JP5114705B2 - Bipyridine-modified nucleoside or nucleotide and method for detecting methylcytosine using the same - Google Patents

Description

  The present invention relates to bipyridine modified nucleosides or nucleotides. The present invention also relates to a method for detecting methylcytosine that uses bipyridine-modified nucleotides to determine whether the target base in a DNA sample is cytosine or methylcytosine. Furthermore, the present invention relates to a probe, a kit, a nucleic acid chip, and a methyl cytosine detection device used in the methyl cytosine detection method.

  Epigenetics consists of DNA methylation that physiologically modifies the genome, chromatin, which is a complex of DNA and protein, and post-translational modification of many proteins that make up chromatin. I have control.

  Regarding the modification of histones in chromatin, chromatin structural conversion by histone modification plays an important role in the induction of transcription. For example, acetylation by histone acetylase triggers chromatin remodeling, and transcription by basic transcription factors and RNA polymerase begins. Histone methylation and phosphorylation also cause transcriptional control, silencing, and chromatin condensation.

  In many eukaryotes, 60 to 90% of CpG dinucleotides in the genome undergo methylation at the 5th carbon atom of cytosine. Methylated CpG is found in heterochromatin and transposons containing many repetitive sequences, and is thought to suppress the activation of viruses and transposons. CpG methylation and histone modification are coordinated with each other.

  Exceptionally, the CG rich region (CpG island) in the promoter region of many genes is not methylated. Furthermore, as an exception, CpG islands are methylated in the imprinted gene, the female inactivated X chromosome. CpG islands are also methylated in the promoter region of a tumor suppressor gene in cancer cells. Therefore, methylation of cytosine can be used as a marker for cancer occurrence, recurrence, and metastasis, and there is a need for a simple method for detecting whether cytosine in a gene is methylated.

  In Patent Documents 1 and 2, DNA samples are treated with bisulfite to change unmethylated cytosine to uracil, and then PCR is performed using primers that preferentially amplify methylated cytosine over uracil. It teaches how to determine whether cytosine has been methylated by detecting the presence or absence of amplification. However, since this method changes the cytosine that occupies the majority instead of methylcytosine, the conversion efficiency of all cytosines affects the determination result, and the determination accuracy decreases accordingly.

  Patent Document 3 teaches a method for determining the DNA methylation rate by contacting an antibody that specifically binds to 5-methylcytosine with single-stranded DNA and measuring the amount of antibody bound to the DNA strand. ing. However, this method requires preparation of antibodies and amplification of DNA samples by PCR, which is troublesome.

  Non-Patent Document 1 teaches that it is possible to detect whether or not a mismatch exists in double-stranded DNA by oxidizing a double-stranded DNA sample with potassium permanganate and hydroxylamine and then treating with piperidine. Yes. This method requires hydroxylamine (see FIGS. 2 and 3). However, this method can only detect the presence of mismatches and cannot distinguish between cytosine and methylcytosine in DNA samples.

  Non-Patent Document 2 determines whether the target base in a DNA sample is cytosine or methylcytosine by inducing a reaction specific to methylcytosine or cytosine and detecting the presence or absence of the reaction. A method is disclosed. In Non-Patent Document 2, regarding a specific reaction to methylcytosine or cytosine in a DNA sample, a guide probe (bulge-forming probe) complementary to the regions on both sides adjacent to it excluding nucleotides having the target base or the target It teaches how to use a guide probe (mismatch forming probe) in which only the base and the corresponding base are mismatched. Specifically, Non-Patent Document 2 discloses a bulge structure or target base in which only nucleotides having a target base jump out of a double helix by hybridizing a bulge-forming probe or mismatch-forming probe and a DNA sample. It teaches that base pairs containing nucleotides can form a loose structure in the double helix, which can cause a specific reaction to the base of interest. However, in the method of Non-Patent Document 2, when methylcytosine is present in addition to the target base, it may be erroneously detected. Therefore, in order to suppress erroneous detection in the method of Non-Patent Document 2, it is essential to remove the single-stranded portion of the hybridized product using exonuclease or the like. For these reasons, the method of Non-Patent Document 2 is desired to be further improved in terms of simplification of operation and shortening of measurement time.

Against the background of such conventional technology, it is desired to establish a method for distinguishing tilcytosine and cytosine more easily and with high accuracy, and to provide a more useful and practical method for detecting methylcytosine. It was.
JP-T-2004-527241 JP-T-2004-500892 Special table 2004-347508 Chinh Bui et al., “Chemical cleavage reactions of DNA on solid support: application in mutation detection”, BMC Chemical Biology, 2003, Vol.3 Akimitsu Okamoto et al., “Sequence-selective osmium oxidation of DNA: ef.cient distinction between 5-methylcytosine and cytosine”, Organic & Biomolecular Chemistry., 2006, 4, 1638-1640

  An object of the present invention is to provide a novel bipyridine-modified nucleoside or nucleotide that is suitably used for determining whether a target base in a DNA sample is cytosine or methylcytosine. Furthermore, the present invention provides a methyl cytosine detection method for determining whether a target base in a DNA sample is cytosine or methyl cytosine, and a probe, kit, nucleic acid chip, and methyl cytosine detection used in the DNA sample. An object is to provide an apparatus.

  In order to solve the above-mentioned problems, the present inventors diligently studied. According to a bulge-forming probe or a mismatch-type probe containing a nucleotide modified with a bipyridine group, these probes are separated from a DNA sample. It has been found that in the hybridized state, the target base on which the bipyridine group can act is limited, so that the selectivity of methylcytosine to be reacted with the bipyridine group can be increased. That is, the present inventors have obtained the following knowledge regarding a novel bipyridine-modified nucleoside or nucleotide and a method for determining whether a target base in a DNA sample is methylcytosine or cytosine.

  (i) In the nucleoside or nucleotide represented by the following general formula (1), the 4,4′-bipyridine group can be selectively bonded to methylcytosine via osmic acid.

[In the formula (1), n represents an integer of 0 to 3,
R ₁ represents a hydrogen atom, a halogen atom, a hydroxyl group, or an amino group,
Z may be substituted with at least one selected from the group consisting of a halogen atom, an alkyl group having 1 to 10 carbon atoms, a carboxyl group, an amino group, a dimethylamino group, and a carbamoyl group. A bipyridine group represents a nucleobase bonded via a linker. ]

(ii) By using the following guide probe (mismatch formation probe) of (a) to hybridize with a DNA sample, a base pair containing a nucleotide having a target base takes a loose structure in a double helix, Other nucleotides in the DNA sample are buried in the double helix structure, so that the reaction can be caused only by the target base.
(a) Guide probe
(1) can hybridize with DNA sample,
(2) the base pairing with cytosine or methylcytosine for detection of DNA sample is mismatched; and
(3) It contains a nucleotide residue represented by the following general formula (1-1).

(iii) Furthermore, by using the following guide probe (bulge forming probe) of (b) to hybridize with the DNA sample, a bulge structure in which only nucleotides having the target base jump out of the double helix is formed. The other nucleotides in the DNA sample are buried in the double helix structure, so that the reaction can be caused only by the target base.
(1) can hybridize with DNA sample,
(2) a deletion of a nucleotide pairing with cytosine or methylcytosine for detection of a DNA sample; and
(3) It contains a nucleotide residue represented by the above general formula (1-1).

  (iv) By hybridizing a DNA sample and a guide probe as described in (ii) or (iii) above to form a structure that can cause a reaction only to the target base, by allowing osmate to act, Different reactants are generated depending on whether the target base is methylcytosine or cytosine. That is, if the target base is methylcytosine, the methylcytosine and the 4,4′-bipyridine group in the nucleotide residue represented by the general formula (1-1) inserted into the guide probe are osmium. Binding through an acid forms a crosslink between the DNA sample and the guide probe. On the other hand, if the target base is cytosine, no crosslink is formed between the DNA sample and the guide probe.

  (v) Therefore, the base in question is cytosine by detecting the presence or absence of the formation of a cross-link between the DNA sample and the guide probe after the osmate is acted as in (iv) above. Or methylcytosine can be determined.

The present invention has been completed based on the above findings, and provides the following novel bipyridine-modified nucleoside or nucleotide, methylcytosine detection method and the like.
Item 1. A nucleoside or nucleotide represented by the general formula (1).

[In the formula (1), n represents an integer of 0 to 3,
R ₁ represents a hydrogen atom, a halogen atom, a hydroxyl group, or an amino group,
Z may be substituted with at least one selected from the group consisting of a halogen atom, an alkyl group having 1 to 10 carbon atoms, a carboxyl group, an amino group, a dimethylamino group, and a carbamoyl group. A bipyridine group represents a nucleobase bonded via a linker. ]
Item 2. Item 2. The nucleoside or nucleotide according to Item 1, wherein the nucleobase is a purine base, a 7-deazapurine base, or a pyrimidine base.
Item 3. Item 3. The nucleoside or nucleotide according to Item 1 or 2, wherein the linker has any one of the following structures.

— (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 − [m1 and m2 are the same or different and represent an integer of 1 to 20]
_{- (O-CH 2 -CH 2} ) l -O- [l represents an integer of 1 to 100]
− (CH ₂ ) _k − [k represents an integer of 1 to 20]
Item 4. A 4,4′-bipyridine group in which Z is substituted with a methyl group is bonded to — (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 — (m1 and m2 are the same as described above) as a linker. Item 2. The nucleoside or nucleotide according to Item 1, which is a purine base bonded thereto.
Item 5. Item 2. The nucleoside or nucleotide according to Item 1, which is a compound represented by the following general formula (1-a).

[Wherein, m1 and m2 are the same as described above. ]
Item 6. A method for detecting the presence or absence of cytosine methylation in a DNA sample,
A DNA sample and a guide probe containing a nucleotide residue represented by the following general formula (1-1) are hybridized so that cytosine or methylcytosine for detection purposes forms a bulge structure or mismatch. 1 process,

[Wherein, R ₁ and Z are the same as described above. ]
It is represented by the general formula (1-1) inserted in the guide probe and methylcytosine formed with a bulge structure or mismatch by allowing osmate to act on the hybridized product obtained in the first step. A second step of cross-linking a 4,4′-bipyridine group in the nucleotide residue to be detected, and a third step of detecting the presence or absence of cross-linking between the guide probe and the DNA sample, Methylcytosine detection method.
Item 7. The guide probe forms a mismatch with cytosine or methylcytosine to be detected and hybridizes with a DNA sample. In the guide probe, the nucleotide residue represented by the general formula (1-1) is: Item 7. The detection method according to Item 6, wherein the detection method is contained in a site that matches and mismatches with the target cytosine or methylcytosine.
Item 8. The guide probe is one in which cytosine or methylcytosine to be detected forms a bulge structure and hybridizes with a DNA sample. In the guide probe, the nucleotide residue represented by the general formula (1-1) is Item 7. The detection method according to Item 6, which is contained in a site that is paired with a base adjacent to cytosine or methylcytosine for detection purposes.
Item 9. Item 9. The detection method according to any one of Items 6 to 8, wherein the residue of the nucleotide represented by the general formula (1-1) is a nucleotide residue represented by the following general formula (1-1-a) .

[Wherein, m1 and m2 are the same as described above. ]
Item 10. Item 10. The detection method according to any one of Items 6 to 9, wherein the guide probe has a length of 20 to 100 bases.
Item 11. Item 11. The detection method according to any one of Items 6 to 10, wherein the guide probe is bound to a solid phase carrier.
Item 12. A probe for detecting the presence or absence of cytosine methylation in a DNA sample, comprising a nucleic acid having the following characteristics:
(1) can hybridize with DNA sample,
(2) the base pairing with cytosine or methylcytosine for detection of DNA sample is mismatched; and
(3) A nucleotide residue represented by the general formula (1-1) is included.

[Wherein, R ₁ and Z are the same as described above. ]
Item 13. A probe for detecting the presence or absence of cytosine methylation in a DNA sample, comprising a nucleic acid having the following characteristics:
(1) can hybridize with DNA sample,
(2) a deletion of a nucleotide pairing with cytosine or methylcytosine for detection of a DNA sample; and
(3) It contains a nucleotide residue represented by general formula (1-1).

[Wherein, R ₁ and Z are the same as described above. ]
Item 14. Item 14. A hybridization product of the probe according to Item 12 or 13 and a DNA sample.
Item 15. Item 14. A kit for detecting methylcytosine comprising the probe according to Item 12 or 13 and osmate.
Item 16. Item 14. A nucleic acid chip in which one or more probes according to Item 12 or 13 are immobilized on a substrate.
Item 17. Item 17. A methylcytosine detection device comprising the nucleic acid chip according to Item 16 and a device capable of detecting a color development pattern on the chip.

Hereinafter, the present invention will be described in detail.
(I) Nucleoside or nucleotide represented by general formula (1) The present invention provides a nucleoside or nucleotide represented by general formula (1).

  In the formula (1), n represents an integer of 0 to 3. When n is 0, the compound corresponds to a nucleoside, and when n is an integer of 1 to 3, the compound corresponds to a nucleotide.

In the formula (1), R ₁ represents a hydrogen atom, a halogen atom, a hydroxyl group, or an amino group. Specific examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. In formula (1), R ₁ is preferably a hydrogen atom.

  In the formula (1), Z represents a nucleobase having a substituted or unsubstituted 4,4′-bipyridine group bonded via a linker.

  Here, specific examples of the nucleobase include a purine base, a 7-deazapurine base, and a pyrimidine base. Among these, a purine base is preferable.

  When the nucleobase is a purine base, specific examples of the purine base include the group of the following general formula (2).

In the formula (2), X ₁ represents O, NH, CH ₂ or S, and is preferably NH. In the group of the general formula (2), the a side is a site that binds to the pentose in the general formula (1), and the b side is a site that binds to the linker.

  Further, when the nucleobase is a 7-deazapurine base, specific examples of the 7-deazapurine base include a group of the following general formula (3).

In formula (3), X ₂ is the same as X ₁ . In the group of the general formula (3), the a side is a site that binds to the pentose in the general formula (1), and the b side is a site that binds to the linker.

  Furthermore, when the nucleobase is a pyrimidine base, specific examples of the pyrimidine base include groups of the following general formula (4) or (4 ′).

In the formulas (4) and (4 ′), X ₃ is the same as X ₁ . In the group of the general formula (3), the a side is a site that binds to the pentose in the general formula (1), and the b side is a site that binds to the linker.

  In Z in formula (1), the linker for linking the substituted or unsubstituted 4,4′-bipyridine group and the nucleobase is not particularly limited, but as a specific example, a linker having the following structure: Is mentioned.

— (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 — [m1 and m2 are the same or different and represent an integer of 1 to 20, preferably an integer of 1 to 5]
_{- (O-CH 2 -CH 2} ) l -O- [l 1-100 integer, preferably an integer of 1 to 10]
_{- (CH 2) k - [} k is an integer of 1 to 20, preferably an integer of 1 to 10]
Among these linkers, preferably _{- (CH 2) m1 -NH-} CO- (CH 2) m2 - , and the like. The linker is not particularly limited, but it is desirable that the linker is configured such that a nucleobase binds to the left binding site and a substituted or unsubstituted 4,4′-bipyridine group binds to the right binding site.

  In Z in formula (1), the substituted or unsubstituted 4,4′-bipyridine group specifically includes a halogen atom, an alkyl group having 1 to 10 carbon atoms, a carboxyl group, an amino group, dimethyl group. It is a 4,4′-bipyridine group which may be substituted with at least one selected from the group consisting of an amino group and a carbamoyl group.

  Here, specific examples of the alkyl group having 1 to 10 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and an isobutyl group. As a C1-C10 alkyl group, Preferably a C1-C5 alkyl group, More preferably, a C1-C3 alkyl group is mentioned.

  Specific examples of the substituted or unsubstituted 4,4′-bipyridine group include groups of the following general formula (5) or (5 ′).

In the formulas (5) and (5 ′), R _{11 to} R ₁₇ and R _{21 to} R ₂₂ are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a carboxyl group, an amino group, A dimethylamino group or a carbamoyl group is shown. In the groups of the formulas (5) and (5 ′), the c side is the site that binds to the linker.

In Z in the formula (1), the substituted or unsubstituted 4,4′-bipyridine group is preferably a group of the general formula (5). Among the groups of the general formula (5), when R _{11 to} R ₁₆ are hydrogen atoms and R ₁₇ is an alkyl group having 1 to 10 carbon atoms (preferably a methyl group), the reaction to osmate The characteristics are high, which is preferable.

As a preferred example of Z in the formula (1), a 4,4′-bipyridine group substituted with one methyl group is represented by — (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 — (m1 And m2 is the same as described above) and purine bases bonded via a linker. Specific examples of more preferable Z include a group represented by the following general formula (Za).

  In the formula (Za), m1 and m2 are the same as described above.

  The nucleoside or nucleotide represented by the general formula (1) of the present invention can be easily produced by chemical synthesis as long as it is an expert in this technical field. As a method for synthesizing the nucleoside or nucleotide represented by the general formula (1), specifically, a nucleoside or nucleotide represented by the following general formula (11) and a substituted or unsubstituted 4,4 having the structure described above A method of linking '-bipyridine with the above-described linker is exemplified.

In the formula (11), R ₁ is the same as described above, and Z ′ represents a nucleobase.

  The nucleoside or nucleotide represented by the general formula (11) and the substituted or unsubstituted 4,4′-bipyridine having the structure described above are known compounds or compounds easily derived from known compounds. In addition, in order to link the nucleoside or nucleotide represented by the general formula (11) and the substituted or unsubstituted 4,4′-bipyridine having the above-described structure with the above-mentioned linker, the type of linker to be used is changed. Accordingly, reaction conditions may be set as appropriate. Such a linker connection condition can also be easily set by a specialist in this technical field.

  In the nucleoside or nucleotide represented by the general formula (1) of the present invention, a substituted or unsubstituted 4,4′-bipyridine can be selectively bonded to methylcytosine via an osmate. Therefore, the nucleoside or nucleotide represented by the general formula (1) is, for example, a structure of a probe used for determining whether a target base in a DNA sample is cytosine or methylcytosine, as described later. Useful as nucleotides.

  Polynucleotides such as probes containing the nucleotide represented by the general formula (1) are prepared according to a known DNA synthesis method using the nucleotide and other nucleotides as raw materials and controlling them to have a predetermined base sequence. it can.

(II) Methylcytosine detection method The methylcytosine detection method of the present invention detects the presence or absence of cytosine methylation in a DNA sample, that is, the target base in the DNA sample is cytosine or is methylcytosine. This is a method for detecting this.

  In the methylcytosine detection method of the present invention, the DNA sample to be detected for the presence or absence of cytosine methylation is not particularly limited in terms of its origin. The DNA sample may be, for example, a biological sample itself such as blood, urine, sputum, semen, hair, skin, etc., or may be isolated from a biological sample. Is preferably used.

  The method for detecting methylcytosine according to the present invention includes the following first to third steps.

A first sample in which a DNA sample and a guide probe containing a nucleotide residue represented by the above general formula (1-1) are hybridized so that cytosine or methylcytosine to be detected forms a bulge structure or mismatch. Process,
By reacting the hybridized product obtained in the first step with osmate, methylcytosine formed with a bulge structure or mismatch, and the general formula (1-1) inserted in the guide probe A second step of cross-linking the 4,4′-bipyridine group in the nucleotide residue represented, and a third step of detecting the presence or absence of cross-linking between the guide probe and the DNA sample.

Hereinafter, the methylcytosine detection method of the present invention will be described for each step.
In order to induce a reaction specific to the detection target base in the first step DNA sample, it is necessary that the drug used for the reaction approaches the detection base, but each nucleotide is usually a double-stranded DNA helix. It is buried in the structure and the drug cannot be accessed.

  Therefore, in the first step of the method of the present invention, cytosine or methylcytosine for detection in a DNA sample (hereinafter sometimes referred to as “target base”) forms a bulge structure or mismatch. A DNA sample is hybridized with a guide probe containing a residue of the nucleotide represented by the general formula (1-1).

  Here, the “bulge structure of the target base” refers to a target base (cytosine or methyl) that has jumped out of the double strand without forming a base pair in a double-stranded nucleic acid formed by a DNA sample and a probe. A bulge composed of nucleotides containing cytosine).

  Thus, by forming a bulge structure or a mismatch with the target base in the DNA sample, it becomes possible to cause a specific reaction with respect to the target base.

In the present invention, as a guide probe (bulge generating probe) for forming a bulge structure with a target base, one having the following characteristics is used:
(1) can hybridize with DNA sample,
(2) A nucleotide that is paired with cytosine or methylcytosine for detection of a DNA sample is deleted, that is,
(3) A nucleotide residue represented by the general formula (1-1) is included.

  In the bulge generating probe, the insertion position of the residue of the nucleotide represented by the general formula (1-1) is particularly limited as long as it is in a position capable of binding to the target base that formed the bulge structure via osmic acid. Although it is not a thing, Preferably, the position (pairing) with respect to the base adjacent to the target base of a DNA sample is illustrated.

  In addition, as a more suitable bulge-generating probe, a DNA sample, except that the nucleotide paired with the target base is deleted and the nucleotide residue represented by the general formula (1-1) is included. And those that are completely complementary to the base sequence. By using such a bulge generating probe, erroneous detection of methylcytosine can be more effectively suppressed.

In the present invention, a guide probe (mismatch generation probe) that forms a mismatch with a target base is one having the following characteristics:
(1) can hybridize with DNA sample,
(2) The base paired with cytosine or methylcytosine for detection of DNA sample is mismatched; and
(3) It contains a nucleotide residue represented by general formula (1-1).

  In the mismatch generating probe, the insertion position of the residue of the nucleotide represented by the general formula (1-1) is not particularly limited as long as it is in a position capable of binding to the mismatched target base via osmic acid. However, Preferably, the position (pairing) with respect to the target base of a DNA sample is illustrated.

  In principle, as a preferred mismatch generating probe, a nucleotide mismatched with a target base is a nucleotide represented by the general formula (1-1), and other nucleotides are completely complementary to a DNA sample. Some are listed. By using such a mismatch generation probe, erroneous detection of methylcytosine can be more effectively suppressed.

  The suitable length of the guide probe differs depending on the method, and is usually preferably about 20 to 100 bases, more preferably about 20 to 50 bases. If it is this length, while being able to form a double strand stably, it is compoundable with a DNA automatic synthesizer.

  In addition, there is no particular limitation as to where the guide probe is designed to form a bulge or mismatch. Preferably, it may be designed so that the bulge or mismatch of the target base can be formed near the center of the double strand.

  The guide probe only needs to be complementary so that it can hybridize with the above region, and may be any of DNA, RNA, peptide nucleic acid (PNA), modified nucleic acid, and the like. When a DNA sample containing a nuclease such as a cell extract is used, it becomes difficult to be degraded by a nuclease by using a modified probe as a guide probe. Examples of the modified nucleic acid include phosphorothioate modified nucleic acid, morpholino modified nucleic acid, 2′-O-alkyl nucleic acid, 2′-N-alkyl nucleic acid, 2′-S-alkyl nucleic acid, 2′-halogen nucleic acid and the like. In addition, in order to stabilize hybridization, nucleotides having a modified base such as 2-aminoadenine and 5-alkynyluridine are arranged in the probe sequence, or the end or the inside of the probe is modified with amine, polyamine or the like. Alternatively, a dideoxynucleotide can be added to the end of the probe.

  In addition, the guide probe is preferably fixed to a bead-like or flat plate-like carrier, whereby the DNA sample bound to the probe is sequentially subjected to different treatments or the DNA sample is washed between treatments. Can do. In addition, the probe can be reused. Examples of such a carrier material include polystyrene, gold, glass, magnetic iron oxide fine particles, and zinc sulfide fine particles having quantum dot properties.

  In the first step, hybridization conditions are not particularly limited. Depending on the type of DNA sample, the length of the guide probe, etc., for example, after treatment with a hybridization solution such as sodium phosphate buffer pH 7 at room temperature to about 100 ° C. for about 5 minutes to 1 hour, pH 7 A condition of washing at about 5 to 25 ° C. for about 1 to 5 minutes using a sodium phosphate buffer or the like is mentioned.

Second Step In this second step, a selective reaction is induced with respect to the target base formed with the bulge structure or mismatch formed in the first step. Specifically, in this second step, the hybridization product obtained in the first step is inserted into a guide probe and methylcytosine in which a bulge structure or mismatch is formed by acting osmate. The substituted or unsubstituted 4,4′-bipyridine group in the nucleotide residue represented by the general formula (1-1) is cross-linked.

  Specific examples of the osmic acid used in the second step include potassium osmate.

  In the second step, in order to form a cross-link between methylcytosine and a 4,4′-bipyridine group, for example, osmate is present in the presence of about 0.1 to 1000 mM, preferably about 1 to 100 mM. What is necessary is just to process for 30 second-about 1 hour at about 0-40 degreeC. Such treatment is desirably performed under pH conditions of about pH 6-7. Furthermore, in this treatment, the cross-link formation rate can be increased by adding an oxidizing activator such as potassium ferricyanide or methylmorpholine oxide to the extent of 0.1 to 100 mM, preferably about 0.1 to 50 mM. .

  In this second step, when the target base is methylcytosine, the methylcytosine, which is the target base in the DNA sample, and the bipyridine group in the guide probe are bonded via osmic acid, and the crosslink having the structure shown below Is formed.

[In the above cross-linked structure, x represents a methylcytosine moiety as a target base in a DNA sample, y represents an osmium acid moiety, and z represents a bipyridine group moiety of a guide probe. In the above cross-link structure, the bipyridine group portion is described as an example having no substituent. ]
On the other hand, in the second step, when the target base is cytosine, the above-described crosslink is not formed.

  The second step may be performed simultaneously with the first step. That is, a 1st process and a 2nd process can also be implemented simultaneously by mixing a guide probe, a DNA sample, an osmate, and the oxidation activator as needed, and making it react on predetermined conditions.

Third Step In the third step, the presence / absence of a cross-link between the guide probe and the DNA sample is detected. When a cross link between the guide probe and the DNA sample is detected, it is determined that the target base is methylcytosine. On the other hand, if the cross-link structure between the guide probe and the DNA sample is not detected, it is determined that the target base is cytosine.

  In the third step, the specific detection method is not particularly limited as long as the cross-link between the guide probe and the DNA sample can be detected.

  As an example of the detection method of the crosslink structure, a method of detecting the labeling substance after labeling the crosslink portion with a labeling substance is exemplified. Here, examples of the label with a labeling substance include enzyme labels, fluorescent labels, electrochemical labels, and radiolabels. Further, the detection of the labeling substance is performed by a known method according to the type of the labeling substance.

  In addition, examples of the method for detecting the cross-link structure include molecular weight measurement of a DNA sample by mass spectrometry or gel electrophoresis. The details are as follows. If the guide probe and the DNA sample are cross-linked, they exist in a bound state even under conditions where hybridization is not possible. On the other hand, if the guide probe and the DNA sample are not cross-linked, they are separated and exist under conditions where hybridization is not possible. Therefore, if the DNA sample is subjected to mass spectrometry analysis or gel electrophoresis under conditions where hybridization cannot be performed, and the weight or molecular weight of the DNA sample is increased, the guide probe and the test DNA are cross-linked. If there is no change in weight or molecular weight, it is determined that the guide probe and the DNA sample are not cross-linked.

Furthermore, as another method for detecting the cross-link structure, a method for measuring the thermal stability of the hybridized product of the guide probe and the DNA sample is exemplified. More specifically, the melting point of the hybridized product is higher when the crosslink is formed than when the crosslink is not formed. Therefore, the presence / absence of crosslink formation can also be determined by measuring the melting point of the hybridized product of the guide probe and DNA sample. Moreover, since the thermal stability of the hybridized product is improved when crosslinks are formed, in addition to the above, the following method can be adopted as a method for measuring the thermal stability:
(1) First, the guide probe is designed so that, in the hybridized product of the guide probe and the DNA sample, a region of the DNA sample that is not hybridized with the guide probe is formed. That is, the number of bases of the guide probe is set to be smaller than the number of bases of the DNA sample.
(2) The hybridized product of the guide probe and DNA sample after the second step is stable when the crosslink is formed, but is hybridized when the crosslink is not formed. Washing is performed after treatment under conditions where the soy product becomes unstable and the double-stranded structure is impaired. As an example of such conditions, conditions for boiling at 95 ° C. for 10 minutes are exemplified. By performing such a treatment, a hybrid product in which a cross-link is formed can maintain a double-stranded structure, whereas a hybrid product in which a cross-link is not formed is double-stranded. The structure is lost and only the guide probe is left.
(3) Separately, a cross-link detection probe that can hybridize to a region that does not hybridize with the guide probe in the DNA sample and has a labeling substance bound thereto is prepared. By causing the cross-link detection probe to act on the processed product of the step (2), it is hybridized with a region in the DNA sample that is not hybridized with the guide probe.
Since the hybridized product in which the crosslink is not formed is only the guide probe by the step (2), the crosslink detection probe cannot be hybridized. On the other hand, since the hybridized product in which the crosslink is formed maintains the double-stranded structure even in the step (2), the probe for detecting the crosslink is in contact with a predetermined region of the DNA sample. Can be hybridized.
(4) After the step (3), the presence or absence of a labeling substance is detected. If the labeling substance is detected, it can be determined that a crosslink is formed in the hybridized product. If the labeling substance is not detected, it can be determined that no crosslink is formed in the hybridized product.

(III) Kit for detecting methylcytosine A kit for detecting methylcytosine of the present invention is a kit for carrying out the above-described method for detecting methylcytosine, and comprises the above-mentioned guide probe and an osmate. This methyl cytosine detection kit may further contain an oxidation activator. The methyl cytosine detection kit may include a reagent for detecting a cross link formed between the guide probe and the DNA sample.

(IV) Using a nucleic acid chip having one or more guide probes immobilized on a nucleic acid chip substrate for detecting methylcytosine, the presence or absence of cytosine methylation at various positions for various genes can be detected at a time. Can do.

  The probe is the guide probe described above, but different probes may be used so that a plurality of target bases can be determined.

  The material of the base of this nucleic acid chip is not particularly limited, and known materials such as glass, silica, and gold can be used. The spot diameter of the probe on the substrate can be about 50 to 200 μm, for example, and the spot pitch can be about 100 to 500 μm, for example.

  What is necessary is just to perform each operation of the method of this invention demonstrated above with respect to the probe group on this nucleic acid chip.

(V) Methylcytosine Detection Device The methylcytosine detection device of the present invention is a device comprising the nucleic acid chip described above and a device capable of detecting a color development pattern on the chip. Examples of the color pattern detection device include a microplate reader, a microchip reader, a fluorescence microscope, and a fluorescence imager that can measure fluorescence, absorption, fluorescence polarization, fluorescence lifetime, and the like.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to these.

Production Example 1
According to the following steps, a phosphoramidite compound of 4′-methyl-2,2′-bipyridine-modified purine nucleoside was synthesized. A specific synthesis method is as follows.

Synthesis of N- (2-aminoethyl)-(4'-methyl-2,2'-bipyridin-4-yl) hexanamide (hereinafter referred to as Compound 1)
(4'-Methyl-2,2'-bipyridin-4-yl) hexanoic acid (568 mg, 2.0 mmol) and PyBOP (Benzotriazole-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate) (1.14 g, 2.2 mmol) are combined with DMF ( Dimethylformamide) (10 ml) and stirred at room temperature for 30 minutes. To this solution was then added 1,2-diaminoethane (222 μL, 2.2 mmol) and the mixture was stirred at room temperature for 2 hours. The resulting solution was concentrated under reduced pressure and diluted with chloroform. The organic phase was washed with 1N sodium hydroxide, washed with sodium chloride solution, and dried with a desiccant (magnesium sulfate), and further concentrated under reduced pressure to give brown oily compound 1 (603 mg, 1.7 mmol, Yield 85%) was obtained. ¹ H NMR (CDCl ₃ ) δ 8.46 (t, 2H, J = 5.4 Hz), 8.20 (d, 2H, J = 6.8 Hz), 7.12-7.08 (m, 2H), 2.66 (quartet, 2H, J = 7.8 Hz), 2.42 (s, 3H), 2.20-2.12 (m, 2H), 1.72-1.52 (m, 4H), 1.42-1.39 (m, 4H), 1.32-1.25 (m, 2H); ¹³ C NMR (CDCl ₃ ) δ155.9, 152.4, 148.8, 148.7, 148.0, 124.5, 123.8, 121.9, 121.1, 38.8, 36.2, 35.1, 29.9, 28.7, 26.7, 20.1; FABMS (NBA / CHCl ₃ ) m / z327 ([ (M + H) ⁺ ]), HRMS calcd. For C ₁₉ H ₂₇ ON ₄ ([(M + H) ⁺ ]) 327.2185, found 327.2184.
(6- (2- (6- (4'-Methyl-2,2'-bipyridin-4-yl) hexanamido) ethylamino-9H-purin-9-yl) -5'-O-dimethoxytrityl-2 Synthesis of '-deoxyriboside (hereinafter referred to as Compound 2)
To DMF (50 ml) containing (6-chloro-9H-purin-9-yl) -5′-O-dimethoxytrityl-2′-deoxyriboside (11.0 g, 19.2 mmol), diisopropyleneamine (23 ml) and DMF (50 ml) containing compound 1 (11.0 g, 55.4 mmol) was added and the mixture was stirred at 75 ° C. for 12 hours. After dilution with ethyl acetate (1 L), the resulting mixture was subjected to a washing treatment with a sodium chloride solution, a drying treatment with a desiccant (magnesium sulfate), a filtration treatment, and a vacuum distillation treatment. The crude product thus obtained was subjected to silica gel column chromatography (chloroform: methanol = 50: 1, containing 1% NH ₄ OH) to obtain white solid compound 2 (15.8 g, 18.3 mmol, yield). 95%). ¹ H NMR (CD ₃ OD) δ8.41 (dd, 2H, J = 4.8, 10.1 Hz), 8.17 (s, 1H), 8.12 (s, 1H), 8.04 (s, 2H), 7.33 (d, 2H , J = 7.3 Hz), 7.20 (d, 4H, J = 8.8 Hz), 7.15-7.08 (m, 5H), 6.71 (dd, 4H, J = 4.0, 8.4 Hz), 6.38 (t, 1H, J = 6.2 Hz), 4.60 (quartet, 1H, J = 4.2 Hz), 4.10 (quartet, 1H, J = 4.4 Hz), 3.66 (s, 6H), 3.43 (t, 2H, J = 5.9 Hz), 3.32-3.27 (m, 2H), 2.83 (dt, 1H, J = 6.2, 13.5 Hz), 2.56 (t, 2H, J = 7.5 Hz), 2.44 (ddd, 1H, J = 4.6, 6.2, 13.4 Hz), 2.36 ( s, 3H), 2.11 (t, 2H, J = 7.1 Hz), 1.61-1.50 (m, 4H), 1.29-1.23 (m, 2H); ¹³ C NMR (CD ₃ OD) δ176.3 160.0, 157.0, 156.3, 154.7, 153.8, 150.3, 150.0, 149.9, 146.2, 140.4, 137.10, 137.06, 131.23, 131.20, 129.3, 128.7, 127.8, 126.0, 125.4, 123.6, 122.9, 114.0, 87.9, 87.6, 85.8, 72.6, 65.0, 55.7, 40.8, 40.3, 36.9, 36.0, 30.9, 29.6, 26.5, 21.2; FABMS (NBA / CH ₃ OH) m / z 863 ([(M + H) ⁺ ]), HRMS calcd.for C ₅₀ H ₅₅ O ₆ N ₈ ([(M + H) ⁺ ]) 863.4245, found 863.4235.
Synthesis of phosphoramidite compound of compound 2 (hereinafter referred to as compound 3) To a solution of compound 2 (47 mg, 54 μmol) and tetrazole (3.8 mg, 54 μmol) in anhydrous acetonitrile (500 μL), 2-cyanoethyltetra Isopropyl phosphorodiamidite (17.1 μl, 54 μmol) was added under a nitrogen atmosphere. The mixture was stirred at room temperature for 30 minutes. The resulting reaction product was filtered to obtain Compound 3.
Deprotection (dimethoxytrityl group) of compound 2 from dimethoxytritylated compound 2 eliminates the nucleoside of the present invention (wherein n in general formula (1) is 0, R1 is a hydrogen atom, Z is the following general formula (Za-1) represented by 4'-methyl-2,2'-bipyridine modified purine).
Further, by removing a protecting group (dimethoxytrityl group) from compound 2 and further adding a predetermined number of phosphoric acids, the nucleotides of the present invention (wherein n in general formula (1) is 1-3 and R1 is hydrogen). A compound in which the atom and Z are 4′-methyl-2,2′-bipyridine-modified purines represented by the general formula (Za-1) is obtained.

Production Example 2
Except for using 1,5-diaminopentane instead of 1,2-diaminoethane, the nucleoside of the present invention (wherein n in the general formula (1) is 0, R1 is A hydrogen atom, a compound wherein Z is the following 4′-methyl-2,2′-bipyridine-modified purine) and a nucleotide (n in the general formula (1) is 1 to 3, R1 is a hydrogen atom, Z is the following general formula ( A compound that is a 4′-methyl-2,2′-bipyridine-modified purine represented by Za-4) was synthesized.

Test Example 1 Detection of methylcytosine
As a DNA sample, 20mer DNA (5′-GGTGGGGGCAGNGCCTCACA-3 ′; N represents cytosine or methylcytosine) (SEQ ID NO: 1) was used, and the 5 ′ end was radiolabeled ( ³² P) and purified.

  Separately, as a mismatch formation probe, 20mer DNA (5′-TGTGAGGCNCTGCCCCCACC-3 ′; N represents 4′-methyl-2,2′-bipyridine-modified purine represented by the general formula (Za-1)) ( SEQ ID NO: 2) was prepared.

  Next, the labeled DNA sample (0.5 μM, 50 μL) and the mismatch forming probe (0.5 μM, 50 μL) are added to a Tris-HCl buffer solution (pH 7.0, containing 1 mM EDTA) containing 5 mM potassium osmate and 100 mM potassium ferresocyanide. And left at 50 ° C. for 10 minutes. The reaction solution thus obtained was subjected to polyacrylamide electrophoresis, and the presence or absence of the formation of a cross-linked body was confirmed by analyzing the increase and decrease in the molecular weight of the product after the reaction. Also, for comparison, using 20mer DNA with an unsubstituted purine without adding potassium osmate or as a mismatch-forming probe instead of 4'-methyl-2,2'-bipyridine modified purine The test was carried out under the following conditions.

  The results are shown in FIG. From the results of lanes 1 to 6 in FIG. 1, it was confirmed that a cross-linked body was not formed under the condition where potassium osmate was not added. It was also confirmed that a cross-linking body was not formed with a mismatch-forming probe that did not have 4′-methyl-2,2′-bipyridine-modified purine. On the other hand, from the results of lanes 7 and 8, it was shown that the DNA sample whose target base is methylcytosine detects a different band from the DNA sample whose target base is cytosine.

  In addition, in order to clarify that a crosslink is formed between a DNA sample whose target base is methylcytosine and a mismatch forming probe, the sample after the above reaction is purified and identified by MALDI-TOF MS. went. From the results, it was suggested from the mass data that the target base was bound to a DNA sample whose methyl cytosine was the target via a mismatch forming probe osmium complex.

  Furthermore, in order to examine whether the crosslink formed between the DNA sample whose target base is methylcytosine and the mismatch-forming probe is a methylcytosine site-selective crosslink reaction, piperidine treatment is performed after the above reaction. The product was analyzed by gel electrophoresis. As a result, since the cleavage was observed only at the methylcytosine site, it was shown that the crosslink was selectively generated with respect to methylcytosine (see FIG. 2).

  That is, from the above results, as shown in FIG. 3, a DNA sample in which the target base is methylcytosine and a mismatch forming probe are selectively cross-linked, and the DNA sample in which the target base is cytosine. It was shown that no cross-link was formed between the probe and the mismatch-forming probe.

  Furthermore, the obtained adduct (DNA sample whose target base is methylcytosine-mismatch formation probe) has a melting point of 79.1 ° C., and is an osmium-untreated adduct (DNA sample whose target base is methylcytosine-mismatch formation probe) Compared with the melting point (54.8 ° C.), the thermal stability was greatly increased. This indicates that methylcytosine can be easily detected using double-stranded stabilization by cross-linking to methylcytosine as an index.

Test Example 2 Detection of methylcytosine The above test example except that the 4′-methyl-2,2′-bipyridine-modified purine represented by the general formula (Za-4) in the mismatch-forming probe is of the following structure The test was conducted in the same manner as in Example 1 to detect methylcytosine.

  The results are shown in FIG. FIG. 4 also shows the results of Test Example 1 described above. From this result, it was revealed that methylcytosine can be selectively detected regardless of the linker length difference (difference of 2 or 5 carbon atoms).

Test Example 3 Detection of methylcytosine using long-chain DNA as a sample To confirm whether methylcytosine can be detected using a long-chain DNA as a sample, the following test was performed.

First, as DNA samples, 30 mer DNA; that 'using (N denotes a cytosine or methylcytosine (SEQ ID NO: 3), its 5 5'-CTCATGGTGGGGGCAGNGCCTCACAACCTC-3)' end was radiolabeled ⁽³² P), purified Prepared. The DNA sample (0.5 μM, 50 μL) and the mismatch forming probe (0.5 μM, 50 μL) used in Test Example 1 are added to a Tris-HCl buffer solution (pH 7.0, containing 1 mM EDTA) containing 5 mM potassium osmate and 100 mM potassium ferresocyanide. And reacted at 50 ° C. for 1 to 20 minutes, and sampled over time. The reaction solution thus obtained was subjected to polyacrylamide electrophoresis, and the presence or absence of the formation of a cross-linked body was confirmed by analyzing the increase and decrease in the molecular weight of the product after the reaction.

  The results are shown in FIG. From this result, it was confirmed that methylcytosine can be detected with high accuracy even for a long-chain (30-mer) DNA sample.

Test Example 4 Detection of methylcytosine using a labeled substance <Test method>
By performing the following steps a to h, methylcytosine was detected using a labeling substance. In addition, it shows in FIG. 6 about the schematic diagram of each process in this test.
Step a: Binding of mismatch-forming probe to chip 20mer DNA (5′-AAACTCCACNCACAAACACG-3 ′; N is 4′-methyl-2,2′-bipyridine represented by the general formula (Za-1)) (Denoting a modified purine) (SEQ ID NO: 4) was prepared and aminated by modifying its 5 ′ end with the group — (CH ₂ ) ₁₈ —NH ₂ .

  Prepare 150 mM Nacaco buffer containing the amination mismatch-forming probe (25 μM) and NaCl (0.1 M), add 2 μL to a chip with aldehyde groups (slide glass coated with aldehyde groups) overnight at room temperature Incubated with. Subsequently, it was washed with an aqueous solution containing 0.15 wt% SDS and then dried to obtain a chip to which the 5 ′ end of the DNA sample was bound.

Step b: Reduction to the chip to which the mismatch-forming probe is bound Next, 4 mL of PBS (phosphate buffered saline; pH 7.0), 10 mg of sodium hydroxide and 1 mL of ethanol are added to the mismatch-forming probe bound to the chip. , Left at room temperature for 5 minutes, washed twice with water and dried to reduce the nitrogen atoms bound between the chip and the mismatch forming probe (ie, between the chip and the mismatch forming probe). The attached group was converted from the group —N═C— to the group —NH—CH ₂ —).

Step c: Hybridization of mismatch-forming probe and DNA sample Next, 50 mer DNA (5′-TTTGAGGTGNGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGA-3 ′; N represents cytosine or methylcytosine) (SEQ ID NO: 5) is used as the DNA sample. A sample (predetermined amount of a mixture of N and methylcytosine in SEQ ID NO: 5) and a mismatch-forming probe bound to a chip were hybridized. Specifically, 2 μL of a 50 mM sodium phosphate solution (pH 7.0) containing a DNA sample (1 μM) and NaCl (0.1 M) is added to the mismatch-forming probe bound to the chip, and left at room temperature for 30 minutes. I put it. Then, after washing with 1 × SSC, further washing with 0.1 × SSC and drying, a hybridized product of a mismatch forming probe and a DNA sample was obtained on the chip.

Step d: Formation of crosslinks in hybridized product Further, 2 μL of a tris-hydrochloric acid buffer solution (pH 7.7, containing 1 mM EDTA) containing 5 mM potassium osmate and 100 mM potassium ferreocyanide is added to the hybridized product obtained above. And allowed to react at room temperature for 1 hour. Subsequently, it was washed with an aqueous solution containing 0.15 wt% SDS and then dried to obtain a reaction product in which a crosslink was formed in the hybridized product.

Step e: Decomposition of the hybridized product in which no crosslink is formed Next, the reaction product subjected to the above treatment is immersed in water at 95 ° C. for 10 minutes and then dried to hybridize in which no crosslink is formed. The product was degraded.

Step f: Hybridization of cross-link detection probe Furthermore, as a cross-link detection probe, 20mer DNA (3'-TCTCTGGCCGCGTGTCTCCT-5 ') in which biotin is bound to the 3' end via triethylene glycol (SEQ ID NO: 6) was prepared. 2 μL of a 50 mM sodium phosphate solution (pH 7.0) containing this crosslink detection probe (10 μM) and NaCl (0.1 M) was added to the reaction product subjected to the above treatment, and left at room temperature for 30 minutes. . Then, after washing with 1 × SSC, further washing with 0.1 × SSC and drying, a reaction product in which the DNA sample and the crosslink detection probe were hybridized on the chip was obtained.

Step g: Blocking of chip Next, 1.5 mL of PBS containing calf serum albumin (BSA) (50 mg / mL) was added to the reaction product on the chip obtained above and allowed to stand at room temperature for 5 minutes. . Thereafter, the chip to which the reaction product was bound was blocked by washing with PBS.

Step h: Binding of Cy3-avidin conjugate Furthermore, to the reaction product on the chip as described above, BSA (50 mg), PBS (1.5 mL) and Cy3-avidin conjugate (15 μL) were added, and at room temperature for 5 minutes. Reacted. Then, after washing with PBS and drying, a reaction product in which Cy3-avidin conjugate was bound to biotin present in the reaction product on the chip was obtained.

Step g: Measurement of fluorescence intensity of Cy3 The fluorescence intensity of Cy3 of the reaction product on the thus treated chip was measured (excitation wavelength: 532 nm, detection wavelength: 585 ± 45 nm).

<Result>
As a result, strong fluorescence luminance was detected according to the proportion of DNA whose target base was methylcytosine in the DNA sample (see FIG. 7). That is, based on this result, according to the present invention, a calibration curve showing the relationship between the methylation frequency and the detected fluorescence brightness is prepared in advance, so that a methyl sample of cytosine is methylated with respect to a DNA sample whose unknown frequency is unknown. It was confirmed that the quantification amount could be quantified.

2 is a diagram showing the results of polyacrylamide electrophoresis performed in Test Example 1. FIG. In Figure 1, the column of K ₂ OsO ₄ is - is intended, it shows the results when no added K ₂ OsO _4, those該欄is "+" with the addition of K ₂ OsO ₄ "" The results are shown. In the figure, the ODN1 '( ^Bpy A) column with "-" is the result when using a mismatch-forming probe that does not have a 4'-methyl-2,2'-bipyridine-modified purine. The column with “+” indicates the result when a mismatch forming probe having 4′-methyl-2,2′-bipyridine-modified purine is used. Further, in the figure, the column “N” in N indicates the result when the target base is cytosine in the DNA sample, and the column “M” indicates that the target base is methyl cytosine in the DNA sample. The result of the case is shown. In Experiment 1, it is a figure which shows the result of having performed the piperidine process after the cross-linking reaction and analyzing the product by gel electrophoresis. In FIG. 2, the “-” in the probe column indicates the result when no mismatch forming probe is added, and the “+” column indicates the 4′-methyl-2,2′-bipyridine modification. Results are shown when using mismatch-forming probes with purines. In the figure, the column of Os is - results of those represents the results obtained without the addition of K ₂ OsO _4, those該欄is "+" is the addition of K ₂ OsO ₄ "" Indicates. Further, in the figure, the column “N” in N indicates the result when the target base is cytosine in the DNA sample, and the column “M” indicates that the target base is methyl cytosine in the DNA sample. The result of the case is shown. It is a figure which shows typically the knowledge clarified from the result obtained in Test Example 1. That is, 4′-methyl-2,2′-bipyridine group and methylcytosine form a crosslink via osmic acid, but in 4′-methyl-2,2′-bipyridine group and cytosine, the crosslink is It is a figure which shows typically not forming. FIG. 6 is a diagram showing the results of polyacrylamide electrophoresis performed in Test Example 2. In FIG. 4, m1 = 1 shows the result when 4′-methyl-2,2′-bipyridine modified purine represented by the general formula (Za-1) is bound to the mismatch forming probe, The case of m1 = 4 shows the result when 4′-methyl-2,2′-bipyridine-modified purine represented by the general formula (Za-4) is bound to the mismatch forming probe. In the figure, the column “N” in N indicates the result when the target base is cytosine in the DNA sample, and the column “M” indicates that the target base is methyl cytosine in the DNA sample. The result of the case is shown. FIG. 6 is a diagram showing the results of polyacrylamide electrophoresis performed in Test Example 3. In the figure, the column “N” in N indicates the result when the target base is cytosine in the DNA sample, and the column “M” indicates that the target base is methyl cytosine in the DNA sample. Results are shown. It is a figure which shows typically for every process about the detection of the methylcytosine performed in Test Example 4. FIG. In the figure, X represents 4′-methyl-2,2′-bipyridine-modified purine, and M represents methylcytosine. It is a figure which shows the test result conducted in Test Example 4. In the figure, [M] / ([M] + [C]) is the ratio (%) of DNA in which N is methylcytosine in the DNA shown in SEQ ID NO: 5 used as the DNA sample, that is, the target base is methyl It shows the ratio (%) of DNA having the target base methylcytosine to the total amount of the DNA having cytosine and the DNA having the target base cytosine. In the figure, the left figure shows the results observed with a fluorescence microscope (results of four experiments; results of one experiment per row). Moreover, in the figure, the right figure plots the average of the luminance of the fluorescence of the four experimental results shown in the left figure.

Claims (15)

A nucleoside or nucleotide represented by the general formula (1).

[In the formula (1), n represents an integer of 0 to 3,
R ₁ represents a hydrogen atom, a halogen atom, a hydroxyl group, or an amino group,
Z may be substituted with at least one selected from the group consisting of a halogen atom, an alkyl group having 1 to 10 carbon atoms, a carboxyl group, an amino group, a dimethylamino group, and a carbamoyl group. Purine base represented by the general formula (2) to which a bipyridine group is bonded via a linker

[In the formula (2), X ₁ represents O, NH, CH ₂ or S, and the group of the general formula (2) is a site where the side a is bonded to the pentasaccharide in the general formula (1). Yes, the b side is the site that binds to the linker. ]. ] The nucleoside or nucleotide according to claim 1, wherein the linker has any one of the following structures.
— (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 − [m1 and m2 are the same or different and represent an integer of 1 to 20]
_{- (O-CH 2 -CH 2} ) l -O- [l represents an integer of 1 to 100]
− (CH ₂ ) _k − [k represents an integer of 1 to 20] A 4,4′-bipyridine group substituted with a methyl group is — (CH ₂ ) _m1 —NH—CO— (CH ₂ ) _m2 — (m1 and m2 are as defined in claim 2 ; The nucleoside or nucleotide according to claim 1, wherein the nucleoside or nucleotide is the purine base represented by the general formula (2) in which the same is bound as a linker. The nucleoside or nucleotide according to claim 1, which is a compound represented by the following general formula (1-a).

[ Wherein m1 and m2 are the same as defined in claim 2.] ] A method for detecting the presence or absence of cytosine methylation in a DNA sample,
A DNA sample and a guide probe containing a nucleotide residue represented by the following general formula (1-1) are hybridized so that cytosine or methylcytosine for detection purposes forms a bulge structure or mismatch. 1 process,

[Wherein R ₁ and Z are the same as defined in claim 1 . ]
It is represented by the general formula (1-1) inserted in the guide probe and methylcytosine formed with a bulge structure or mismatch by allowing osmate to act on the hybridized product obtained in the first step. A second step of cross-linking a 4,4′-bipyridine group in the nucleotide residue to be detected, and a third step of detecting the presence or absence of cross-linking between the guide probe and the DNA sample, Methylcytosine detection method. The guide probe forms a mismatch with cytosine or methylcytosine to be detected and hybridizes with a DNA sample. In the guide probe, the nucleotide residue represented by the general formula (1-1) is: The detection method according to claim 5, wherein the detection method is contained in a site that is mismatched by pairing with cytosine or methylcytosine to be detected. The guide probe is one in which cytosine or methylcytosine to be detected forms a bulge structure and hybridizes with a DNA sample. In the guide probe, the nucleotide residue represented by the general formula (1-1) is The detection method according to claim 5, wherein the detection method is contained in a site paired with a base adjacent to cytosine or methylcytosine to be detected. The detection according to any one of claims 5 to 7, wherein the residue of the nucleotide represented by the general formula (1-1) is a nucleotide residue represented by the following general formula (1-1-a): Method.

[ Wherein m1 and m2 are the same as defined in claim 2.] ] The detection method according to claim 5, wherein the guide probe has a length of 20 to 100 bases. The detection method according to claim 5, wherein the guide probe is bound to a solid phase carrier. A probe for detecting the presence or absence of cytosine methylation in a DNA sample, comprising a nucleic acid having the following characteristics:
(1) can hybridize with DNA sample,
(2) the base pairing with cytosine or methylcytosine for detection of DNA sample is mismatched; and
(3) A nucleotide residue represented by the general formula (1-1) is included.

[Wherein R ₁ and Z are the same as defined in claim 1 . ] A probe for detecting the presence or absence of cytosine methylation in a DNA sample, comprising a nucleic acid having the following characteristics:
(1) can hybridize with DNA sample,
(2) a deletion of a nucleotide pairing with cytosine or methylcytosine for detection of a DNA sample; and
(3) It contains a nucleotide residue represented by general formula (1-1).

[Wherein R ₁ and Z are the same as defined in claim 1 . ] A kit for detecting methylcytosine comprising the probe according to claim 11 or 12 and osmate. A nucleic acid chip on which one or more of the probes according to claim 11 or 12 are fixed on a substrate. An apparatus for detecting methylcytosine comprising the nucleic acid chip according to claim 14 and an apparatus capable of detecting a color development pattern on the chip.

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