efficient labeling.

In formula (M1), R ¹ and R ² each independently represent a hydrogen atom or a hydrocarbon group, Y represents an electron-withdrawing group, and * represents a bonding site with an adjacent atom.

The hydrocarbon group represented by R ¹ and R ² includes a substituted or unsubstituted alkyl group.
The alkyl group is preferably an alkyl group having 1 to 10 carbon atoms, more preferably an alkyl group having 1 to 5 carbon atoms, and more preferably an alkyl group having 1 or 2 carbon atoms. The alkyl group may be a linear alkyl group or a branched alkyl group.
When the alkyl group has a substituent, the substituent is preferably a substituent that has no reactivity during labeling of an inosine base, and examples of the substituent include an alkoxy group (e.g., a methoxy group, an ethoxy group), a halogeno group (e.g., a chloro group, an iodo group), etc.

Examples of the electron-withdrawing group represented by Y include an oxygen atom, and an atom or atomic group having an unsaturated electron pair other than an oxygen atom.
Examples of atoms or atomic groups having an unsaturated electron pair other than an oxygen atom include nitrogen and a nitro group.
From the viewpoint of availability, Y is preferably an oxygen atom.

The * indicates a bonding site with an adjacent atom, and the inosine base labeling site represented by formula (M1) bonds to the additional chemical structure via the *.

(B) Additive Chemical Structure The additive chemical structure is a site having a desired function, which is directly or indirectly bound to the nitrogen atom of the inosine base at the site to which it is added by reaction with a labeling agent. Here, the term "function" refers to any function intended to be imparted to the labeled inosine base, such as detection and/or quantification of a molecule by the labeled chemical structure, interaction and/or binding with other molecules by the labeled chemical structure, or a function of causing a chemical reaction or an enzymatic reaction with other molecules, which is possessed by the chemical structure added by labeling.

Examples of the added chemical structure include a detection label, an identification label, a purification label, a reaction label, and the like.
In the present disclosure, an adduct chemical structure that is a detection label means a chemical structure that has properties that enable the presence and/or quantification of an inosine base having an adduct site or a nucleic acid molecule containing the same by detection using an appropriate method.
In the present disclosure, the additional chemical structure that is an identifying label means a chemical structure that has properties that enable improved sensitivity and/or accuracy in detecting an inosine base having an additional site or a nucleic acid molecule containing the same, or that enable clarification of the difference from a comparison target.
In the present disclosure, the additional chemical structure that is a purification label means a chemical structure having a property that enables the purification of an inosine base having an additional site or a nucleic acid molecule containing the same. Specifically, for example, a nucleic acid molecule containing an inosine base having an additional site is captured by a compound having a specific ability to interact or form a bond (affinity) with the inosine base or nucleic acid molecule, or a carrier (resin, magnetic beads, etc.) to which the compound is immobilized, and after washing away non-specific and non-binding molecules, the nucleic acid molecule containing the inosine base can be released or purified to a state that can be used for the next reaction.
In the present disclosure, an addition chemical structure that is a reaction label means a chemical structure that has properties that enable an analytical technique specific to the site where the inosine base is present by chemically reacting an inosine base having an addition site or a nucleic acid molecule containing the same with a substrate compound.

Additional chemical moieties that are detectable labels include fluorescent dyes, visible light dyes, luminescent substrates, and the like.
Examples of fluorescent dyes include structures that are used in research in the field of molecular and cell biology, have excitation or fluorescence wavelengths in the ultraviolet to near-infrared range, and can be detected by a fluorescent microscope, a fluorescent detection device, etc. Specific examples of fluorescent dyes include fluoresceins, azos, rhodamines, coumarins, pyrenes, cyanines, etc. In addition, examples of fluorescent dyes include fluorescently labeled antibodies such as FITC, Cy5, Alexa, and DyLight, and fluorescent dye crosslinkers with maleimides for fluorescent labeling of proteins.
Visible light dyes include compounds that absorb visible light, such as blue, and exhibit color when viewed with the naked eye or with a conventional microscope or detection device in molecular and cell biology experiments.
Examples of luminescent substrates include compounds that serve as substrates for luciferase, such as luciferin.

Additional chemical structures that are identification markers include chemical structures that increase the molecular weight, such as alkyl groups (methyl groups, ethyl groups, etc.) and phenol ring derivatives (phenol groups, etc.); bulky structures that cause steric hindrance; structures with high ionization efficiency (chemical structures such as cyano groups that improve ionization efficiency during analysis in a mass spectrometer); and chemical structures that significantly change the current transmittance/resistance value in nanopores, etc.

Examples of additional chemical structures that are purification labels include affinity tags used in tag-ligand affinity purification; structures that react with specific functional groups to form strong bonds such as covalent bonds or ionic bonds; and the like.
Examples of affinity tags used in tag-ligand affinity purification include chemical structures that are captured by a chemical structure immobilized on a carrier such as a resin or magnetic beads due to affinity or bond formation, such as a biotin structure such as biotin-streptavidin, and a chemical structure that forms a covalent bond such as NHS ester.

Additional chemical structures of reaction labels include chemical structures that interact with specific compounds by forming a complex; chemical structures that have catalytic activity against specific compounds; one of the fluorescent labels used in FRET assays; chemical structures that emit fluorescence after cleavage by an enzyme such as a protease; chemical structures that enable complementary strand extension by a DNA primer cross-linked with streptavidin and a polymerase after biotin labeling; chemical structures that enable secondary or tertiary signal enhancement by an antibody cross-linked with streptavidin after biotin labeling; and other chemical structures that specifically bind to additional chemical structures and show a color reaction due to their activity acting on a specific substrate.

In one embodiment, the additional chemical structure may be a functional group capable of linking other chemical structures (such as a label). In other words, even if the additional chemical structure itself does not have the function of a label, if it is capable of further linking other labels, it can exert a similar function by linking the label to the inosine base labeling site. Examples of functional groups capable of linking other chemical structures include amines, carboxyl groups, hydroxyl groups, thiol groups, alkyne groups, etc. Alternatively, the functional group may be a chemical structure that allows the use of a unique tag and ligand set, such as a FLAG tag or an HA tag.

When the inosine base labeling site (A) and the additional chemical structure (B) are indirectly bound, the inosine base labeling site and the additional chemical structure may be bound via any linking group. The linking group is not particularly limited as long as it does not impair the purpose of the labeling method of the present disclosure, and may be any hydrocarbon group or a combination of any hydrocarbon group and any heteroatom or group. Examples of the linking group include an alkylene group having 1 to 6 carbon atoms; a combination of an alkylene group having 1 to 6 carbon atoms and one or more of -O-, -NR- (R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), -C(O)O-, -OCO-, -CO-, -CONH-, -SO-, and -SO ₂ -; -C=C-, -C≡C-, and the like.

[Labeling method]
The labeling of inosine bases is carried out by contacting a nucleic acid with an inosine base labeling agent.
Labeling of inosine bases can be carried out in a solvent, which may include water, an organic solvent, an aqueous solvent, an acid or base for adjusting the pH, or any mixture thereof.
Examples of the organic solvent include alcohols (such as ethanol), amines (such as triethylamine), and aprotic polar solvents (such as dimethylformamide (DMF), dimethylimidazolidinone (DMI), dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidinone, and sulfolane).
Examples of the aqueous solvent include a phosphate buffer solution, a mixture of a phosphate buffer solution and NaCl, and the like.
The acid includes hydrochloric acid, acetic acid, and the like.
Examples of the base include sodium hydroxide, potassium hydroxide, and aqueous ammonia.
In order to provide versatility in selecting a method for purifying nucleic acid after the labeling reaction, it is preferable that everything other than the nucleic acid and the labeling agent is volatile.

In one embodiment, it is preferable to use a solution in which triethylamine (TEA) is dissolved in ethanol as a solvent. A solution in which triethylamine (TEA) is dissolved in ethanol has affinity for both water-soluble molecules and organic compounds, and is therefore suitable for the reaction of nucleic acid with an inosine base labeling agent. In addition, triethylamine is a tertiary amine, and is preferable because it has the effect of stably retaining an inosine base labeling agent activated in a solvent. Furthermore, a solution in which triethylamine (TEA) is dissolved in ethanol contains ethanol, so that the higher-order structure, such as a double-stranded structure, which a nucleic acid can have in an aqueous solution at room temperature, exists in a dissolved state, and the labeling of the inosine base occurs uniformly regardless of the structure of the nucleic acid.
Since triethylamine is alkaline as it is, it is preferable to titrate it with an acid such as acetic acid to adjust the pH to the pH described below. Hereinafter, the solvent whose pH has been adjusted with acetic acid is also referred to as a "TEAA buffer solution."
In one embodiment, the TEAA buffer can activate the reaction efficiency of the inosine base labeling agent at around 70°C.

The alcohol concentration in the TEAA buffer solution is, for example, preferably 30 to 60% by volume, more preferably 40 to 50% by volume, and may be, for example, 50% by volume.
The pH of the solvent is preferably from 7.0 to 10.0, more preferably from 7.0 to 9.0, and even more preferably from 8.0 to 9.0, and may be, for example, 8.6.

In one embodiment, a mixed solvent of phosphate buffer, NaCl, and an aprotic polar solvent (dimethylformamide (DMF), dimethylimidazolidinone (DMI), dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidinone, sulfolane, etc.) may be used. From the viewpoint of performing an inosine base-specific reaction well, it is preferable to use a mixed solvent of 0.01 to 0.02 M phosphate buffer, 0.15 to 0.5 M NaCl, and 2.7 to 5.4 M dimethylformamide (DMF) or dimethylimidazolidinone (DMI) at final concentrations.

In addition to the nucleic acid and inosine base labeling agent, the reaction system may contain polyethylene glycol to improve the reaction.

From the viewpoint of facilitating the reaction, the reaction temperature is preferably 50°C to 90°C, more preferably 60°C to 80°C, and even more preferably 65°C to 75°C, and may be, for example, 70°C.

From the viewpoint of carrying out a sufficient reaction, the reaction time is preferably 5 minutes or more, more preferably 15 minutes or more, may be 30 minutes or more, or may be 60 minutes or more. On the other hand, since the inosine base labeling agent used in the present disclosure has a high reaction rate, labeling can be carried out by a relatively short reaction time. From the viewpoint of shortening the process, the reaction time is preferably 180 minutes or less, more preferably 120 minutes or less, even more preferably 90 minutes or less, or may be 60 minutes or less. From the above viewpoints, the reaction time is preferably 5 to 180 minutes, more preferably 5 to 120 minutes, even more preferably 15 to 90 minutes, and particularly preferably 15 to 60 minutes.

The concentration of the inosine base labeling agent in the reaction system is preferably 1 to 100 mM, more preferably 5 to 80 mM, and even more preferably 10 to 50 mM, from the viewpoint of facilitating the reaction.

If the additional chemical structure is a functional group capable of binding other labels such as detection labels, identification labels, purification labels, and reaction labels, a reaction to add a further detection label, identification label, purification label, reaction label, and the like may be carried out before, in parallel with, and/or after the above reaction.

<Method for detecting inosine base>
In one embodiment, there is provided a method for detecting an inosine base in a nucleic acid, comprising labeling an inosine base in a nucleic acid by the method for labeling an inosine base, and detecting the labeled inosine base.
The detection may be detection of the presence or absence of an inosine base, detection of the amount of an inosine base, or detection of the position of an inosine base (including a region containing an inosine base). Thus, the method for detecting an inosine base may include quantifying the labeled inosine base. The method for detecting an inosine base may include identifying the position of the labeled inosine base.
Detection of inosine bases may be performed by comparing unlabeled nucleic acid as a control with the labeled nucleic acid.

When the additional chemical structure (B) contained in the inosine base labeling agent is a detection label, the detection of the inosine base may be performed by detecting the detection label. For example, when the detection label is a fluorescent dye, the presence or absence, amount, etc. of the inosine base can be detected by detecting the fluorescent dye.

Detection of inosine bases may be performed by subjecting the labeled nucleic acid to mass spectrometry. Mass spectrometry can detect the presence or absence, amount, etc. of inosine bases. In mass spectrometry, the labeled nucleic acid may be directly analyzed, or the labeled nucleic acid may be partially fragmented with a nuclease and then analyzed, or the labeled nucleic acid may be decomposed into nucleosides with a nuclease and then analyzed. For example, the labeled nucleic acid may be decomposed into nucleic acid fragments or nucleosides, and the inosine base can be detected by the increase or decrease in the peak of the nucleic acid fragment or nucleoside containing the inosine base compared to the unlabeled nucleic acid in liquid chromatography/mass spectrometry (LC/MS).

The method for detecting inosine base may include performing a complementary chain extension reaction (for example, reverse transcription reaction when the target nucleic acid is RNA) using a nucleic acid containing a labeled inosine base as a template. When the inosine base is labeled, the label inhibits the incorporation of cytidine, and the extension of the complementary chain stops before that point. Therefore, the detection of the inosine base is possible by examining the synthesized complementary chain (for example, cDNA when the target nucleic acid is RNA).
After synthesizing a complementary strand by a complementary strand extension reaction, an amplification reaction may be carried out using the complementary strand as a template.

The complementary strand extension reaction can be carried out by mixing a template nucleic acid, a primer, dNTPs, a nucleic acid extension enzyme, etc. in an appropriate reaction solution (e.g., a buffer solution) and incubating at a predetermined temperature for a certain period of time. When detecting inosine sites using the Sanger sequencing method or direct sequencing method, a primer having a gene-specific sequence (GSP primer) may be used as the primer. On the other hand, in next-generation sequencing (NGS) analysis, etc., a random primer may be used as the primer. By using a random primer, even if multiple inosine bases are present in one nucleic acid molecule, the detection of the multiple inosine bases becomes easier.

For example, inosine bases may be detected by examining the length of the synthesized complementary strand. This allows the presence, absence, amount, position, etc. of inosine bases to be detected. The length of the synthesized nucleic acid can be examined by standard methods such as electrophoresis.

Also, inosine bases may be detected by analyzing the base sequence of the synthesized complementary strand. This allows the presence, absence, amount, position, etc. of inosine bases to be detected.

The synthesized complementary strand may also be detected using a probe. As described above, the complementary strand whose extension has stopped before the inosine base site becomes a short complementary strand lacking the upstream sequence of the template nucleic acid. Therefore, if a probe containing only the sequence upstream of the inosine base position is used, it will react with the complementary strand of an unlabeled nucleic acid but will not react with the complementary strand of a labeled nucleic acid. Therefore, the presence or absence, amount, etc. of the inosine base can be detected by detecting the presence or absence or intensity of the signal of the probe that has reacted with the nucleic acid. The position of the inosine base can also be identified by designing the probe.

When the nucleic acid of interest is RNA, the method for detecting the inosine base may include subjecting the labeled RNA to a reverse transcription reaction to synthesize cDNA. The inosine base may be detected based on the synthesized cDNA.
The reverse transcription reaction can be carried out by a conventional method. For example, the reverse transcription reaction can be carried out by mixing template RNA, primer, dNTP, reverse transcriptase, etc. in an appropriate reaction solution (e.g., buffer solution) and incubating at a predetermined temperature for a certain period of time. A random primer may be used as the primer.
After synthesis of the cDNA, a cDNA amplification reaction may be carried out. In this case, for example, reverse transcription and amplification may be carried out in a series of operations by RT-PCR, or transcription amplification may be carried out using the reverse transcribed cDNA as a template.
As described above, the synthesis of cDNA can be inhibited specifically at the labeled site of the inosine base in the reverse transcription reaction, and therefore the inosine base may be detected by the above-mentioned method using the synthesized cDNA.
As a control, unlabeled RNA may be subjected to reverse transcription reaction to synthesize cDNA, and inosine bases may be detected by comparing with cDNA synthesized from labeled RNA.
When the target nucleic acid is RNA, the method described in WO 2007/018169 can be used to detect inosine.

Sequencing Method
In one embodiment, a method for determining the sequence of a nucleic acid is provided, which includes detecting an inosine base by the inosine detection method. According to the inosine detection method, it is possible to detect an inosine base in any nucleic acid, including RNA or DNA expressed in trace amounts, which has been difficult to do so far, and the detection method can be applied to nucleic acid sequencing by identifying the position of inosine. In one embodiment, the method for determining the sequence of a nucleic acid includes a sequencing step, and the sequencing step includes determining the sequence of a nucleic acid other than inosine and detecting the inosine base by the inosine detection method. The sequencing of a nucleic acid other than inosine can be performed by a known method.

<Method for concentrating nucleic acids containing inosine bases>
In one aspect, there is provided a method for concentrating a nucleic acid containing an inosine base, the method comprising: labeling an inosine base in a nucleic acid by the inosine base labeling method; and concentrating a nucleic acid containing the labeled inosine base.
The term "enrich" refers to, for example, labeling only inosine bases in a molecular population of nucleic acid strands, and separating the nucleic acid strands containing the labeled inosine from the nucleic acid strands not containing inosine and those in which all the inosine bases present on a single molecule of the nucleic acid strand are not labeled, thereby increasing the proportion of nucleic acid molecules containing the labeled inosine base in the separated product. The enrichment can be performed, for example, by using the inosine base labeling agent described above or an additional label added to the inosine base labeling agent to separate the nucleic acid containing the labeled inosine base from the nucleic acid not containing inosine or the nucleic acid in which all the inosines present on a single molecule are unlabeled.

Concentration of nucleic acids containing inosine bases can be achieved, for example, when a purification label is used as the additional chemical structure of the inosine base labeling agent, by extracting the labeled inosine base-containing nucleic acids using a carrier that binds to the purification label.
For example, if an inosine base labeling agent having biotin as an additional chemical structure is used, the labeled nucleic acid can be purified using streptavidin cross-linked beads, etc. The purification label is not limited to biotin, and the above-mentioned purification labels can be used, and nucleic acids containing inosine bases can be concentrated using known purification techniques.

Combinations of purification labels and carriers include the following:
Biotin and streptavidin, fluorescein and anti-fluorescein antibody, FLAG tag and anti-FLAG tag antibody, HA tag and anti-HA tag antibody, MBP tag and amylose, DIG (digoxigenin) and anti-DIG antibody, other tag functional groups and antibodies against them, other tags that do not cause changes in the inosine labeling reaction and can be prepared as derivatives of inosine base labeling agents and ligands against them

<Kit>
In one embodiment, a kit is provided that includes the inosine base labeling agent stored in a container. The kit may be a kit for labeling, detecting, sequencing, enriching or purifying an inosine base.

The kit may further include any other reagents, etc., depending on the purpose. For example, the kit may include, for the above-mentioned purpose, a reaction solution (buffer solution, etc.), nuclease, nucleic acid elongation enzyme, reverse transcriptase, various reagents for nucleic acid amplification (polymerase, dNTP, etc.), a probe, a washing solution, etc., stored in containers. Each reagent may be stored in a separate container, or multiple reagents may be stored in the same container as long as it does not contradict the desired purpose. The kit may also include instructions instructing any of the methods of use described in this disclosure.

There are no particular limitations on the containers, and containers suitable for storing each reagent may be selected as appropriate.

Next, embodiments of the present disclosure will be described in detail using examples, but the embodiments of the present disclosure are not limited to these examples.

Background Adenosine deamination mechanism in the central dogma of life (Fig. 1)
In the "Central Dogma of Life" of gene expression, which involves transcribing the necessary genes from DNA to RNA and producing proteins, which are functional expressions, various expression regulation mechanisms work at each stage to maintain appropriate gene expression. One of these regulation mechanisms is a mechanism that modifies the chemical structure of the four bases [A, G, C, T (U)] of DNA and RNA, which are the genetic information itself. However, in metazoans, including mammals such as humans and mice, there is a phenomenon in which sequences are edited from the original genome sequence by enzymes in cells. In this disclosure, we focus on a modification called inosine (I) caused by the deamination reaction of adenosine (A). In the Watson-Crick base pairing that is essential in the central dogma, A forms an [A:T (U)] pair with thymine (T) or uridine (U), and guanosine (G) forms a [G:C] pair with cytidine (C). However, I after A deamination forms a base pair with C, just like G. As a result, the base pair is changed from A:T (U) to I:C, which is equivalent to editing from A to G in genetic information, and this mechanism is called the A-to-I editing mechanism. Regarding A-to-I editing in RNA, after the discovery of ADAR (Adenosine deaminases acting on RNA), only mechanisms controlling amino acid substitution or alternative splicing were found in a very limited number of types of mRNA for a dozen years. Since then, the discovery of editing sites has increased dramatically due to the progress of the Human Genome Project and next-generation sequencing (NGS) technology, and A-to-I editing has now been reported in more than 50,000 sites and more than 2,000 types of genes in humans. On the other hand, editing sites present in the amino acid coding region of mRNA are less than 1%, and the function of editing sites is known in less than 10% of the total.

Recently, the inventors' research group and overseas research groups have discovered that ADAR actually has the activity of A-to-I RNA editing and A-to-I DNA editing of RNA and DNA strands using RNA:DNA hybrid strands as substrates. This discovery suggests that an active base editing mechanism from A to G caused by A-to-I DNA editing in genomic DNA is inherent in mammals. Indeed, the presence of the base moiety of inosine (DNA) as hypoxanthine has long been detected as a source of mutation due to spontaneous deamination. However, no attempt has been made to identify the inosine site on DNA. This is because, in addition to the fact that it has not been noticed until now, there are only two molecules of a single region of DNA in one cell, and there is no technology with the sensitivity to analyze it, and furthermore, inosine is replaced by G when it undergoes PCR amplification, making it impossible to detect inosine with existing technology.

principle
Overview of inosine-specific reactions and usage The most basic method for identifying inosine is to perform sequence analysis of the region to be verified on DNA and RNA purified from the same specimen. If the region originates from the same genomic DNA and has a G or a mixed sequence of G and A on the RNA, while it has an A on the DNA, the site may have undergone A-to-I RNA editing. In this method, the comparison of the sequences of DNA (before editing) and RNA (after editing) is the basis for discrimination. Therefore, accurate mapping to the genome sequence from which the RNA originates is essential. However, in general, mapping errors in cases of low sequence specificity and contamination of the G sequence due to noise generated in the experimental reaction are unavoidable, and the detection rate of false positives for inosine is high, and the identification accuracy tends to be low.

To overcome this difficulty, the Inosine Chemical Erasing (ICE) method has been developed, which utilizes a cyanoethyl group addition reaction based on the chemical properties of inosine. With this technique, identification does not require DNA analysis from the same sample, and the addition of a cyanoethyl group to inosine on RNA causes steric hindrance to Watson-Crick base pairing, causing the primary cDNA strand to stop just before inosine during the reverse transcription reaction to obtain cDNA for sequence analysis. The stopped primary cDNA is not increased in the subsequent PCR reaction, and the G base signal reflecting inosine disappears in the sequence analysis results of the amplified cDNA. This principle makes it possible to identify inosine in RNA strands using Sanger sequencing or NGS.

Purpose of Development On the other hand, as mentioned above, the genomic DNA of the target specific region in the cell is basically only two molecules derived from homologous chromosomes, and is extremely small compared to RNA species. In addition, since it is an editing mechanism of the DNA itself, which is the comparison target before editing, it is impossible to detect by sequence comparison between RNA and DNA. In addition, A-to-I RNA editing in non-coding RNA, which is expressed in extremely small amounts compared to general mRNA, has been reported in recent years. However, in the identification of inosine sites on RNA with low expression levels and inosine sites on DNA with low abundance ratios to the entire genome sequence, in normal NGS, the information of nucleic acid sequence species with high expression levels accounts for the majority, so the number of reads is relatively limited, and the identification accuracy tends to decrease or not be detected.

On the other hand, in the editing method of genome DNA using Cas protein and the editing method of transcribed RNA, the development of a single base mutation type that introduces a change to inosine by deamination of adenosine is progressing. In particular, the number of reports of examples is increasing in research into diseases caused by single base mutations in genes and their effects. In addition, in ADAR2, which is one of the ADAR enzyme family that is an editing enzyme, a decrease in editing of glutamate receptors, which are one of the main target mRNAs for editing, causes amyotrophic lateral sclerosis (ALS). Currently, clinical trials are being conducted for this type by introducing the ADAR2 gene. In the above-mentioned artificial DNA editing or RNA editing, there is a possibility of off-target effects of the editing enzyme. In particular, the off-target effects of editing enzymes on DNA have tended to be ignored.

For DNA and RNA species containing inosine, which exists at a very small ratio to the number of nucleic acid bases in the body, concentration or isolation based on the presence or absence of inosine is considered to be effective. However, in a reaction using acrylonitrile, the cyanoethyl group once added to inosine is chemically very stable, and further addition of labels such as fluorescent or affinity tags is impossible. In contrast, the method disclosed herein is a technology that can utilize the added label for analysis after an inosine-specific addition reaction (FIG. 2). In the examples, a chemical reagent that reacts specifically with inosine was found, and labeling by addition of an inosine base-specific fluorescent or affinity tag, which was not possible with the above-mentioned cyanoethylation, was realized (FIG. 3). By utilizing these reactions, the inosine content in cells or samples can be easily measured by fluorescent labeling. In addition, as an example, by labeling biotin specifically with inosine, it has become possible to isolate and purify only nucleic acid chains containing inosine from a pool of non-edited nucleic acids.

ICLAMP method (Inosine Chemical Labeling & Affinity Purification):
Chemical Structure and Properties of Labeling Agent Hereinafter, the method according to the present disclosure will be referred to as the ICLAMP method. Desirable conditions for the labeling agent used in the method according to the present disclosure will be shown below, taking maleimide as an example.
- Being cyclic, the three-dimensional structure of the carbon ring is stable.
- There is a double bond between carbon atoms Cα and Cβ.
Either or both of the 2- and 5-position carbons have a bond to an oxygen atom and an unsaturated electron pair, or have a bond to an atom or atomic group other than an oxygen atom and having an unsaturated electron pair.
The addition reaction to inosine is an electrophilic addition reaction (Michael addition) to the 1-position of the inosine base, and the labeling agent has electron-withdrawing properties due to α,β-unsaturated electron pair transfer in the reaction solution.
The added chemical structure at the N1 position is any chemical structure including a hydrogen atom, an alkyl group, a fluorescent functional group, a biotin structure, etc., and does not undergo any change upon reaction with inosine.
The atom or substituent bonded to carbon atoms Cα and Cβ is any atom or substituent such as a hydrogen atom or an alkyl group, which does not undergo any change upon reaction with inosine.

Below, as an example, a method using a maleimide derivative as a labeling agent is explained. The convenience of using maleimide derivatives is that they are easy to obtain, as they have already been developed and are commercially available as protein modification reagents, and there is a wide range of options for added chemical structures. In this disclosure, fluorescent labeling and biotin labeling were carried out as examples.

Reaction mechanism Among adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), and hypoxanthine (the base moiety of inosine, also called inosine in a broad sense), which constitute the nucleic acid bases in DNA and RNA, inosine has an acid dissociation constant pKa value closest to 7 (FIG. 4). Since the reaction mode of the labeling agent in the present disclosure is an electrophilic addition reaction, inosine, which has a pKa value closest to the pH and whose equilibrium leans toward a negative charge state, has the highest reactivity among the nucleic acid bases having a negative charge in a neutral to weakly alkaline solution. The reaction mechanism of maleimide and maleimide derivatives with inosine is as follows.
In a weakly alkaline solvent containing alcohol such as ethanol, the hydrogen atom (proton) at the N1 position is released into the alcohol due to the unsaturated electron pair and double bond at the N1 position, which are characteristics of inosine, and the nitrogen atom at N1 is highly negatively charged, existing in an equilibrium state.
On the other hand, in maleimides, a positive charge is generated at the 4th (or 3rd) carbon atom through the unsaturated electron pair and the double bond due to the electron-withdrawing property of the oxygen atom. As mentioned above, a characteristic of maleimide-like labeling agents is that, since maleimides are bilaterally symmetrical, a positive charge can also be generated at the 3rd (or 4th) carbon atom.
- An electrophilic addition reaction (Michael addition reaction) occurs from the positively charged carbon atom on the maleimide side to the negatively charged nitrogen atom at the N1 position of inosine.

<Example>
〔material〕
(Maleimide derivatives)
Fluorescein-5-maleimide (FITC-maleimide): Tokyo Chemical Industry Co., Ltd. (TCI). FITC-maleimide was dissolved in DMSO to prepare a 250 mM or 50 mM stock solution. Biotin-PEG6-maleimide (biotin-maleimide): Tokyo Chemical Industry Co., Ltd. (TCI). Biotin-maleimide was dissolved in DMSO to prepare a 250 mM or 50 mM stock solution.

(DNA and RNA oligonucleotides)
RNA-6A: 5'-GGCGAGAGGCAAGAGGCGCGCAGUAGGGCGGCAGAAGCGGCGUAGCGGGCCGCGCGUCGGGC-3' (SEQ ID NO: 1)
RNA-6I: 5'-GGCGAGAGGCAIGAGGCGCGCIGUIGGGCGGCIGAIGCGGCGUIGCGGGCCGCGGUCGGGC-3' (SEQ ID NO: 2)
DNA-6dA: 5'-GGCGAGAGGCAAGAGGCGCGCAGTAGGGCGGCAGAAGCGGCGTAGCGGGCCGCGCGTCGGGC-3'
(SEQ ID NO: 3)
DNA-6dI: 5'-GGCGAGAGGCAIGAGGCGCGCIGTIGGGCGGCIGAIGCGGCGTIGCGGGCCGCGCGTCGGGC-3'
(SEQ ID NO: 4)
DNA-dA: 5'-GACACACAAGCGACACAACGAG-3' (SEQ ID NO: 5)
DNA-dI: 5'-GACACACAAGCGICACAACGAG-3' (SEQ ID NO: 6)
FITC-DNA-dA: 5'-[FITC] GACACACAAGCGACACAACGAG-3' (SEQ ID NO: 7)
FITC-DNA-dI: 5'-[FITC] GACACACAAGCGICACAACGAG-3' (SEQ ID NO: 8)
Cy5-DNA-dA: 5'-[Cy5] GACACACAAGCGACACAACGAG-3' (SEQ ID NO: 9)
Cy5-DNA-dI: 5'-[Cy5] GACACACAAGCGICACAACGAG-3' (SEQ ID NO: 10)
Cy5-DNA-dA-FITC: 5'-[Cy5] GACACACAAGCGACACAACGAG[FITC]-3' (SEQ ID NO: 11)
Cy5-DNA-dI-FITC: 5'-[Cy5] GACACACAAGCGICACAACGAG[FITC]-3' (SEQ ID NO: 12)
Cy5-4dA: 5'-[Cy5]GAACACAAAAAAAAACGAGAC-3' (SEQ ID NO: 13)
Cy5-4dI: 5'-[Cy5]GAACCAIAIAIAIAIACGAGAC-3' (SEQ ID NO: 14)
Cy5-4dU: 5'-[Cy5]GAACACAUAUAUAUACGAGAC-3' (SEQ ID NO: 15)
Cy5-4dT: 5'-[Cy5]GAACACATATATACGAGAC-3' (SEQ ID NO: 16)
Cy5-36nt-dI: 5'-[Cy5]GACAGACAGCCAICACAACAAGAGACCAGACACAGAG-3' (SEQ ID NO: 17)
FAM-20nt-dA: 5'-[FAM] GACAGACAGCCACACGACAG-3' (SEQ ID NO: 18)
FAM-30nt-dU: 5'-[FAM] GACAGACAGCCUCACAACAAGAGAAGAGAG-3' (SEQ ID NO: 19)
Cy5-24nt-dI-Bio: 5'-[Cy5] GACAGACAGCCICACAACAGACAG[Bio]-3' (SEQ ID NO: 20)

(Reaction solution)
TEAA buffer [50% (v/v) ethanol, 3M or 1.1M triethylamine, titrated to pH 8.6 with acetic acid]

(Purification Reagent Kit)
QIAquick Nucleotide Removal kit (QIAGEN)
MinElute PCR Purification kit (QIAGEN)
Phenol-chloroform-isoamyl alcohol mixture (PCI), pH 7.7-8.3 (Sigma)
RNeasy Maxi kit (QIAGEN)
miRNeasy Mini kit (QIAGEN)
Blood & Cell Culture DNA Midi kit (QIAGEN)

(Affinity purification reagents)
Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific)
2x Binding Buffer (50mM HEPES buffer pH 7.5, 2M NaCl, 25mM EDTA, 0.01% Tween 20)
1x Blocking Buffer (25mM HEPES buffer pH 7.5, 1M NaCl, 12.5mM EDTA, 0.005% Tween 20, 1% BSA)
1x Binding Buffer (25mM HEPES buffer pH 7.5, 1M NaCl, 12.5mM EDTA, 0.005% Tween 20)
1x Washing Buffer (5mM HEPES buffer pH 7.5, 200mM NaCl, 2.5mM EDTA, 0.005% Tween 20)
Formamide (Nacalai Tesque)

(Primer extension reagent)
Q/R A-to-I 5'-end ³² P-labeled DNA primer (sequence: 5'-GATCTTTGCGAAATCGCATC-3' (SEQ ID NO: 21)) designed downstream of the RNA editing site (99% of A's are edited to inosine)
T4 polynucleotide kinase (NEB)
2x LS (80% formamide, 0.025% BPB, 0.025% xylene cyanol, 20% glycerol)
5x Glycerol RT buffer (250mM Tris-HCl PH 7.5, 375mM KCl, 15mM _MgCl2 , 50% Glycerol)
SuperScript III reverse transcriptase (Invitrogen)
5 M Betaine (Merck-Sigma)
2x Loading solution (RI) (7 M urea, 0.05% bromophenol blue, 0.05% xylene cyanol, 1x TBE)
5x Glycerol RT buffer (250mM Tris-HCl pH 7.5, 375mM KCl, 15mM _MgCl2 , 50% Glycerol)
SuperScript III reverse transcriptase (Invitrogen)
Elution Buffer (0.3 M sodium acetate pH 5.2, 0.1% SDS)

(Device)
iBright (Thermo Scientific)
Concentration dryer (Eppendorf)
Tube penetration mixer (Intellimixer RM-2M (ELMI))
Amersham Typhoon scanner (Cytiva)

[Inosine-specific green fluorescent labeling with FITC-maleimide]
(Labeling of synthetic oligonucleotides with FITC-maleimide)
DNA or RNA oligonucleotides were added at 50 pmol to 38 μL of TEAA buffer solution prepared in a 1.5 mL tube so that the final concentration was 1 pmol/μL, and the mixture was vigorously suspended and dissolved by vortexing. FITC-maleimide was added to the reaction solution so that the final concentration was 10 mM or 50 mM, or 0 mM for comparison, and the mixture was vigorously suspended and dissolved by vortexing. The final volume was adjusted to 50 μL with DMSO. The reaction solution was incubated at 70° C. in the dark using a heat lid type solid phase incubator for the reaction time (5 minutes to 60 minutes) under each condition. After the reaction time had elapsed, the tube was immediately placed on ice and rapidly cooled to stop the reaction.

(Purification after oligonucleotide labeling)
After the labeling reaction, the excess maleimide was removed and the nucleic acid was purified as follows. The DNA oligonucleotides after the reaction were purified using the QIAquick Nucleotide Removal kit and the MinElute PCR Purification kit. The reaction solution was mixed with 10-fold volume of Buffer PNI (QIAquick Nucleotide Removal kit). The mixture was loaded onto a spin column. The subsequent adsorption, centrifugation, washing, and elution processes were basically performed according to the QIAquick Nucleotide Removal Kit. After washing the column five times with 750 μL Buffer PE, the DNA oligonucleotides were eluted with 200 μL of water, and the resulting 200 μL solution was concentrated to 20-70 μL using a freeze-concentration dryer (Eppendorf). Then, 10-fold volume of Buffer PNI was added to the solution, and purification was performed according to the MinElute PCR Purification protocol. One additional washing step was added to the prescribed procedure, for a total of two washings.

PAGE Analysis of Labeled DNA Oligonucleotides
The purified labeled DNA oligonucleotides were electrophoresed on a 15% denaturing polyacrylamide gel. 7 pmol of DNA was loaded per lane per reaction. Bands were detected using iBright, and quantitative analysis was performed using Image J software. The amount of labeled DNA was obtained from the fluorescence intensity of FITC in each band. The intensity of DNA amount was measured using a nucleic acid stain (toluidine blue or GelRed), or the fluorescence intensity of Cy5 was measured using DNA previously labeled at the 5' end with Cy5. Each sample was normalized by comparing the fluorescence intensity of bands with known amounts of FITC-DNA-dI or Cy5-DNA-dI-FITC oligonucleotide. The percentage conversion was calculated as the molar ratio of fluorescein to nucleic acid stain or Cy5 per well. Reactions were analyzed in triplicate, and the average and standard deviation were obtained.

[Purification technique using biotinylation labeling of inosine]
(Usage sample)
Synthetic RNA oligonucleotides, DNA oligonucleotides, and total RNA fractions or genomic DNA (gDNA) extracted and purified from cultured cells or mouse tissues were used.

(Extraction and purification of total RNA fraction)
Total RNA fractions from mouse tissues were extracted using the RNeasy Maxi kit after tissues were homogenized. Total RNA fractions from cultured cells were purified using the miRNeasy Mini kit. Total RNA (40 μg) was dissolved in 100 μL of reaction solution containing 100 mM Tris-HCl and 2 mM _MgCl2 , and fragmented by incubation at 95°C for 8 minutes. The fragmented total RNA was purified by ethanol precipitation.

(Extraction and purification of genomic DNA)
Genomic DNA from mouse tissues and cultured cells was purified using the Blood & Cell Culture DNA Midi kit.

(Labeling of Nucleic Acid Samples)
When synthetic oligonucleotides were used, 50 pmol was reacted with biotin-maleimide under the same reaction conditions as with FITC-maleimide and purified. Total RNA fraction or gDNA (30 μg) was dissolved in water, the volume was adjusted to 2 μL in a 1.5 mL tube, 38 μL of 1.1 M TEAA buffer was added, and the mixture was vigorously suspended. Next, 10 μL of 250 mM biotin-maleimide (final concentration: 50 mM) was added and suspended again. As a control, 10 μL of DMSO was used as 0 mM. The final solution volume was 50 μL. Labeling with maleimide was performed by incubation at 70° C. for 1 hour. Then, the reaction was stopped by adding 400 μL of cold water to the solution. Next, removal of unreacted excess maleimide and nucleic acid purification were performed as follows. After isopropanol precipitation of the nucleic acid fraction, separation using PCI (Phenol/Chloroform/Isoamyl alcohol) (pH 7.9) was performed twice to remove the remaining maleimide. Next, the aqueous phase containing the nucleic acid was suspended in an equal amount of diethyl ether, and after standing and centrifugation, the upper ether layer containing the remaining phenol was removed. The ether remaining in the aqueous phase was removed by evaporation using a freeze-concentration dryer, and finally the target nucleic acid was purified by ethanol precipitation. When the nucleic acid sample was used for affinity purification, the oligonucleotide was dissolved in 20 μL of water, and the total RNA fraction or gDNA was dissolved in 240 μL of water. On the other hand, when the nucleic acid sample was used for the primer extension method, the RNA was dissolved in 3 μL of water.

(Affinity purification)
To separate and concentrate the nucleic acid fraction containing biotin-maleimide-labeled inosine from the nucleic acid fraction not containing inosine, we used magnetic beads Dynabeads MyOne Streptavidin C1, whose surface is cross-linked with streptavidin, which has a strong affinity for biotin. The beads were used according to the recommended protocol with the following modifications.

In order to remove impurities during the separation and purification process, the compositions of the suspension, washing, and nucleic acid elution solutions for the beads were optimized as follows. The amount of beads used was 10 μL when the target of purification was oligonucleotide, and 120 μL when the target was total RNA fraction or gDNA. The beads (50% by volume slurry) well suspended in the storage solution were transferred to a tube. After adding 500 μL of 1x Blocking buffer to the tube, the beads were washed by gently stirring the tube upside down for 2 minutes using a tube osmotic stirrer. The tube was immediately placed on a magnetic stand for 2 minutes, and the supernatant was collected. To prevent the beads from drying, 500 μL of 1x Blocking buffer was immediately added, and the above stirring, standing, and supernatant collection were repeated. The above washing operation was repeated a total of three times. Finally, 2x Binding Buffer was added to the washed beads (20 μL for oligonucleotides, 240 μL for total RNA fraction or gDNA) so that the beads had a final concentration of 5 μg/μL (volume to give a 25% by volume slurry), and the beads were suspended and then allowed to stand.
On the other hand, in order to avoid non-specific adhesion to the inner wall of the tube during the suspension operation of the beads and nucleic acid after washing, the tube to be used was pretreated. 1.5 mL of 1x Blocking Buffer was added to a 2 mL tube, and the inner wall and the back of the lid were blocked by stirring for about 1 hour using a tube permeation stirrer. 20 μL of oligonucleotide dissolved in water after labeling purification, or 240 μL of total RNA or gDNA, was transferred to a tube whose inner wall had been washed beforehand. To these, an equal volume of the washed beads, which are the above 25% by volume slurry, was added. The mixture of beads and nucleic acid sample was gently stirred at room temperature for 60 minutes using a tube permeation stirrer while turning the tube upside down, to promote the binding reaction of biotin-streptavidin. After that, the solution containing the beads was left to stand on a magnetic stand for 2 minutes, and the supernatant was collected (FT: collected as flow-through and stored in a separate tube). Next, to wash the beads, 500 μL of 1x Washing Buffer was added, and the tube was gently stirred upside down using a tube osmotic stirrer, then left on a magnetic stand for 2 minutes, and the supernatant was removed. This washing operation was repeated a total of three times. Next, to elute the target nucleic acid fraction bound to the magnetic beads, 20 μL (in the case of oligonucleotides) or 100 μL (in the case of total RNA or gDNA) of 100% formamide was added to the beads and incubated at 70 ° C. for 5 minutes. The tube was immediately left on a magnetic stand for 1 minute, and the supernatant containing the eluted fraction was transferred to a new tube. The nucleic acid was then recovered by ethanol precipitation together with glycogen, which is a coprecipitant, and further rinsed with 80% ethanol and air-dried. The total RNA fraction and gDNA fraction were then dissolved in 3 μL of water.

(Detection of inosine sites by primer extension method)
Next, we verified whether regions containing inosine were purified and detected as expected after inosine-specific labeling and affinity purification for synthetic DNA or RNA containing inosine, in which the presence of inosine is known, or for glutamate receptor mRNA in a total RNA fraction extracted from mouse brain tissue.

Primer extension by reverse transcription reaction using RNA as a template was performed according to the method described in the following literature [Higuchi et al., 1993; Sakurai et al., 2010; Seeburg et al., 1998]. Mouse glutamate receptor B mRNA contains a site where A-to-I RNA editing occurs at 99% or more (Q/R site). A Q/R A-to-I detection DNA primer (sequence: 5'-GATCTTTGCGAAATCGCATC-3': SEQ ID NO: 22) was designed to detect this site.

Labeling of DNA primers with radioisotopes was carried out as follows: 4 pmol of DNA primer was mixed with 1 μL of 10× T4 PNK buffer, 1 μL (3.3 pmol) of γ- ³² P ATP (3000 Ci/mmol, 10 μCi/μL), water, and 10 units of T4 polynucleotide kinase to make a total volume of 10 μL.

The solution was reacted at 37°C for 30 minutes, and then immediately boiled at 95°C for 3 minutes to heat inactivate the enzyme. Then, 10 μL of 2x LS was added to the solution, and the whole solution was used for electrophoresis in a state where the secondary structure of the nucleic acid was dissolved by heating at 95°C for 5 minutes. For electrophoresis, a 10% polyacrylamide, 7M urea-1x TBE (Tris-borate EDTA Buffer) gel of 10 cm x 10 cm size was used. The gel after electrophoresis was detected by a Typhoon scanner, and the actual position on the gel was identified. ^{The 32} P-labeled primer was fragmented into a rectangular parallelepiped of 1 to 2 mm each with the gel, and each primer type was transferred to a 2 mL tube. Then, 400 μL of Elution Buffer was added, and the solution was shaken at room temperature for 90 minutes using a tube osmotic stirrer so as not to foam. The supernatant from which the DNA had been eluted was then transferred to a 1.5 mL tube, taking care not to collect gel debris, and the DNA pellet was collected by ethanol precipitation with the addition of glycogen as a co-precipitant. The pellet was rinsed with 80% ethanol, air-dried, and dissolved in 10 μL of water. The radioactivity of the labeled DNA primer was measured using a scintillation counter. The labeled DNA primer was reconstituted to a molar concentration of 0.4 μM and 1,000,000 to 1,500,000 cpm per μL, and used in the following primer extension method.

30 μg of purified brain total RNA was dissolved in 3 μL of water, to which 1 μL of 5'-end ³² P-labeled Q/R A-to-I detection DNA primer was added. 2 μL of 5x Glycerol RT buffer and 2 μL of 5M Betaine were added to this solution, mixed, incubated at 70 ° C for 2 minutes, and cooled to room temperature. Next, reverse transcription reaction was performed. 0.5 μL of 0.1M DTT, 1 μL of 1.5 mM dNTPs, and 100 units of SuperScript III reverse transcriptase were added to the cooled solution to make a total reaction volume of 10 μL, and reverse transcription was performed at 50 ° C for 60 minutes. After the reaction, 10 μL of 2x Loading solution (RI) and 20 μL of 100% formamide were added, boiled at 95 ° C for 5 minutes, and 10 μL was used for electrophoresis. Electrophoresis was performed on a gel of 15% polyacrylamide, 7 M urea, and 1× TBE. After electrophoresis, the gel was transferred to an Imaging plate, and bands were detected and analyzed using an Amersham Typhoon scanner (Cytiva).

On the other hand, detection of inosine at the Q/R site of mouse Gria2 mRNA was performed by a method in which only the extended strand with α- ^32p dCTP, which is incorporated during primer extension, was radioactively labeled without using a 5'-end labeled primer. Sample RNA was adjusted to 2 μL, and 0.5 pmol primer, 2 μL of 5x glycerol RT buffer, and 2 μL of 5 M betaine were added, incubated at 70° C. for 2 minutes, and then cooled to room temperature. Then, 0.5 μL of 0.1M DTT, 0.5 μL of 3 mM dNTP (mixture of dATP, dTTP, dGTP), 2 μL of 3.3 μM α- ^32p dCTP (3000 Ci/mmol, 10 μCi/μL), and 100 units of SuperScript III reverse transcriptase were added to make a total solution volume of 10 μL. This was incubated at 50 ° C for 60 minutes to perform a reverse transcription reaction. After the reaction, 10 μL of 2x Loading solution and 20 μL of 100% formamide were added, and the mixture was boiled at 95 ° C for 5 minutes, and 10 μL was used for electrophoresis. Electrophoresis was performed on a gel of 15% polyacrylamide, 7 M urea, and 1x TBE. After electrophoresis, the signals from the gel were transferred to an Imaging plate, and bands were detected and analyzed using an Amersham Typhoon scanner (Cytiva).

Quantification of inosine using FITC-maleimide
In this test, it was verified whether inosine can be quantified using FITC-maleimide.

　Inosine-containing oligonucleotides (5'-[Cy5]GACACACAAGCGICACAACGAG-3' (SEQ ID NO: 10)) of known concentration were added to 38 μL of TEAA buffer solution prepared in a 1.5 mL tube so that the final concentrations were 0.03 μM, 0.06 μM, 0.1 μM, 0.6 μM, 0.8 μM, and 1 μM, respectively, and suspended and dissolved vigorously by vortexing. FITC-maleimide was added to the reaction solution so that the final concentration was 10 mM, and suspended and dissolved vigorously by vortexing. The final volume was adjusted to 50 μL with DMSO. The reaction solution was incubated at 70°C for 15 minutes in the dark using a heat lid type solid phase incubator. After the reaction time had elapsed, the tubes were immediately placed on ice to rapidly cool and stop the reaction.

After the labeling reaction, excess maleimide was removed and the nucleic acid was purified as described above. Since the PAGE analysis described above does not have sufficient sensitivity for detecting trace amounts of labeled oligonucleotide, a Typhoon scanner was selected as a more sensitive detection method. The purified labeled oligonucleotide was transferred to a membrane (Amersham Hybond-N+) using a BIO-RAD BioDot device: 1706545. The fluorescence intensity was then confirmed using a Typhoon scanner, and after measurement, quantification was performed using Image J software according to the PAGE analysis method described above. The concentration of inosine-containing oligonucleotide and the fluorescence intensity of FITC were plotted to create a standard curve.

[Study of aqueous reaction solvents]
Based on the literature reports on the reaction of inosine with acrylonitrile, an aqueous solution of 0.01M phosphate buffer-2.5M NaCl solution (final concentration, pH 8.6) and 2.7M dimethylformamide (DMF) is sometimes used as the reaction solvent. In this case, the reaction is generally carried out at 40°C for 3 hours. However, as a result of carrying out a reaction in this aqueous solution using a nucleic acid containing inosine and acrylonitrile, the reaction efficiency was poor, and impurities and decomposition of the nucleic acid, which are thought to be derived from side reactions, were observed. Therefore, in this test, the relationship between an aqueous reaction solution in which a phosphate buffer-NaCl solution and dimethylformamide (DMF) or the like are mixed and an inosine-specific reaction was examined in the reaction of a nucleic acid containing inosine with maleimide.

The following buffer solutions were prepared:
Buffer A: 0.01M phosphate buffer-0.5M NaCl/5.4M DMF (40%)
Buffer B: 0.02M phosphate buffer-0.15M NaCl/2.7M DMF (20%)
Buffer C: 0.5M TEA/0.02M phosphate buffer-0.15M NaCl/2.7M DMF (20%)
Buffer D: 0.02M phosphate buffer-0.5M NaCl/5.4M DMF (40%)
Buffer E: 0.5M TEA/0.02M phosphate buffer-0.5M NaCl/5.4M DMF (40%)
Buffer F: 0.02M phosphate buffer - 0.5M NaCl/DMI (40%)
Buffer G: 0.02 M phosphate buffer-0.5 M NaCl/ethanol (40%)

DNA oligonucleotides containing deoxyadenine (A) or deoxyinosine (I) labeled with Cy5 (SEQ ID NO: 9: 5'-[Cy5] GACACACAGCGACACAACGAG-3'; SEQ ID NO: 10: 5'-[Cy5] GACACACAGCGICACAACGAG-3') were added at 50 pmol to 38 μL of each buffer solution prepared in a 1.5 mL tube to a final concentration of 1 pmol/μL, and suspended and dissolved vigorously by vortexing. FITC-maleimide was added to the reaction solution to a final concentration of 10 mM, and suspended and dissolved vigorously by vortexing. The final volume was adjusted to 50 μL with DMSO. The reaction solution was incubated at 70°C for 3 hours in the dark using a heat lid type solid phase incubator. After the reaction time had elapsed, the tube was immediately placed on ice to rapidly cool and stop the reaction. The reaction conditions are shown in the table below.

After the labeling reaction, excess maleimide was removed and the nucleic acid was purified as described above. The purified labeled oligonucleotide was subjected to PAGE analysis as described above.

<Results and Commentary of the Example>
[Optimization of reaction solution composition, pH, and temperature for RNA labeling with FITC-maleimide]
In this development, first, the composition, pH, and temperature of the reaction solution used were optimized using an analysis experiment of the labeling efficiency of inosinated RNA oligonucleotides with FITC-maleimide as a model.

To confirm the reaction efficiency, two types of RNA oligonucleotides containing adenosine (RNA-6A) or inosine (RNA-6I) at six specific positions were synthesized, and after the labeling reaction under each condition, purification was performed and the labeling efficiency was quantitatively compared by electrophoresis. The I/A ratio was calculated by dividing the fluorescence intensity of FITC labeled RNA-6I by that of RNA-6A to determine the optimal reaction conditions. As mentioned above (Figure 4), based on the pKa value of each nucleic acid base from the published literature [Gillingham et al., 2016; Luyten et al., 1998; Thaplyal & Bevilacqua, 2014], the labeling efficiency was examined between pH 7.8 and pH 9.2, within the range in which the reaction efficiency of inosine is maximized compared to other bases, and it was confirmed that pH 8.6 was optimal. In addition, the reaction temperature was also compared under conditions changed in increments of 10°C, and it was confirmed that the maximum reaction efficiency and specificity were obtained at 70°C. Next, the composition of the reaction solution was examined. As a result, both 3M TEAA buffer (50% (v/v) ethanol, 3M triethylammonium) and 1.1M TEAA buffer (50% (v/v) ethanol, 1.1M triethylammonium) improved the reaction efficiency. Inosine showed the highest reaction rate when combined with maleimide in 3M TEAA buffer. From the above verification, it was found that the optimal reaction conditions for inosine-specific maleimide are TEAA buffer (oligonucleotide: 3M TEAA, total RNA and genomic DNA: 1.1M TEAA), pH 8.6, and temperature 70°C.

[Verification of DNA labeling reaction efficiency with FITC-maleimide]
Next, the reaction efficiency including DNA oligonucleotides containing inosine was verified. The inosine labeling conditions optimized using RNA oligonucleotides were applied to DNA oligonucleotides containing six deoxyadenosines (DNA-6dA) and six deoxyinosines (DNA-6dI). The difference in the reaction between RNA and DNA was verified at maleimide concentrations of 2 mM (mild condition) and 10 mM (medium condition) for reaction times of 15 minutes, 30 minutes, and 60 minutes. As a result, RNA and DNA showed significant signals in 15 minutes of reaction with FITC-maleimide under both mild and medium conditions. As the reaction time progressed, the signal significantly improved after 30 minutes and reached a maximum at 60 minutes (Figure 5).

Next, to measure the labeling efficiency and intensity per inosine base, an oligonucleotide (DNA-dI) containing only one deoxyinosine in a 22-base DNA strand was prepared. For comparison with this oligonucleotide, a DNA-dA oligonucleotide was prepared in which the deoxyinosine site was replaced with a deoxyadenosine. First, to examine the relationship between reaction time and the degree of labeling, a time course measurement was performed for DNA-dI and DNA-dA in the presence of 10 mM FITC-maleimide for up to 8 hours (Figure 6). As a result, the DNA-dI-specific FITC labeling signal increased and reached a peak within 1 hour, and no increase in the signal was observed for reaction times of 1 hour or more.

Next, to more accurately investigate the maximum labeling rate achievable with 10 mM FITC-maleimide, DNA-dI (Cy5-DNA-dI) and DNA-dA (Cy5-DNA-dA) pre-labeled with Cy5 at the 5' end were prepared. Based on the previous results, a time course was performed at 0, 15, 30, and 60 minutes with N=3 (Figure 7). The fluorescein labeling in Cy5-DNA-dI increased clearly at 15 minutes. On the other hand, only a faint signal was detected for Cy5-DNA-dA. In addition, with increasing reaction time, the FITC-maleimide-labeled DNA among all DNAs observed with Cy5 labeling showed an upward shift in the electrophoretic gel due to its addition. After electrophoresis, the intensities of the bands detected by fluorescence with FITC and Cy5 were quantified. Furthermore, the ratio of maleimide-labeled DNA to the total amount of DNA used (% Labeling) was calculated by dividing the fluorescence intensity of FITC by that of Cy5, and plotted against the time axis. This labeling ratio was further calculated as the ratio of DNA-dI/DNA-dA to calculate an index of reaction specificity to inosine (I/A). From these results, it was found that the reaction efficiency of DNA-dI to 10 mM FITC-maleimide reached a maximum of 40% at 60 minutes, and the reaction rate was highest from 0 to 15 minutes, which was the range in which non-specific reactions to A were suppressed to the minimum. Similarly, the relationship between reaction efficiency and reaction time depending on the concentration of maleimide was examined at three concentrations of FITC-maleimide (2 mM, 10 mM, 50 mM). From the I/A ratio, the condition in which the specific reactivity to inosine was maximized was 10 mM and 15 minutes of reaction. On the other hand, the inosine labeling efficiency was highest under 50 mM maleimide conditions, and in particular, after 60 minutes of reaction, the labeling efficiency with FITC-maleimide reached nearly 100% (Figure 8).

Next, in order to perform reproducible and highly accurate quantification of the labeling efficiency with 50 mM FITC-maleimide, DNA previously labeled with Cy5 at the 5' end (DNA-dI) was used in the same manner as above, and a time course analysis was performed with N=3. As a result, it was confirmed that the reaction efficiency of about 80% was indeed reproducibly observed under 50 mM FITC-maleimide conditions (Figure 9). The difference in efficiency from the results described above (Figure 8) was thought to be due to the staining or detection method of the total DNA amount. In conclusion, from the above verification results, it was found that the most inosine-specific reaction conditions were a 15-minute reaction in the presence of 10 mM maleimide, and the conditions with the highest inosine labeling efficiency were a 1-hour reaction with 50 mM maleimide.

[Investigation of reaction specificity using FITC-maleimide]
Next, under the condition of this reaction solution at pH 8.6, the reactivity with other nucleic acid bases A, U, and T, which may cause an addition reaction of maleimide, was verified. In this verification, in order to detect and quantify nonspecific labeling signals assumed to be low, four types of oligonucleotides containing four deoxyinosines (Cy5-4dI), four deoxyadenosines (Cy5-4dA), four deoxyuridines (Cy5-4dU), and four deoxythymidines (Cy5-4dT) were prepared in synthetic DNA fluorescently labeled with Cy5 at the 5' side. As a result of analyzing the reactivity of FITC-maleimide with these four oligonucleotides, the degree of fluorescent labeling was approximately 60 to 20 times more efficient with Cy5-4dI than with other bases, as expected from the pKa value. From this result, the reactivity of maleimide with inosine in the experimental system described below was evaluated to be sufficiently significant and specific (FIG. 10).

Affinity purification of DNA oligonucleotides using biotin-maleimide
Next, the inosine labeling reaction using biotin-maleimide, which constitutes the principle of inosine base-specific nucleic acid purification technology, was verified. For the verification, a DNA oligonucleotide containing 5'Cy5 fluorescently labeled inosine (Cy5-35nt-dI) was prepared as the main target for biotin labeling and purification by biotin-streptavidin binding. As negative controls to verify non-specific labeling and purification of bases other than inosine, a DNA oligonucleotide containing 5'FAM fluorescently labeled deoxyuridine (FAM-30nt-dU) and a DNA oligonucleotide containing deoxyadenosine (FAM-24nt-dA) were prepared. On the other hand, as a positive control for calculating the purification efficiency by streptavidin via biotin when the biotin-maleimide labeling efficiency is assumed to be 100%, in addition to the 5'Cy5 fluorescent label, a DNA oligonucleotide (Cy5-24nt-dI-Bio) containing inosine whose 3' end had been 100% biotinylated at the time of synthesis was prepared. The above oligonucleotides were designed to have different base lengths in order to enable simultaneous detection by electrophoresis. After reacting equimolar amounts of Cy5-35nt-dI, FAM-30nt-dU, and FAM-24nt-A at 70°C for 15 minutes under biotin-maleimide concentration conditions of 0, 2 mM, and 10 mM, the excess biotin-maleimide was removed and purified, and then Cy5-24nt-dI-Bio, which is a streptavidin purification control oligonucleotide with an equimolar amount to the previous oligonucleotide, was added. This mixture was subjected to affinity binding using streptavidin-crosslinked magnetic beads, and then pull-down purification was performed using the magnetic bead technique. The finally eluted nucleic acid fraction was electrophoresed, and the fluorescent bands were detected and quantified, resulting in a purification yield of 90-100% using biotin-streptavidin binding. Based on this, it was confirmed that the labeling fluorescence using biotin-maleimide conforms to the characteristics confirmed with FITC-maleimide, and that the purification efficiency dependent on biotin-labeled inosine was also 90-100% (FIG. 11). It was confirmed that the difference maintained sufficient specificity even against the negative control oligonucleotide.

[Affinity purification of mouse total RNA using biotin-maleimide]
Next, after inosine-specific labeling with biotin-maleimide and purification with streptavidin magnetic beads, it was verified whether base editing of inosine generated in cells can be detected while avoiding the contamination of various nucleic acids that do not contain inosine. As a target sample, a total RNA fraction extracted and purified from an adult mouse brain was prepared. The inosine to be detected was the Q/R site that was almost 100% A-to-I edited on the glutamate receptor B (Gria2) mRNA. The mouse brain total RNA fraction was fragmented to about 800 nucleotides, labeled with biotin-maleimide (MI+), and then subjected to affinity pull-down purification with streptavidin-crosslinked magnetic beads (MI+PD+). Washing and elution after pull-down were performed as follows. The primary solution from the first wash was designated as flow-through fraction FT1, and the eluted fraction was designated as E1. Next, FT1 was purified again by pull-down using streptavidin magnetic beads, and the primary solution from the second wash was designated as the flow-through fraction FT2, and the eluted fraction as E2. As a positive control for inosine detection, cyanoethylated total RNA (CE+) treated with acrylonitrile was prepared based on [Sakurai et al., 2010]. For each of the above RNA samples, primer extension was performed using a radioisotope ³² P-labeled primer (Figure 12A, B).

As a result, when primer extension was performed using total RNA (MI-) that had not reacted with maleimide as a template, cDNA synthesis passed through the inosine site by incorporating C, and no bands due to elongation termination at the inosine site were detected. On the other hand, in CE+, a band corresponding to cDNA terminated just before the cyanoethylated inosine site at the Q-to-R site of Gria2 mRNA was observed. In the case of MI+ reacted with maleimide, a band was observed at the same position as in CE+, and the base pairing of inosine and C was inhibited by the same principle as cyanoethylation of inosine, causing cDNA elongation termination just before that point. Next, under the MI+PD+ condition, the strongest elongation termination band was observed in E1, with a yield of 55% compared to MI+. FT1 contained a small amount of inosine labeled, but most of it was recovered in E2 by another pull-down purification, and the yield of inosine-labeled RNA from E1 + E2 was calculated to be 70%. Furthermore, no bands due to inosine were detected in FT2. These results demonstrated that it is possible to specifically label and concentrate and purify inosine-containing nucleic acids contained in total RNA fractions obtained from actual tissues (Figure 12B).

On the other hand, to confirm that affinity pull-down of inosine with biotin-maleimide can purify and enrich inosine-containing RNA, a primer extension experiment was performed on the same RNA sample as above using a 5'-unlabeled primer in the presence of radioisotope α- ³² P dCTP (Figure 13). Radioisotope labeling results in two ³² P signals per molecule of RNA due to the incorporation of C into two Gs before reaching inosine. As a result, clear bands corresponding to cDNA terminated at the inosine site were observed in CE+, MI+, and MI+PD+. Furthermore, in the pull-down samples E1 and E2, since all RNA molecules other than those containing inosine were removed by affinity purification, the background band noise was very low, and the bands indicating extension termination at maleimidized inosine were more clearly detected. The above results indicate that inosine-containing RNA can be specifically purified and enriched by biotin-maleimide labeling.

[Quantitative determination of inosine]
We verified whether inosine can be quantified using inosine-containing oligonucleotides of known concentrations and FITC-maleimide. As shown in the standard curve (FIG. 14), the fluorescence intensity of FITC increases depending on the substrate concentration (inosine concentration). This demonstrated that inosine can be quantified using FITC-maleimide.

[Study of aqueous reaction solvents]
The reaction efficiency between nucleic acid containing inosine and maleimide was examined when an aqueous reaction solution was used in which a phosphate buffer-NaCl solution, which is sometimes used in the reaction of inosine and acrylonitrile, and dimethylformamide (DMF) were mixed instead of the TEAA buffer. Also, the reaction efficiency was examined when dimethylimidazolidinone (DMI) or ethanol was used instead of DMF. FIG. 15A shows the results of PAGE analysis. FIG. 15B shows the fluorescence intensity ratio (FITC/Cy5) of FITC and Cy5 in each sample. The horizontal axis represents the sample number. FIG. 15C shows the fluorescence intensity ratio (I/A) of deoxyinosine-containing oligonucleotide and deoxyadenine-containing oligonucleotide in each buffer. The test results showed that the specific reaction of inosine was particularly favorable in 0.01-0.02M phosphate buffer-0.15-0.5M NaCl solution (final concentration, pH 8.6) and 2.7-5.4M dimethylformamide (DMF) aqueous reaction solvent. It was confirmed that in aqueous reaction solvents of the same concentration, solvents that improve the solubility of FITC-maleimide, such as DMF, DMI, and ethanol, support the reaction between inosine and maleimide well. In particular, it was confirmed that the reaction efficiency was improved by increasing the concentrations of the phosphate buffer and NaCl solution, and the specific reaction of inosine was strongest in 0.02M phosphate buffer-0.5M NaCl solution (final concentration, pH 8.6) and 5.4M dimethylformamide (DMF) aqueous reaction solvent.

[Other label examples]
(Inosine-specific red fluorescent labeling with Cy5-maleimide)
The basic conditions were as described in the section "Labeling of synthetic oligonucleotides with FITC-maleimide", but Cy5-maleimide was used instead of FITC-maleimide, and it was confirmed that the Cy5 labeling reaction to inosine occurred in the same way as with FITC.

(Comparison with inosine-specific cyanoethylation reaction using acrylonitrile)
In order to confirm the reaction mechanism of maleimide in this development, a comparative analysis was performed with the inosine-specific cyanoethylation reaction using acrylonitrile in the existing ICE method. The reaction conditions for cyanoethylation alone were basically the same as those for the ICE (Inosine Chemical Erasing) method [Sakurai et al., 2010]. The DNA oligonucleotide used in the comparative analysis was dissolved in 4 μL or less of water to a final concentration of 1 pmol/μL, and then dissolved in 30 μL of 1.1 M TEAA buffer. Then, 4 μL of 15.2 M acrylonitrile (Tokyo Kasei) was added, and the reaction was performed at 70° C. for 15 or 30 minutes in the dark. The QIAquick Nucleotide Removal kit (QIAGEN) and MinElute PCR Purification kit (QIAGEN) were used to purify the DNA oligonucleotide after the reaction. On the other hand, nucleic acid cyanoethylated with acrylonitrile was purified once, and this sample was subjected to an additional labeling reaction with FITC-maleimide (Figure 16). In addition, in order to compare the strength of the reaction activity of maleimide and acrylonitrile, a competitive reaction was performed in the presence of both reagents. The conditions were set by combining the ICLAMP method and the ICE method. 50 pmol of DNA oligonucleotide was dissolved in 38 μL of 3 M TEAA buffer, and then 1.6 M of acrylonitrile and 10 mM of FITC-maleimide were added, and the final volume was adjusted to 50 μL with DMSO, and the reaction was performed at 70°C for 15 minutes or 30 minutes in the dark.

The results of electrophoretic analysis (Figure 16) showed that when a cyanoethylation reaction with acrylonitrile was performed first, followed by a reaction with FITC-maleimide, the FITC labeling efficiency was reduced by approximately one-third. On the other hand, when acrylonitrile and FITC-maleimide were reacted simultaneously, the FITC labeling efficiency was only slightly reduced compared to the reaction with FITC-maleimide alone. From the above, it was shown that the addition site of maleimide to inosine is the first position of inosine, just like cyanoethylation with acrylonitrile. Furthermore, the results of the competitive reaction confirmed that the reaction of maleimide with inosine is stronger than that of acrylonitrile.

(Compared to acrylamide derivatives)
Next, a group overseas recently reported the development of a derivative of acrylamide containing fluorescein (FITC-acrylamide) and its reaction with inosine [Knutson et al., 2018]. Therefore, we used FITC-acrylamide to compare the inosine-specific fluorescent addition reaction with the ICLAMP method. FITC-acrylamide (acrylamide fluorescein) was synthesized organically as described in [Knutson et al., 2018].

In the literature, RNA oligonucleotides were used, but in this comparison, we started with a 22-base-long DNA oligonucleotide containing 5' Cy5-labeled inosine. The labeling reaction with FITC-acrylamide was carried out at 70°C for 15 minutes to 24 hours in a TEAA buffer containing 250 mM FITC-acrylamide and 10 pmol/μL DNA oligonucleotide, according to the conditions in the literature. On the other hand, the labeling reaction with FITC-maleimide was carried out for 15 minutes to 1 hour in a TEAA buffer containing 10 mM FITC-maleimide and 1 pmol/μL DNA oligonucleotide. After the reaction, the oligonucleotides were purified, and an equal amount was prepared for the total amount of oligonucleotide and analyzed by electrophoresis. The inosine labeling efficiency was determined by the fluorescence intensity of FITC, and the total DNA amount was quantified by the fluorescence intensity of Cy5. As a result of the comparison (Figure 17), as shown above, with FITC-maleimide, inosine-specific FITC labeling was observed after 15 minutes, and the labeling efficiency reached its maximum after 1 hour. On the other hand, with FITC-acrylamide, weak inosine-selective FITC labeling was observed after 8 hours of reaction, and as per the literature, the labeling efficiency had not reached its maximum even after 24 hours. After 1 hour, the fluorescence intensity ratio of FITC-maleimide to inosine was approximately 50 times that of FITC-acrylamide.

Next, a comparison was made under the same reaction conditions, using the same amount of terminally unlabeled oligonucleotide containing inosine as in the ICLAMP method, and using the same molar concentration (10 mM) of FITC-acrylamide as FITC-maleimide. In both cases, the reaction was carried out at 70°C for 15 minutes to 24 hours in a TEAA buffer solution containing 1 pmol/μL of DNA oligonucleotide. After the reaction, the oligonucleotide was purified, and an equal amount was analyzed by electrophoresis, with the total amount of oligonucleotide being equal. The inosine labeling efficiency was measured by the fluorescence intensity of FITC, and the total DNA amount was quantified by the staining reagent. As a result of the comparison (Figure 18), even when the concentration of DNA oligonucleotide was the same in both reagent reactions, almost no fluorescent labeling of FITC-acrylamide was observed after 1 hour, weak fluorescence was confirmed from 8 hours, and the maximum labeling efficiency was not reached even after 24 hours.

The above verifications, when comparing FITC-maleimide with FITC-acrylamide, confirmed that, firstly, in terms of reaction time, the optimal labeling reaction time for FITC-acrylamide is 24 to 48 hours, whereas for FITC-maleimide it is 15 minutes to 1 hour, showing a large difference in reaction efficiency. Secondly, in terms of specificity for inosine as well, focusing on the reaction after 8 hours as an example, it was confirmed that FITC-maleimide shows approximately six times the inosine specificity confirmed in this study with FITC-acrylamide.

(Labeling with streptavidin-maleimide)
Currently, many types of maleimide derivatives are commercially available as research reagents. Among them, in addition to the above-mentioned FITC, Cy5, and biotin derivatives, streptavidin-maleimide was used to carry out an inosine-specific labeling reaction in the same manner as in the past, and it was confirmed that labeling occurred.

<Comparison with similar technologies>
The following methods for chemically modifying inosine have been reported in the field of existing molecular biology.

[Matched-tissue method]
A-to-I The most common method for detecting the presence of inosine at an RNA editing site before the establishment of the ICE method was the matched-tissue method. This method is a method of comparing the sequence of RNA (amplified as cDNA) derived from the same tissue of the same individual with the sequence of the corresponding region in the genome. Since inosine (I) forms a base pair with C, G is incorporated into the I-equivalent site in cDNA after reverse transcription from mRNA and PCR amplification. Therefore, even though the base on the genome is A at the editing site, G or A/G is mixed on the cDNA. However, with this method, it is difficult to distinguish between the mixture of G due to false signals caused by non-specific amplification of PCR, genome contamination, sequence errors, noise, SNP between alleles, pseudogenes, gene copies, splice variants, etc., and the mixture of G derived from the desired inosine. In addition, in the case of exon boundary regions or regions where pseudogenes exist, it is difficult to distinguish the genome region corresponding to the RNA, so this method itself cannot be used. Furthermore, since the inosine site identified by this method is detected as G in the sequence, the presence of inosine is not strictly proven. In addition, since it is necessary to prepare both genome and RNA from the same individual, tissue, cell, and/or condition, there is also a drawback in that the samples that can be analyzed are limited.

The advantages of the ICLAMP method over the above techniques are as follows.
- In the ICLAMP method, analysis is possible with only one sample, either RNA or DNA, which is the subject of analysis.
When searching for and identifying inosine sites in RNA, in analyses using next-generation sequencing, the detection sensitivity and accuracy usually depend on the number of reads, which correlates with the expression level of each RNA species. However, the ICLAMP method makes it possible to concentrate and purify nucleic acids containing inosine, enabling the analysis of RNA species with low expression levels.
When searching for and identifying inosine sites in DNA, analysis using next-generation sequencing usually requires analysis of the entire genome sequence, but the ICLAMP method makes it possible to concentrate and purify nucleic acids containing inosine, so the required analysis depth is less.
In the matched-tissue method, when searching for and identifying inosine sites in DNA, it is impossible to prepare DNA before inosinization as a comparison subject of the same origin, and an experiment proving inosine is not possible. On the other hand, by using the ICLAMP method, it is possible to identify inosine sites without requiring DNA before inosinization.

[Inosine site cleavage method using glyoxal and RNase T1]
In this method, after treatment with glyoxal reagent, which utilizes the chemical properties of inosine and guanosine, only inosine is easily reversible to the original base and is subjected to base-selective cleavage by RNase T1. One is an inosine-specific cleavage method aimed at identifying inosine sites in in vivo RNA [Morse, 2004; Morse & Bass, 1997, 1999]. This method consists of the following three steps.
(1) Using glyoxal as a chemical modification reagent, the bases of guanosine (G) and inosine (I) in the RNA chain are modified. In the case of guanosine (G), glyoxal is added to the 1-position and ^N2- position to form a cis-diol, which is then combined with boric acid to form a compound (G*). On the other hand, in the case of inosine (I), since there is no amino group at the 2-position, glyoxal is added only to the 1-position. Furthermore, since the added structure is unstable, it easily undergoes a reverse reaction and returns to the original inosine.
(2) The RNA after treatment with glyoxal and boric acid is cleaved by RNase T1, which is specific to G and inosine. At this time, cleavage does not occur at the G site because G has been modified to G*. On the other hand, cleavage occurs at the inosine site because inosine has not been modified (it has returned to its original state). As a result, inosine site-specific cleavage is possible.
(3) The adduct is removed from the G* in the cleaved RNA, an anchor sequence is attached to the RNA fragment containing inosine, and reverse transcription and PCR are carried out from this site to analyze the sequence.

Using this method, Morse et al. have identified inosine sites in several mRNAs (Morse and Bass, 1999). In this analysis method, it is difficult to adjust the RNase T1-insensitive modification of G by glyoxal and boric acid treatment, and it is difficult to modify all G, which accounts for 1/4 of the bases that make up RNA, so it is inevitable that some unmodified G will remain. Furthermore, inosine is present in such small amounts that only 0-30 bases are present in a single mRNA consisting of several thousand to tens of thousands of bases, and the background caused by cleavage of unmodified G sites makes it extremely difficult to detect cleavage at the target inosine site. In addition, an analysis has been reported in which this reaction was applied to a library preparation method for next-generation sequencing [Cattenoz et al., 2013].

The advantages of the proposed ICLAMP method over the above techniques are as follows:
The ICLAMP method requires only one inosine-specific chemical and enzymatic reaction step, the Michael addition reaction, and is therefore simpler and more reproducible than the above-mentioned techniques which require multiple steps.
In the ICLAMP method, the treatment specific to inosine bases is an intermolecular chemical reaction, such as with maleimide derivatives, and does not generally require an enzymatic reaction. Therefore, there is less variation in activity, deterioration, and lot-to-lot differences compared to enzymes, which are proteins. In addition, changing to a new lot is easy and inexpensive.

[Method of cleaving at inosine site using endonuclease V]
The enzyme endonuclease V (eEndoV) of Escherichia coli is a deoxyinosine 3' endonuclease that recognizes double-stranded and single-stranded DNA (dsDNA and ssDNA) containing deoxyinosine and cleaves the second and third phosphodiester bonds 3' from the deoxyinosine mismatch, leaving a nick at the 3' hydroxyl and 5' phosphate groups. Kuraoka et al. showed that human EndoV (hEndoV) prefers RNA substrates over DNA substrates. hEndoV preferentially binds RNA and efficiently hydrolyzes the second phosphodiester bond located 3' to inosine in unpaired inosine-containing single-stranded RNA regions in dsRNA [Morita et al., 2013]. Although hEndoV prefers single-stranded substrates, eEndoV is equally active on both single-stranded and double-stranded RNA [Vik et al., 2013]. To date, the following three methods for identifying inosine sites using endonuclease V have been reported.

(1) EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) [Knutson, Arthur, et al., 2020]. In this method, Ca ²⁺ ions are added instead of Mg ²⁺ ions required for cleavage activity to promote the inosine binding activity of EndoV without cleaving RNA. Furthermore, by using an EndoV enzyme recombinant tagged with maltose binding protein (MBP), it is possible to select RNA containing inosine using anti-MBP-bound magnetic beads. The EndoVIPER method cannot capture inosine contained in double-stranded RNA structures. Therefore, the aforementioned glyoxal modification is used to change the base structure of G to make the RNA single-stranded. At this time, inosine returns to its original structure by a reversible reaction, so the EndoVIPER method is performed using this as a target to select RNA containing inosine. Subsequently, the glyoxal is removed from the modified G, allowing the RNA to be analyzed by next-generation sequencing. Compared to conventional RNA-seq, this method makes it possible to avoid the effect of the expression level of the RNA itself and increase the number of reads resulting from RNA containing inosine.

(2) The EndoV-seq method simply utilizes the properties of endonuclease V to cleave RNA two bases downstream of inosine on the RNA strand, and then adds a poly(U) sequence to the 3' end of the RNA fragment. By selectively amplifying such fragments and creating a library, inosine can be detected by next-generation sequencing [Chen et al., 2022].

(3) In the EndoV-induced biotin-dATP labeling method, double-stranded DNA containing fragmented inosine is treated with the EndoV enzyme of Escherichia coli to introduce a cleavage downstream of the inosine base in the DNA strand containing the inosine. The inosine site is then replaced with biotinylated dATP by treatment with E. coli DNA pol I containing biotinylated dATP as a substrate and E. coli DNA Ligase. After that, affinity purification with biotin is performed, and the resulting DNA fragments are sequenced. The site where the G signal, where inosine has been replaced without the series of treatments, is detected as an A signal reflecting the replacement with biotinylated dATP, is determined to be the inosination site [Zheng et al., 2022].

The advantages of the ICLAMP method over the above techniques are as follows.
The ICLAMP method requires only one inosine-specific chemical and enzymatic reaction step, the Michael addition reaction, and is therefore simpler and more reproducible than the above-mentioned techniques which require multiple steps.
・In the ICLAMP method, the specific treatment of inosine bases is a chemical reaction between molecules such as maleimide derivatives, and does not basically require an enzyme reaction, so there is less variation in activity, deterioration, and difference between lots compared to enzymes, which are proteins.In addition, unlike proteins, new preparation does not require time and a certain level of technical skill, so changing to a new lot is easy and low cost.
In the ICLAMP method, the secondary and tertiary structures of the target nucleic acid are resolved under optimal reaction solution and temperature conditions, so the effect of differences in reaction efficiency due to structure on the detection rate and accuracy of inosine is small. On the other hand, it has been reported that the reaction efficiency of EndoV varies depending on the salt concentration, the secondary and tertiary structures of the nucleic acid, and whether it is a single-stranded region or a double-stranded region, which may lead to missed detection and variation in identification accuracy.
・Because EndoV has different characteristics for RNA and DNA, the principles and techniques of the inosine detection method for RNA and the inosine detection method for DNA are different. On the other hand, the ICLAMP method can be used to analyze both RNA and DNA using reactions under the same conditions.

[ICE (Inosine Chemical Erasing) Method: Cyanoethylation of Inosine with Acrylonitrile]
Inosine-specific chemical modification using acrylonitrile as a modifying agent has been reported by Yoshida et al. (Yoshida et al., 1967). In this report, to investigate the function of inosine present at the anticodon site of yeast transfer RNA, acrylonitrile was used to cyanoethylate the 1-position of inosine to inhibit its base pairing ability. The inosine chemical erasing (ICE) method is a technique that utilizes this reaction mechanism to identify inosinylation sites on transcripts [Sakurai et al., 2010, 2014]. The reaction mechanism of the ICE method is a Michael addition reaction mechanism to the N1 position of inosine via the conjugated state of an unsaturated electron pair, as shown below.

In the case of acrylonitrile, an inosine base labeling agent reported in a paper, the carbon atom at the β position is positively charged due to the electron-withdrawing properties of the cyano group. This carbon atom undergoes electrophilic addition to the nitrogen atom at position 1, which is the active amine of inosine, resulting in the addition of a cyanoethyl group to inosine. Next, a reverse transcription reaction is performed on RNA that has been specifically chemically modified with inosine that has been specifically chemically modified with inosine. If there is no chemical modification, cytidine (C) is incorporated into the inosine site by reverse transcriptase. On the other hand, if chemical modification is performed, the incorporation of C is inhibited by this chemical modification, and the extension of the reverse transcription chain is stopped before that point. After that, the reverse transcription chain whose extension has been inhibited is detected for detection, and the inosine site in the RNA is identified by comparing the presence or absence of inosine chemical modification. The information to be detected at this time includes its length, base sequence, and amount. Other techniques include detection by terminating the extension of the primer during reverse transcription, direct sequencing, in which the cDNA after reverse transcription is amplified using a PCR primer and then analyzed by Sanger sequencing [Sakurai et al., 2010; Sakurai & Suzuki, 2011], and the ICE-seq method, which has been applied to next-generation sequencing [Sakurai et al., 2014; Suzuki et al., 2015].

The advantages of the ICLAMP method over the above techniques are as follows.
In both the ICE and ICLAMP methods, the addition reaction to inosine works even when the target is RNA, DNA, or a mixture of these nucleic acids. As verified in the examples, the maleimide used in the ICLAMP method has a higher reactivity to inosine than the acrylonitrile used in the ICE method.
・As mentioned in the [Development Objectives] section, in the ICLAMP method, the cyanoethyl group once added to inosine in the ICE method is chemically very stable, and it is not possible to add additional chemical structures such as fluorescence or tags. In contrast, the ICLAMP method is a technology that can utilize the added chemical structure for analysis after the inosine-specific addition reaction.
In analyses using next-generation sequencing, detection sensitivity and accuracy usually depend on the number of reads, which correlates with the expression level of each RNA species. However, the ICLAMP method makes it possible to concentrate and purify nucleic acids that contain inosine, enabling the analysis of RNA species with low expression levels.
When searching for and identifying inosine sites in DNA, analysis using next-generation sequencing usually requires analysis of the entire genome sequence, but the ICLAMP method makes it possible to concentrate and purify nucleic acids containing inosine, so the required analysis depth is smaller.
As the additional chemical structure, FITC, Cy5, biotin, streptavidin, and other chemical structures with various uses and characteristics can be used. Furthermore, maleimide and its derivatives are currently known as labeling reagents for sulfur atoms in amino acids of proteins, and a wide variety of derivatives are being developed, so they can be purchased relatively cheaply for use in the ICLAMP method.
- The ICE and ICLAMP methods share some of the same reaction principles, but ICLAMP has advantages in terms of development and applicability.

[Labeling with acrylamide derivatives]
A derivative of acrylamide with fluorescent or biotin groups has been developed that has a reaction mechanism similar to the Michael addition reaction of inosine with acrylonitrile and is used as a material for gels for nucleic acid electrophoresis analysis [Knutson et al., 2018]. In the examples, the ICLAMP method and acrylamide derivatives were compared in terms of the labeling efficiency of inosine. As reported in the literature, the time required for inosine labeling when acrylamide fluorescein was used at 250 mM was more than 24 hours (Figure 17). Next, it was reported that when acrylamide fluorescein was used at 10 mM, the same as FITC-maleimide in the ICLAMP method, FITC-maleimide showed the maximum amount of labeling at 1 hour, while acrylamide fluorescein was barely detectable even after 24 hours (Figure 18). In addition, N-(4-ethynylphenyl)acrylamide (EPhAA) was later developed based on acrylamide fluorescein [Knutson, Korn, et al., 2020], but its reaction efficiency is about four times that of acrylamide fluorescein, and the required labeling reaction time is 6 to 12 hours.

The advantages of the ICLAMP method over the above techniques are as follows.
As shown in the Examples (FIGS. 17 and 18), the ICLAMP method has higher inosine specificity.
As shown in the examples, the ICLAMP method achieves higher labeling efficiency with a shorter reaction time than the inosine labeling reaction using acrylamide fluorescein. Since the optimal reaction temperature for acrylamide derivatives and the ICLAMP method is 70°C, a shorter reaction time avoids unexpected chemical changes. This means that the ICLAMP method can suppress side reactions and decomposition of samples to a lower level.
As shown in the examples, in the ICLAMP method, maleimide derivatives with various additional chemical structures, including fluorescent compounds of various colors and labels such as biotin and streptavidin, already exist as protein-targeting labeling agents, and are readily available on the market at low cost as research reagents, making them extremely versatile and convenient.

<Suitable applications>
[Direct sequencing method]
Even in the absence of a derivative having an additional chemical structure, maleimide acts in the same manner as acrylonitrile in the ICE method. In particular, in analyses using RNA as a sample, detection by primer extension termination, as in the ICE method, and detection by a direct sequencing method in which cDNA after reverse transcription is amplified with a PCR primer and analyzed by Sanger sequencing are possible.

The verification in this example revealed that both acrylonitrile in the ICE method and maleimide derivatives in the ICLAMP method act on inosine in RNA and DNA. However, when DNA is the target, the sequence analysis results show that the inosine conversion efficiency is 0%, 50%, and 100% for a specific DNA site per cell, i.e., one site on each homologous chromosome, for a total of two sites that can be inosinated. It is thought that this efficiency is mixed for the entire cell population. In that case, by comparing the presence and absence of maleimide (or acrylamide) reaction, a G signal reflecting inosine is detected under the absence condition, and the disappearance of the G signal is detected under the reaction condition, it is possible to prove that the site is inosine. In this way, when the presence rate of the nucleic acid containing inosine to be analyzed in the population sample is low, the ICLAMP method allows high sensitivity by concentration and purification.

[ICLAMP-seq: Next-generation sequencing]
In the ICLAMP method, inosine is labeled with biotin-maleimide, allowing selective enrichment and purification of inosine-containing nucleic acids. The recovered nucleic acids can then be used in next-generation sequencing analysis, allowing comprehensive identification and verification of inosine-conjugated sites in the target nucleic acid with higher sensitivity and accuracy than existing methods, and can be applied to both RNA and DNA.

When DNA is the subject of analysis, the number of molecules of a specific sequence per cell is two, and as mentioned above, typical next-generation sequencing analysis methods are insufficiently sensitive to detect inosinylation sites, which are rare compared to the number of bases in the entire genome. Also, unlike the case of inosinylation in RNA, the bases of DNA, which is the original blueprint of genes, are edited, so there is usually no comparison target to verify the base change, especially to prove the inosinylation site caused by the endogenous DNA editing mechanism. For the same reason, it is unavoidable to detect base changes other than inosinylation, such as single nucleotide polymorphisms, DNA mutations, and mapping errors between similar sequences, as false positives, and verification is almost impossible by sequencing alone. On the other hand, the ICLAMP method is a detection method based on the chemical characteristics of the inosine base, just like the ICE method, so it is possible to detect inosine and verify its authenticity even with only a DNA sample.

When targeting RNA, in typical next-generation sequencing analysis methods, the sensitivity and accuracy of sequence detection depends on the proportion of each RNA species in the total RNA population. Therefore, it is difficult to detect and identify inosinylation sites in RNA species with low expression levels, such as functional non-coding RNA, lincRNA, microRNA and its precursors, pre-spliced mRNA precursors, and rare mRNA. In contrast, the ICLAMP method enriches and purifies RNA species containing inosine from the RNA population that serves as the basis for the next-generation sequencing library, making it possible to detect inosine with high sensitivity and accuracy.

In recent years, the "Base Editor" method, which artificially mutates A in a target site in DNA or RNA to G via inosinylation by adding an active domain of adenosine deaminase to the Cas protein used in genome editing, has been frequently used in basic and medical research. When implementing such technology, it is generally essential to verify the presence or absence of off-target effects of the mutation introduction. However, as mentioned above, it is very difficult to detect inosine on RNA or genomic DNA that is expressed in low amounts, so there may be off-target sites that are overlooked. Even in such cases, the ICLAMP method improves sensitivity and accuracy while keeping the scale of analysis small, making it possible to verify this.

[Rapid measurement of inosine content in sample specimens using ICLAMP method]
When A-to-I RNA/DNA editing is the onset and underlying cause of a disease, early diagnosis before onset or at the time of risk emergence is most desirable. In such cases, urine or blood collected during health checkups are considered to be samples that can be collected and that are less resistant to from people undergoing the test. By purifying a certain amount of nucleic acid from the urine or blood and subjecting it to fluorescent labeling by the ICLAMP method, it becomes possible to evaluate the inosine content per unit sample by fluorescence measurement quickly, conveniently, and at low cost. If an abnormality is detected, additional diagnosis by the above-mentioned ICLAMP-seq becomes possible.

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The disclosure of Japanese Patent Application No. 2023-175606, filed on October 10, 2023, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims (18)

contacting a nucleic acid with an inosine base labeling agent;
The inosine base labeling agent is
(A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
A method for labeling an inosine base in a nucleic acid, comprising: The method for labeling an inosine base according to claim 1, wherein the added chemical structure is selected from the group consisting of a detection label, an identification label, a purification label, a reaction label, and a functional group capable of binding these. The method for labeling an inosine base according to claim 1, wherein the added chemical structure is a detection label selected from the group consisting of a fluorescent dye, a visible light dye, and a luminescent substrate; or a purification label which is an affinity tag. The method for labeling an inosine base according to claim 1, wherein the cyclic structure of the inosine base labeling site is a five-membered ring structure. The method for labeling an inosine base according to claim 1, wherein the cyclic structure of the inosine base labeling site includes a maleimide ring. The method for labeling an inosine base according to claim 1, wherein the contact is carried out in a solution of triethylamine dissolved in ethanol, or in a mixed solvent of a phosphate buffer, NaCl, and an aprotic polar solvent. Labeling an inosine base in a nucleic acid by the labeling method according to any one of claims 1 to 6;
detecting the labeled inosine base; and
A method for detecting an inosine base in a nucleic acid, comprising: The method for detecting an inosine base according to claim 7, further comprising quantifying the labeled inosine base. The method for detecting an inosine base according to claim 7, further comprising identifying the position of the labeled inosine base. The method for detecting an inosine base according to claim 7, wherein detecting the labeled inosine base includes performing a complementary strand extension reaction using a nucleic acid containing the labeled inosine base as a template. A method for determining a nucleic acid sequence, comprising detecting an inosine base by the detection method according to claim 7. Labeling an inosine base in a nucleic acid by the labeling method according to any one of claims 1 to 6;
concentrating nucleic acids containing labeled inosine bases;
A method for concentrating a nucleic acid containing an inosine base, comprising: (A) an inosine base labeling site including a cyclic structure including a carbon-carbon double bond, two carbon atoms adjacent to the carbon-carbon double bond, and a nitrogen atom, and an electron-withdrawing group bonded to each of the two carbon atoms adjacent to the carbon-carbon double bond;
(B) an additional chemical structure that is directly or indirectly attached to the nitrogen atom of the inosine base labeling site; and
13. An inosine base labeling agent comprising: The inosine base labeling agent according to claim 13, wherein the added chemical structure is selected from the group consisting of a detection label, an identification label, a purification label, a reaction label, and a functional group capable of binding these. The inosine base labeling agent according to claim 13, wherein the additional chemical structure is a detection label selected from the group consisting of a fluorescent dye, a visible light dye, and a luminescent substrate; or a purification label which is an affinity tag. The inosine base labeling agent according to claim 13, wherein the cyclic structure of the inosine base labeling site is a five-membered ring. The inosine base labeling agent according to claim 13, wherein the cyclic structure of the inosine base labeling site includes a maleimide ring. A kit comprising the inosine base labeling agent according to any one of claims 13 to 17 stored in a container.