JP2012060901A - Oligonucleotide and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detective system easily detectable of base's deletion and insertion.SOLUTION: The detective system is provided, which uses an oligonucleotide having two kinds of units selected from a first unit represented by formula (1) (wherein, R1 is a 2-4C alkynylene group; R2 is a phenylene group; A1 is H, a phosphate group, or nucleotide or oligonucleotide linked via a phosphodiester linkage, and B1 represents a hydrogen atom, phosphate group, nucleotide or oligonucleotide), a second unit represented by formula (2) (wherein, R2 is H or a 1-4C alkylene group) and a quencher unit; wherein, the two kinds of units are set apart from nucleotide(s) of one or two or more bases.

Description

  The present invention relates to oligonucleotides and uses thereof.

  It is preferable from the viewpoint of medical economics as well as improving the patient's QOL to know in advance the possibility of developing a disease in the future or the effectiveness and side effects of drugs. Genetic testing is suitable for testing such unique characteristics of humans and animals. Currently, genetic testing is primarily directed to single nucleotide polymorphisms (SNPs) in which one base is replaced with another base. However, gene polymorphisms in humans and the like are not limited to SNPs, but are widely related to deletions and insertions of one base and insertions and deletions of two or more bases. There is also a report that polymorphisms other than SNPs account for 75% of gene polymorphisms in humans (Non-patent Document 1).

  On the other hand, various methods for detecting gene polymorphisms and mutations including SNPs are known (Non-patent Documents 2 and 3). In addition, it has been reported that perylene is used as a fluorescent dye, and that 1-base deletion and 2-base deletion can be detected by wavelength shift due to excimer emission by bimolecular association from monomer emission of each of two perylenes (non-patent document). 4)

J.C.Venter et l., Plos Biol., 2007, 5, e266 B. Eshaque, et al., Biotechonology Advances. 2006, 24, 86 Saito, I, et, al., J. AM. CHEM. SOC. 2004, 126, 8364 H. Kashida, et al., Tetrahedron Lett., 2007, 48, 6759-6762.

  However, in the conventional method, it has not always been possible to easily and accurately detect base deletion and insertion. There were also no oligonucleotides that could effectively cope with such mutations.

  The present inventors tried to construct a mutation detection system by new derivatization of perylene as a fluorescent dye. As a result, by using a combination of two perylene derivatives with different structures between the oligonucleotide main chain and the perylene group, not only one base but also one or more base deletions or insertion mutations I found that I can be targeted. Further, it has been found that such a perylene derivative itself is useful as a signal site. According to the disclosure of the present specification, the following means are provided based on such findings.

According to the disclosure herein, an oligonucleotide, the first unit being separated by one or more nucleotides;
(In the formula (1), R1 represents an alkynylene group having 2 to 4 carbon atoms, R2 represents a phenylene group, and A1 is a nucleotide linked through a hydrogen atom, a phosphate group, or a phosphodiester bond, or Represents an oligonucleotide, B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide, and Perylene represents a perylene group.)
The second unit:
(In Formula (2), R3 represents an alkynylene group having 2 to 4 carbon atoms, R4 represents an alkylene group having 1 to 4 carbon atoms, and A1 represents a hydrogen atom, a phosphate group, or a phosphodiester bond. B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide, and Perylene represents a perylene group.) And two types selected from a quencher unit. Oligonucleotides are provided.

The first unit may be represented by the following formula.

The second unit may be represented by the following formula.

  The oligonucleotide can be used as a probe for detecting a deletion mutation or insertion mutation of 1 base or 2 bases or more. In particular, it can be used as a probe for detecting deletion mutations or insertion mutations of 3 or more bases. In addition, a molecular beacon can be formed, and the first unit and the second unit are provided on one strand of the stem when the molecular beacon is formed, and the first unit and the second unit are coupled to the unit. Char may be provided on the other strand of the stem.

According to the disclosure of the present specification, compounds of the following formulas (3) and (4) are also provided. Any of these compounds are useful as amidite monomers for synthesizing the oligonucleotides disclosed herein.
(In the formula (3), R1, R2 and Perylene have the same meaning as in the formula (1). In the formula (4), R3, R4 and Perylene have the same meaning as in the formula (2). In 3) and formula (4), C1 represents a hydrogen atom or a hydroxyl protecting group, and D1 represents a hydrogen atom, a hydroxyl protecting group, a phosphoramidite group, or a linking group bonded to or bonded to a solid phase carrier. In the present invention, the term “phosphoamidite group” includes all those that can be used in the phosphoramidite method.

According to the disclosure of the present specification, an oligonucleotide probe immobilized body comprising:
There is provided an immobilized body immobilized on a solid phase carrier using the oligonucleotide according to any one of claims 1 to 5 as a probe.

  According to the disclosure of the present specification, there is provided a method for testing a deletion mutation or an insertion mutation, wherein the presence or absence of the deletion mutation or the insertion mutation is distinguishable from each wild type. There is provided a detection method comprising the step of bringing the disclosed oligonucleotide into contact with a test DNA-containing sample. The test DNA-containing sample may contain DNA collected from a human or an animal. Moreover, the inspection method of the presence or absence of the onset possibility of a disease may be used.

  According to the disclosure of the present specification, it is a test kit for a deletion mutation or an insertion mutation, and the presence or absence of the deletion mutation or the insertion mutation having one or more bases can be distinguished from each wild type. A kit is provided comprising the oligonucleotide probe of claims 1-8 configured as above.

  According to the disclosure of the present specification, an oligonucleotide comprising one or both of the first unit and the second unit is provided.

It is a figure which shows the mechanism which identifies 1 base deletion by light emission. It is a figure which shows the mechanism which identifies 3 base deletion by light emission. Emission spectra of various perylene derivatives in the double chain Fluorescence spectrum when FAF is used as a probe Fluorescence spectrum when LAL is used as a probe Fluorescence spectrum when FAL is used as a probe Fluorescence spectrum when used for FCTTF probe Fluorescence spectrum when used with LCTTL probe Fluorescence spectrum when used with FCTTL probe

  The disclosure herein relates to oligonucleotides and uses thereof. The oligonucleotide disclosed in the present specification comprises two types selected from a first unit, a second unit, and a quencher separated by one base or two or more bases. That is, the present disclosure discloses an oligonucleotide comprising a first unit and a second unit, an oligonucleotide comprising a first unit and a quencher unit, and an oligonucleotide comprising a second unit and a quencher unit. Relates to nucleotides.

  As shown in FIG. 1 and FIG. 2, for example, when an oligonucleotide comprising a first unit and a second unit is used as a probe, it is hybridized with a target having a deletion of 1 base or 2 bases or more. When a bulge containing a base or two or more bases is formed and the signal site linked to the threoninol chain in the first unit and the signal site linked to the threoninol chain in the second unit are normally hybridized Different from the orientation, a predetermined arrangement state is formed. Each of the first unit and the second unit has a signal site containing a perylene group, but has a predetermined structure between the first unit and the main chain. Therefore, the first unit and the second unit hybridize with a target that does not have the deletion. It will be in a different arrangement state than when you do. At the same time, a unique excitation light based on such a sequence state can be emitted, and a unique signal different from that when hybridizing with a target not having the deletion is exhibited.

  In addition, when an oligonucleotide having a first unit and a quencher unit separated by 1 base or 2 bases or more is used as a probe and hybridized with a target having a deletion of 1 base or 2 bases or more, 1 in the probe A bulge containing a base or two or more bases is formed. At this time, the signal portion of the first unit and the quencher of the quencher unit are oriented along with bulge formation to form a predetermined arrangement state. On the other hand, when hybridizing with a target not having the deletion, such a bulge is not formed, and the first unit and the quencher unit are arranged separated by one base or two or more bases. As a result, when the bulge is formed and when the bulge is not formed, the arrangement state of the quencher with respect to the signal site of the first unit is different, and the action of the quencher with respect to the signal site of the first unit is different. For this reason, such a probe will exhibit a unique signal different from that at the time of hybridization with a target having no deletion against a target having a deletion. A similar phenomenon can occur in an oligonucleotide having a second unit and a quencher unit separated by one base or two or more bases.

  From the above, according to the oligonucleotide probe disclosed in the present specification, a target having a deletion of one base or two or more bases can be distinguished from a target having no such deletion. In other words, a target having an insertion of one base or two or more bases can be distinguished from a target having no such insertion.

  Each of the first unit and the second unit has a signal site containing perylene, but each of these signal sites functions as a dye. These can be said to be different pigments. There are only very limited reports on the structure of exciplexes (hetero excimers) in which different dyes form excited dimers, and it is difficult to control exciplex or excimer formation at present. However, as for the structure of perylene excimer, it has been clarified that pyrenes are arranged with an offset of 1.4 cm, as described in “Organic Quantum Chemistry and Photoscience”, page 287 by Kei Yamazaki. Although different from the excimer, according to Chemistry A European Journal, Vol. 9, pp. 3466 to 3471, the dye in the H aggregate that has a layered structure without shifting the dyes. The J-aggregate having a shifted structure emits light more strongly on the longer wavelength side than the monomer.

  The present inventors hypothesized that it is important to take a structure in which the dyes deviate in the bulge in order to form an exciplex (or excimer) by double strand formation with the deletion polymorphism. . In order to take such a deviated structure, as a result of diligently considering that it is more advantageous for two dyes having different structures to associate (exciplex) than to associate two identical dyes (excimer), As a result of the above, that is, the knowledge that the presence or absence of the deletion can be identified by providing the specific deletion unit corresponding to the specific first unit and the second unit apart from each other. It was.

  At the same time, the present inventors have found that both the first unit and the second unit are useful as a fluorescent unit. Hereinafter, the disclosure of this specification will be described in detail.

(Oligonucleotides and probes)
The length of the oligonucleotide disclosed in the present specification is not particularly limited, but is preferably about 10 to 100 bases. The oligonucleotide may have a length exceeding 100 bases.

  In the present oligonucleotide, the first unit and the second unit can be provided separated by 1 base or 2 bases or more. A first unit and a second unit separated by 3 or more bases or 4 or more bases may be provided. Even when the present oligonucleotide is separated by 3 or more bases, it can emit specific excitation light during bulge formation. Preferably it is 3 bases or more. Further, they are preferably separated by 6 or less bases. This is because if too few bases are separated, the orientation of the first unit and the second unit during bulge formation is limited, and the intended discernible signal is not exhibited. This is because if too many bases are separated, it becomes difficult to provide a signal that can be discriminated whether or not a bulge is formed.

  The first unit and the second unit, the first unit and the quencher unit, and the second unit and the quencher unit are all provided separated by one nucleotide or two or more nucleotides. The sequence of these two units in the oligonucleotide is not limited. That is, any of the two units may be located on the 5 ′ end side. In formula (1) or formula (2), A1 usually corresponds to the 5 'side of the oligonucleotide, and B1 corresponds to the 3' side. Specifically, a configuration in which the second unit is arranged on the 3 'side of the first unit can be mentioned.

(First unit)
A 1st unit is represented by Formula (1). The first unit has a perylene group. The perylene group is advantageous in that it is photochemically stable and has high quantum efficiency. In the perylene group, hydrogen atoms bonded to one or more carbon atoms other than the carbon atom linked to the R1 group may be appropriately substituted. Although the substituent is not particularly limited, for example, each independently a hydrogen atom; an alkyl group or alkoxy group having 1 to 20 carbon atoms that is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, or a carboxyl group An unsubstituted or substituted alkenyl or alkynyl group having 2 to 20 carbon atoms substituted with a halogen atom, hydroxyl group, amino group, nitro group or carboxyl group; hydroxyl group; halogen atom; amino group; nitro group; or carboxyl group be able to.

  In formula (1), R1 is an alkynylene group having 2 to 4 carbon atoms. Alkynylene groups include ethynylene groups (—C≡C—), propynylene groups (—C≡C—C—, —C—C≡C—), butynylene groups (—C≡C—C—C—, —C). -C≡C-C-, -C-C-C≡C-). Preferably, it is an ethynylene group.

  Although the connection position of R1 to perylene is not particularly limited, it may be any of 1-position, 2-position and 3-position of perylene. Preferably, it is any one of the 2nd, 4th, 9th and 10th positions.

  In the formula (1), R2 represents a phenylene group. The phenylene group may not be substituted, but a hydrogen atom bonded to one or more carbon atoms not involved in the connection may be substituted. Although the substituent is not particularly limited, for example, each independently a hydrogen atom; an alkyl group or alkoxy group having 1 to 20 carbon atoms that is unsubstituted or substituted with a halogen atom, a hydroxyl group, an amino group, a nitro group, or a carboxyl group An unsubstituted or substituted alkenyl or alkynyl group having 2 to 20 carbon atoms substituted with a halogen atom, hydroxyl group, amino group, nitro group or carboxyl group; hydroxyl group; halogen atom; amino group; nitro group; or carboxyl group be able to.

  Although the connection bonding site in the phenylene group of R2 is not particularly limited, it may be connected to the main chain at any of the ortho, meta, and para positions from the connection position with respect to R1. The para position is preferred.

As the first unit, for example, a unit represented by the following formula is suitable.

(Second unit)
A 2nd unit is represented by Formula (2). Similar to the first unit, the second unit has a perylene group. As with the first unit, the perylene group may be optionally substituted.

  In Formula (2), R3 is an alkynylene group having 2 to 4 carbon atoms. Alkynylene groups include ethynylene groups (—C≡C—), propynylene groups (—C≡C—C—, —C—C≡C—), butynylene groups (—C≡C—C—C—, —C). -C≡C-C-, -C-C-C≡C-). Preferably, it is an ethynylene group.

  The connection position of R3 with respect to the perylene group is not particularly limited, and may be any of the 1-position, 2-position, 3-position, etc. of the perylene group. Preferably, it is any one of the 2nd, 4th, 9th and 10th positions.

  In the formula (2), R4 is an alkylene group having 1 to 3 carbon atoms. For example, methylene group, ethylene group, and propylene group. Preferably, it is an ethylene group.

As the second unit, for example, a unit represented by the following formula is suitable.

  In the formulas (1) and (2), A1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide linked via a phosphodiester bond, and B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligo. Represents a nucleotide. Nucleotides and oligonucleotides have a phosphate group at the carbon atom at the 5 'position of the 5 monosaccharide. When B1 is a nucleotide and an oligonucleotide, they are linked by a phosphodiester bond through their phosphate group.

  The main chain portion in the first unit and the second unit of the present oligonucleotide has a D-threoninol-phosphate structure as shown in the formulas (1) and (2). . By having the main chain, it is considered that an excited state based on exciplex is easily formed in the first unit and the second unit. In other units of the oligonucleotide, a sugar-phosphate backbone can be provided. In units other than the first unit and the second unit, known modifications and alterations may be made in the base and sugar moieties.

  The quencher unit includes a quencher in the main chain part that can constitute a part of the main chain of the oligonucleotide. The main chain portion in the quencher unit may be a normal sugar-phosphate main chain, but may have a D-threoninol-phosphate structure. For example, a unit represented by the following formula may be used.

  In the above formula, A1 and B1 have the same meaning as in formula 1, and Y represents a quencher.

  The quencher is not particularly limited as long as it can quench the fluorescence derived from these units at the time of bulge formation with respect to the first unit or the second unit.

  For example, the quencher (Y) includes the following. In addition, the various compounds of the following examples are illustrations, and all the hydrogen atoms may be appropriately substituted with a substituent. Further, the quencher atom linked to the main chain portion is not particularly limited.

  A preferable quencher includes a compound having an anthraquinone skeleton. As the compound having an anthraquinone skeleton, in addition to anthraquinone, one or two or more hydrogen atoms bonded to the carbon atom constituting the benzene ring of anthraquinone may be a hydroxyl group, a nitro group, an amino group, a sulfonic acid group, a halogen atom, Examples include various anthraquinone derivatives substituted with one or more functional groups selected from an alkylamino group and the like. Various anthraquinone derivatives can be easily obtained from, for example, Tokyo Chemical Industry Co., Ltd. For example, natural examples of the anthraquinone derivative include compounds of cochineal dyes, red dyes such as alizarin, and rack dyes. Anthraquinone is preferred. An example of a quencher unit is shown in the following formula.

  The oligonucleotide can be configured to detect deletion mutations or insertion mutations of one or more bases. The intended mutation can be detected by bringing the oligonucleotide into contact with the target DNA under conditions that allow hybridization. In order to detect the mutation, the portion other than the mutation is configured to have a base sequence complementary to the target DNA at least within a certain range.

  This oligonucleotide is configured to have a base sequence complementary to either a wild type or a mutant type. That is, in order to detect a deletion mutation of one base or two or more bases, a probe is designed so as to have a base sequence complementary to a wild-type base sequence that does not have the deletion mutation. A first unit and a second unit are provided separated by a base that is deleted in the variant. Similarly, the first unit and the quencher unit may be provided. Furthermore, a second unit and a quencher unit may be provided.

  On the other hand, in order to detect an insertion mutation of one base or two or more bases, the probe base sequence is designed so as to have a base sequence complementary to the mutant base sequence of the insertion mutation. That is, the first unit and the second unit are provided apart from each other by the base inserted in the mutant type. Similarly, the first unit and the quencher unit may be provided. Furthermore, a second unit and a quencher unit may be provided.

  The oligonucleotide can also be configured to form a molecular beacon. The molecular beacon is a single-stranded DNA and is configured to be able to form a stem-loop structure. The molecular beacon can detect the target by opening the stem and hybridizing with the target DNA. When this oligonucleotide probe is used as a molecular beacon, the first unit and the second unit are provided on one strand of the stem, and the quencher for the first unit and the second unit is provided on the other strand of the stem. Can be provided. By doing this, the light emission can be quenched when the stem is closed, and when hybridized with the target DNA, a bulge is formed in the stem and exhibits a characteristic light emission in the first unit and the second unit. Can do. Although it does not specifically limit as a quencher, For example, anthraquinone etc. are mentioned.

  According to the disclosure herein, an oligonucleotide comprising at least one or both of the first unit and the second unit is also provided. The signal sites provided in the first unit and the second unit themselves emit light that is shifted to the longer wavelength side compared to conventional perylene. Therefore, an oligonucleotide comprising one or more of these units as a signal unit is useful as a probe. These oligonucleotides are also useful as precursors of the above-described bright oligonucleotides comprising the first and second units. The first unit and the second unit in the oligonucleotide of this aspect include all the various aspects described above.

  When the oligonucleotide of this embodiment is used as a probe, the first unit and the second unit may be provided, or one of them may be provided. Moreover, the oligonucleotide of this aspect can be equipped with 1 type, or 2 or more types of 1st units and 2nd units, respectively. The arrangement of these units in the oligonucleotide is not particularly limited. The first unit may be continuous or the second unit may be continuous so that a signal can be detected. Further, the first unit and the second unit may be combined and continuously arranged. Furthermore, these units may be located only on one terminal side of the oligonucleotide or on both ends. It may also be inside the oligonucleotide.

(Manufacturing method of oligonucleotide probe)
The present oligonucleotide can be obtained by incorporating the first unit and the second unit, the first unit and the quencher unit, and the second unit and the quencher unit, respectively, into the oligonucleotide. The introduction of the first unit and the second unit and the other combinations of the oligonucleotides already described in this case may not be carried out in the conventionally known DNA solid phase synthesis method in place of the amidite derivative corresponding to the nucleotide. This is possible by using an amidide derivative. As a method for obtaining an amidite derivative, for example, the following scheme is exemplified.

In the above scheme, the amino group of D-threoninol is protected with ethyl trifluoroacetate, and then DMT is introduced and deprotected to obtain a DMT derivative of D-threoninol. Thereafter, this DMT derivative and 4-pentynoic acid are introduced into the amino group of D-threoninol to obtain a pentynoic acid-DMT derivative. A separately synthesized halogen (here Br) perylene is reacted with this DMT derivative to introduce perylene via an ethynylene group to obtain an ethynylperylene DMT derivative. This ethynylperylene DMT derivative is then amidite-ized.

  In the above scheme, after synthesizing 4-ethynylbenzoic acid, a DMT derivative of D-threoninol is introduced into the hydroxyl group of the carboxyl group to obtain a phenylethynyl DMT derivative. Thereafter, it is reacted with a halogenated perylene to introduce perylene via an ethynylene group to obtain a phenylethynylperylene DMT derivative. Next, this phenylethynylperylene DMT derivative is amidated.

  Although not illustrated, after obtaining an amidite derived from D-threoninol, the first unit or the second unit may be formed using the amidite to form a final amidite derivative.

  According to the disclosure of the present specification, amidites useful for the synthesis of the present oligonucleotide represented by the following formulas (3) and (4) are also provided. For the same configurations (R1, R2, R3, R4, Perylene, etc.) as in Formulas (1) and (2) in Formulas (3) and (4) above, the configurations in Formula (1) and Formula (2) It is synonymous. Therefore, the structures applicable to the present specification in Formula (1) and Formula (2) and preferred structures also apply to Formulas (3) and (4).

  In the above formulas (3) and (4), C1 represents a hydrogen atom or a hydroxyl protecting group. The hydroxyl protecting group is not particularly limited, and a conventionally known hydroxyl protecting group can be used. Examples include fluorenylmethoxycarbonyl group (FMOC group), dimethoxytrityl group (DMT group), quaternary butyldimethylsilyl group (TBDMS group), monomethoxytrityl group, trifluoroacetyl group, levulinyl group, or silyl group. It is done. A preferred protecting group is a trityl group, for example selected from monomethyltrityl (MMT) dimethoxytrityl (DMT) and quaternary butyldimethylsilyl group (TBDMS group).

  D1 represents a hydrogen atom, a hydroxyl protecting group, a phosphoramidite group, or a linking group bonded to or bonded to a solid phase carrier. A compound (amidite compound) in which D1 is a phosphoramidite group can be used as a phosphoramidite reagent by the phosphoramidite method to synthesize oligonucleotides. In the present invention, the phosphoramidite group can be represented by the following formula (5).

(In Formula (5), each Q1 may be the same or different and represents a branched or straight chain alkyl group having 1 to 5 carbon atoms, and Q2 represents a branched group. Represents a linear or linear alkyl group having 1 to 5 carbon atoms or an optionally substituted alkoxyl group.)

In the above formula (5), Q1 is especially but not limited to be mentioned as an isopropyl group are preferred, As the _Q2, -OCH 2 _CH 2 CN, and the like. For example, as a phosphoamide group, the compound of a following formula is mentioned as a specific example of Formula (5).

  In addition, in the formulas (3) and (4), the compound in which D1 is a linking group bonded to the solid phase carrier is obtained by binding the linking group to a predetermined functional group on the solid phase carrier such as an amino group, It is held on a solid support. In the formulas (3) and (4), the compound in which D1 is a linking group bound to a solid phase carrier is a combination of various oligonucleotides because the present oligonucleotide is bound to the solid phase carrier via the linking group. It can be used as a starting material for solid phase synthesis. By using this starting material, an oligonucleotide having a unit represented by formula (1) or formula (2) can be produced.

  After obtaining the amidite derivatives of the first unit and the second unit, a conventionally known DNA synthesis method, for example, the method described in Nature Protocols 2007, Vol. 2, pages 203 to 212, is used. Therefore, an oligonucleotide having the first unit or the second unit at a desired site can be synthesized.

  According to such a method, an amidite derivative of a quencher unit can be obtained, and an oligonucleotide having a quencher unit can be produced.

(Immobilized oligonucleotide probe)
According to the disclosure of the present specification, an oligonucleotide probe is immobilized, and the oligonucleotide is immobilized as a probe on a solid phase carrier. The oligonucleotide may be capable of forming a molecular beacon.

  The solid phase carrier may be, for example, a bead or a flat plate, and the material is not particularly limited, but a solid phase carrier made of glass or plastic can be used. Preferably, the solid support is a flat plate and is an array in which two or more kinds of oligonucleotide probes are fixed in a fixed sequence. The array can fix a large number of oligonucleotide probes, and is convenient for comprehensively detecting various polymorphisms and mutations. In addition, the array may include a plurality of partitioned individual array regions on a single solid phase carrier. In these individual array regions, the same set of oligonucleotide probes may be immobilized, or different sets of oligonucleotide probes may be immobilized.

  The immobilization form of the oligonucleotide probe is not particularly limited. It may be covalent or non-covalent. The oligonucleotide probe can be immobilized on the surface of the solid phase carrier by various conventionally known methods. An appropriate linker sequence may be provided on the surface of the solid phase carrier.

  The use of such a probe immobilization body is not particularly limited. Suitable for deletion or insertion mutation of 1 base or 2 bases or more. It is particularly suitable for deletion mutations and insertion mutations of 3 or more bases. Specific examples include testing for genetic diseases caused by deletion mutations such as small mouth disease, complex pituitary hormone deficiency, pustular fibrosis, and recessive congenital myotony. Moreover, the test | inspection use of the diagnosis of the disease with which the variation | mutation on DNA, onset, and severity are known, onset prediction, and prognosis prediction is mentioned.

(Inspection method)
According to the disclosure of the present specification, there is provided a method for examining a deletion mutation or an insertion mutation, wherein the oligonucleotide probe is configured such that the presence or absence of a deletion mutation or an insertion mutation can be distinguished from each wild type, There is provided a detection method comprising a step of contacting a test DNA-containing sample. According to this test method, a mutation associated with a deletion or insertion of one base or two or more bases can be easily detected by a normal hybridization operation. Applications of this test method include diagnosis of various diseases, onset prediction, prognosis prediction and the like, as with the probe-immobilized body.

  The test DNA-containing sample is sufficient if it contains the DNA to be examined, and can be collected from various organisms. Typically, examples include those obtained from body fluids or tissues such as human or animal blood and extracted from nucleic acids, or those obtained by amplifying DNA by PCR or the like.

This inspection method is preferably carried out using an oligonucleotide probe immobilized body such as a probe array. For example, this test method for detecting a three-base deletion mutation is performed as the following steps.
(1) An array is prepared on which oligonucleotide probes are constructed so that the mutant type to be detected can be distinguished from the wild type. The oligonucleotide probe has a base sequence that is complementary to a wild-type base sequence that does not have a deletion, and is separated from the first unit, second by two or more bases that are deleted by mutation. It includes two types selected from a unit and a quencher unit.
(2) A test DNA-containing sample (hereinafter simply referred to as a test sample) is supplied to the array, and a hybridization reaction is performed under predetermined hybridization conditions.
(3) After elapse of a predetermined time, the array is washed, and hybridization between DNA in the test sample and the oligonucleotide probe is performed by detecting a fluorescent signal emitted by perylene.
(4) When the test sample contains target DNA, that is, when it contains mutant DNA, the mutant DNA hybridizes with the oligonucleotide probe. However, a bulge is formed on the probe side at the deletion site of the mutant DNA. As a result, in this probe, fluorescence due to exciplex light emission by the first unit and the second unit can be observed. Exciplex emission is shifted to a longer wavelength side than fluorescence during hybridization with a wild type that does not form a bulge, and both can be distinguished. The fluorescent signal can be detected by a fluorescent scanner or the like.

  In the case of an oligonucleotide probe comprising a first unit (or second unit) and a quencher unit, if a bulge is formed as a result of hybridization, the first unit (or second unit) is formed. When quenching by the quencher with respect to the signal site is enhanced and no bulge is formed, quenching by the quencher is suppressed. Thereby, a mutation can be detected.

(Inspection kit)
The test kit disclosed in this specification is a test kit for a deletion mutation or an insertion mutation, and the presence or absence of the deletion mutation of 1 base or 2 bases or more or the insertion mutation of 3 bases or more is determined for each wild type. The oligonucleotide probe can be included so as to be distinguishable from each other. This kit may contain an immobilized product as an oligonucleotide probe.

  Hereinafter, the disclosure of the present specification will be specifically described with reference to examples. However, the following examples do not limit the disclosure of the present specification.

(1) Synthesis of Ethynylperylene amidite monomer ( 4 ) (Scheme 1)
In this example, an amidite monomer for introducing ethynylperylene (hereinafter also referred to as F) into an oligonucleotide was synthesized according to the following scheme.

Compound 1 was obtained by the method described in Angewandte Chemie International Edition, 2010, Volume 49, page 5502. Place 4-pentynoic acid (0.177 g, 1.80 mmol, 1.2 eq), PyBOP (0.937 g, 1.80 mmol, 1.2 eq) in an eggplant flask, dissolve in CH ₂ Cl ₂ about 15 ml, Et ₃ N about 7.5 ml, Stir for 10 minutes. Next, Compound 1 (0.611 g, 1.50 mmol, 1.0 eq) dissolved in about 15 ml of CH ₂ Cl ₂ was added and stirred overnight. After completion of the reaction, the mixture was washed with a saturated aqueous NaHCO ₃ solution and a saturated aqueous NaCl solution using a separatory funnel. This was purified by silica gel column chromatography (developing solvent composition: hexane: ethyl acetate: Et ₃ N = 50: 50: 3) to obtain Compound 2 . Yield: 0.46 g (0.943 mmol) Yield: 62.9%

  Perylene (1.01 g, 4.0 mmol, 1.0 eq) was placed in the flask, and NBS (0.712 g, 4.0 mmol, 1.0 eq) was placed in the connected dropping funnel, and the atmosphere was replaced with nitrogen. Perylene was dissolved in about 135 ml of dehydrated DMF and NBS was dissolved in about 45 ml of dehydrated DMF, and NBS was slowly dropped from the dropping funnel and allowed to react overnight with the fluorescent lamp turned on. When 1000 ml of water was added after completion of the reaction, bromoperylene precipitated as a solid. Yield: 1.28 g (3.87 mmol) Yield: 96.8%

Put a CuI (0.0036 g, 0.0189 mmol, 0.02 eq) and (Ph ₃ P) ₄ Pd (0.0437 g, 0.0378 mmol, 0.04 eq) into a two-necked flask connected to a Dimroth condenser, and purge with nitrogen. Was dissolved in THF. Next, bromoperylene (0.344 g, 1.04 mmol, 1.1 eq) was placed in a pear-shaped flask and purged with nitrogen, and dissolved in about 20 ml of piperidine and about 10 ml of dehydrated THF. This was dripped at the above-mentioned two neck flask at room temperature, and it stirred at 60 degreeC for 30 minutes. Furthermore, compound 2 (0.46 g, 0.943 mmol, 1.0 eq) was put into another pear flask, and the atmosphere was purged with nitrogen, and dissolved in about 5 ml of dehydrated THF. This was dropped into a two-necked flask in which bromoperylene was being activated, and allowed to react overnight while refluxing. This was purified by silica gel column chromatography (developing solvent composition: chloroform: hexane: Et ₃ N = 50: 50: 3) to obtain Compound 3 . Yield: 0.29 g (0.393 mmol) Yield: 37.8%

Compound 3 (0.140 g, 0.190 mmol, 1.0 eq) was placed in an eggplant flask, purged with nitrogen, dissolved in dehydrated CH ₂ Cl ₂ and dehydrated acetonitrile, azeotroped twice, and about 1 ml of dehydrated CH ₂ Cl ₂ and It was dissolved in about 1 ml of dehydrated acetonitrile and about 0.17 ml of Et ₃ N. 2-Cyanoethyldiisopropylchlorophosphoramidite (0.0947 g, 0.40 mmol, 2.1 eq, 0.0893 ml) was slowly added dropwise on an ice bath and stirred. After 20 minutes, the ice bath was removed and the reaction was allowed to proceed at room temperature for about 40 minutes. After that, chloroform was added, and the mixture was washed twice with a saturated aqueous NaHCO ₃ solution and once with a saturated aqueous NaCl solution using a separatory funnel. This was purified by silica gel column chromatography (developing solvent composition: hexane: ethyl acetate: Et ₃ N = 50: 50: 3) to obtain Compound 4 . After dissolving in dehydrated acetonitrile and azeotroping three times, it was stored in a bottle under a nitrogen environment. Yield: 0.15 g (0.16 mmol) Yield: 84.2%

(2) Synthesis of Phenylethynylperylene Amidite Monomer ( 9 ) (Scheme 2)
In this example, an amidite monomer for introducing phenylethynylperylene (hereinafter also referred to as L) was synthesized according to the following scheme.

Add CuI (0.03 g, 0.158 mmol, 0.0158 eq) and (Ph ₃ P) ₄ Pd (0.03 g, 0.025 mmol, 0.0025 eq) to ethyl 4-iodobenzoate (2.76 g, 10 mmol, 1.0 eq) in a two-necked flask. , 1.66 ml) and purged with nitrogen, dissolved in about 30 ml of Et ₃ N, and ethynyltrimethylsilane (1.28 g, 13 mmol, 1.3 eq, 1.80 ml) was added dropwise. Thereafter, the reaction was continued overnight and stopped 24 hours after the start of the reaction. The solid in the reaction solution was removed by suction filtration, and the solvent of the filtrate was removed by an evaporator, and then purified by silica gel column chromatography (developing solvent composition: hexane: ethyl acetate = 1: 1) to obtain Compound 5 . . Yield: 2.71 g (11.0 mmol) Yield: ~ 100%

Compound 5 (2.71 g, 11.0 mmol) was dissolved in 8 ml of ethanol, and 12 ml of 1N NaOH was slowly added dropwise on an ice bath. The reaction was allowed to proceed for 2 hours while cooling in an ice bath, the ice bath was removed, and the reaction was allowed to proceed overnight. Next, after washing the reaction solution twice with about 20 ml of diethyl ether with a separatory funnel, the aqueous layer was made acidic with 1N HCl and extracted with about 200 ml of diethyl ether, and the diethyl ether layer was then saturated with NaCl. Then, diethyl ether was removed by evaporation to obtain Compound 6 . Yield: 1.35 g (9.22 mmol) Yield: 83.8%

Compound 6 (0.675 g, 4.62 mmol, 1.1eq) was dissolved in about 20 ml of CH ₂ Cl ₂ and approximately 20 ml of Et ₃ N, compounds were dissolved therein of CH ₂ Cl ₂ to about 10 ml 1 ( 1.70 g, 4.17 mmol, 1.0 eq) and PyBop (2.60 g, 5.00 mmol, 1.2 eq) dissolved in about 20 ml of CH ₂ Cl ₂ were added and stirred at room temperature for 24 hours. The reaction solution was then washed 3 times with a separatory funnel with about 70 ml of saturated aqueous NaHCO ₃ solution. This was purified by silica gel column chromatography (developing solvent composition: hexane: ethyl acetate: Et ₃ N = 60: 40: 3) to obtain Compound 7. Yield: 1.67 g (3.12 mmol) Yield: 74.8%

CuI (0.002 g, 9.6 × 10 ^-3 mmol, 0.02 eq) and (Ph ₃ P) ₄ Pd (0.022 g, 19.2 × 10 ^-3 mmol, 0.04 eq) were placed in a two-necked flask connected to a Dimroth condenser. The mixture was purged with nitrogen and dissolved in about 2.5 ml of dehydrated THF. Bromoperylene (0.159 g, 0.48 mmol, 1.0 eq) was placed in another pear-shaped flask and purged with nitrogen, and dissolved in about 10 ml of piperidine and about 5.0 ml of dehydrated THF. This was dripped at the above-mentioned two neck flask at room temperature, and it stirred at 60 degreeC for 30 minutes. Moreover, compound 7 (0.283 g, 0.528 mmol, 1.1 eq) was put into another pear-shaped flask, purged with nitrogen, and dissolved in about 2.5 ml of dehydrated THF. The bromoperylene was dropped into the activated two-necked flask and refluxed for 3 hours. Thereafter, the solvent was removed, and the residue was purified by silica gel column chromatography (developing solvent composition: chloroform: hexane: Et ₃ N = 50: 50: 3) to obtain Compound 8 .
Yield: 0.22 g (0.280 mmol) Yield: 58.3%

Compound 8 (0.19 g, 0.242 mmol, 1.0 eq) was placed in an eggplant flask, purged with nitrogen, and then dissolved in about 5 ml of dehydrated CH ₂ Cl ₂ and about 0.17 ml of Et ₃ N. 2-Cyanoethyldiisopropylchlorophosphoramidite (0.115 g, 0.484 mmol, 2.0 eq, 0.108 ml) was slowly added dropwise on an ice bath and stirred. After 20 minutes, the ice bath was removed and the reaction was allowed to proceed for about 1 hour at room temperature. After completion of the reaction, chloroform was added, the reaction solution was washed twice with a saturated aqueous solution of NaHCO ₃ and twice with a saturated aqueous solution of NaCl, and chloroform was removed by evaporation to obtain Compound 9 . After dissolving in dehydrated acetonitrile and dry CH ₂ Cl ₂ and azeotroping three times, it was placed in a bottle under a nitrogen environment.

  In this example, DNA containing the amidite monomer synthesized in Example 1 and Example 2 was synthesized. The emission spectra of DNA into which ethynylperylene (F) and phenylethynylperylene (L) were introduced were compared with those of conventional perylene (E).

Synthesis of DNA containing these amidite monomers is described in Nature Protocols, Vol. 2, pages 203 to 212 using commercially available phosphoramidite monomers corresponding to four natural bases. It was synthesized and purified by the method. The synthesized DNA is shown below. Conventional perylene-introduced DNA was synthesized by the method described in Tetrahedron Letters 2007, Vol. 49, pages 6759 to 6762. The results are shown in FIG.

E1a: 5'-GGTATCEGCAATC-3 '
F1a: 5'-GGTATCFGCAATC-3 '
L1a: 5'-GGTATCLGCAATC-3 '
So (native): 5'-GATTGCGATACC-3 '

  As shown in FIG. 3, it was found that F and L newly synthesized this time have sufficient luminescence intensity even in double-stranded DNA as compared with conventional perylene E. In addition, the F and L monomer emissions have maximum emission wavelengths of about 480 nm and 500 nm, respectively, and the conjugation system has expanded, resulting in wavelength shifts of about 20 nm and 40 nm longer than the conventional perylene E, respectively.

  In this example, a single base deletion detection probe was designed and evaluated. As a probe for detecting a single base deletion, DNA having L or F introduced on both sides of a single base whose deletion is to be detected was designed as shown below. In this case, as shown in FIG. 1, when the wild type (T1) forms a double strand, it cannot be contacted by a base pair between the fluorescent dyes, so that only monomer emission is observed. However, in the single nucleotide deletion polymorphism, a tribasic bulge is formed and the two fluorescent dyes are contacted in the bulge, so that excimer or exciplex emission can be performed.

Probe DNA
FAF: 5'-GGTATCFAFGCAATC-3
LAL: 5'-GGTATCLALGCAATC-3
FAL: 5'-GGTATCFALGCAATC-3
Target DNA
So: 5'-GATTGCGATACC-3 '
T1: 5'-GATTGC T GATACC-3 '

  First, the stability when double strand formation with wild type (T1) or single nucleotide deletion (So) was examined. As shown in Table 1, the results are shown in Table 1. Compared with the double-stranded formation with T1, the three types of probes have a bulge-type structure when double-stranded with deletion-type So. did.

  Next, when FAF is used as a probe, the fluorescence spectra of 1) probe alone (FAF), 2) wild type and double strand formation (FAF / T1), 3) single nucleotide polymorphism (FAF / So) As shown in FIG.

  As shown in FIG. 4, only monomer luminescence was observed in each of the wild type (T1) single nucleotide polymorphism (So). In FAF / So, only quenching due to the interaction between dyes was observed, and no excimer luminescence was recognized when the deletion target was recognized.

  Next, when using LAL as a probe, the fluorescence spectra of 1) probe alone (LAL), 2) wild type and double strand formation (LAL / T1), 3) single nucleotide polymorphism (LAL / So) As shown in FIG. As shown in FIG. 5, excimer luminescence upon recognition of the deletion DNA could not be observed. Fluorescence intensity increased with LAL / T1 and LAL / So compared to probe single strand (LAL).

  Next, the fluorescence spectrum of the heteroassociation probe FAL of the present invention is as follows: 1) probe alone (FAL), 2) wild type and double strand formation (FAL / T1), 3) single nucleotide polymorphism (FAL / So) Is shown in FIG.

Surprisingly, the fluorescence of F was completely quenched, and only L monomer emission was observed. This is because highly efficient FRET from F to L is occurring. Exciplex emission was observed around 550-600 nm, but it was slightly longer than 570 nm compared to the wild type duplex (FAL / T1). The emission intensity of FAL / So was slightly higher than that of FAL / T1. Therefore, when comparing the ratio of the emission intensity at ₅₀₆ nm based on monomer emission to the emission intensity at 600 nm based on excimer emission (I ₆₀₀ / I ₅₀₆ ), I ₆₀₀ / I ₅₀₆ = 0.168 for the wild type (T1), deletion type ( In S0), I ₆₀₀ / I ₅₀₆ = 0.245, showing a clear difference.

  In this example, a three-base deletion detection probe was designed and evaluated. As shown below, as a probe for detecting a three-base deletion, DNA in which L or F was introduced on both sides of the three bases for which the deletion was to be detected was designed. The principle is exactly the same as in the case of single nucleotide deletion shown in FIG.

Probe DNA
FCTTL: 5'-AATATCATFCTTLTGGTGTTT-3
FCTTF: 5'-AATATCATFCTTFTGGTGTTT-3
LCTTL: 5'-AATATCATLCTTLTGGTGTTT-3
Target DNA
WT (wild type): 5'-CATAGGAAACACCA AAG ATGATATTTTCTTT-3 '
MU (Trinucleotide deletion type): 5'-CATAGGAAACACCAATGATATTTTCTTT-3 '

  First, the stability when double strand formation with wild type (WT) or single nucleotide deletion (MU) was examined. As shown in Table 2, the results are shown in Table 2. Compared to the double-stranded formation with WT, the three types of probes have a bulge-type structure when double-stranded with deleted WT. did.

  Next, when FCTTF is used as a probe, the fluorescence spectrum of 1) probe alone (FCTTF), 2) wild type and double strand formation (FCTTF / WT), and 3) trinucleotide polymorphism (FCTTF / MU) As shown in FIG. As shown in FIG. 7, only monomer luminescence was observed in all cases of wild type (WT) trinucleotide polymorphism (MU), and excimer luminescence upon recognition of the deletion target could not be observed.

  Next, when LCTTL is used as a probe, 1) probe alone (LCTTL), 2) wild type and double strand formation (LCTTL / WT), 3) trinucleotide polymorphism (LCTTL / MU) fluorescence spectrum Is shown in FIG. As shown in FIG. 8, only the monomer luminescence was observed in any of the wild type (WT) trinucleotide polymorphism (MU), and excimer luminescence upon recognition of the deletion target could not be observed.

  Next, when FCTTL is used as a probe, 1) probe alone (FCTTL), 2) wild-type and double-strand formation (FCTTL / WT), and 3) trinucleotide polymorphism (FCTTL / MU) fluorescence spectrum Is shown in FIG.

As shown in FIG. 8, as in the case of FAL, the fluorescence of F was completely quenched, and only L emission was observed. This is because highly efficient FRET from F to L is occurring. A clear exciplex emission was observed from 550 to 650 nm in the triple nucleotide deletion (FCTTL / MU) compared to the wild type duplex (FCTTL / WT). Therefore, the ratio of the emission intensity at ₅₀₆ nm based on monomer emission and the emission intensity at 600 nm based on excimer emission (I ₆₀₀ / I ₅₀₆ ) was compared with FAL, and I ₆₀₀ / I ₅₀₆ = 0.063 in the wild type (WT). In the deletion type (MU), I ₆₀₀ / I ₅₀₆ = 0.35, and a clear difference of 5 times or more was observed. This difference could be sufficiently observed visually, and it could be identified as green emission in the wild type and yellow emission in the deletion type.

Sequence number 1-7: Synthetic oligonucleotide

Claims (13)

An oligonucleotide comprising:
A first unit separated by one or more nucleotides;
(In the formula (1), R1 represents an alkynylene group having 2 to 4 carbon atoms, R2 represents a phenylene group, and A1 is a nucleotide linked through a hydrogen atom, a phosphate group, or a phosphodiester bond, or Represents an oligonucleotide, and B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide.)
The second unit;
(In Formula (2), R3 represents an alkynylene group having 2 to 4 carbon atoms, R4 represents an alkylene group having 1 to 4 carbon atoms, and A1 represents a hydrogen atom, a phosphate group, or a phosphodiester bond. And B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide.), And an oligonucleotide comprising two types selected from a quencher unit. The oligonucleotide according to claim 1, wherein the first unit is represented by the following formula.
The oligonucleotide according to claim 1 or 2, wherein the second unit is represented by the following formula.
  The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide is a probe for detecting a deletion mutation or insertion mutation of 3 or more bases.   A molecular beacon can be formed, and the first unit and the second unit are provided on one strand of the stem at the time of molecular beacon formation, and a quencher for the first unit and the second unit is provided. The oligonucleotide according to any one of claims 1 to 4, which is provided in the other strand of the stem. An oligonucleotide probe-immobilized product comprising:
An immobilized body immobilized on a solid phase carrier using the oligonucleotide according to any one of claims 1 to 5 as a probe. A method for testing a deletion mutation or an insertion mutation, comprising:
A step of contacting the oligonucleotide according to any one of claims 1 to 5 with a test DNA-containing sample, wherein the presence or absence of the deletion mutation or the insertion mutation can be distinguished from each wild type,
A detection method comprising:   The detection method according to claim 7, wherein the test DNA-containing sample includes DNA collected from a human or an animal.   The test method according to claim 7 or 8, which is a test method for the presence or absence of the possibility of developing a disease. A test kit for deletion mutation or insertion mutation, comprising:
The kit containing the oligonucleotide probe in any one of Claims 1-5 comprised so that the presence or absence of the said deletion mutation or the said insertion mutation was distinguishable from each wild type. A compound represented by the following formula (3).

(In Formula (3), R1 represents an alkynylene group having 2 to 4 carbon atoms, R2 represents a phenylene group, Perylene represents a perylene group, C1 represents a hydrogen atom or a hydroxyl protecting group, and D1 Represents a hydrogen atom, a hydroxyl protecting group, a phosphoramidite group, or a linking group bound to or bound to a solid phase carrier.) A compound represented by the following formula (4).
(In Formula (4), R3 represents a C2-C4 alkynylene group, R4 represents a C1-C4 alkylene group, Perylene represents a perylene group. C1 is a hydrogen atom or a hydroxyl group. Represents a protecting group, and D1 represents a hydrogen atom, a hydroxyl protecting group, a phosphoramidite group, or a linking group bound to or bound to a solid phase carrier. An oligonucleotide comprising:
First unit;
(In the formula (1), R1 represents an alkynylene group having 2 to 4 carbon atoms, R2 represents a phenylene group, and A1 is a nucleotide linked through a hydrogen atom, a phosphate group, or a phosphodiester bond, or Represents an oligonucleotide, and B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide.)
And a second unit;
(In Formula (2), R3 represents an alkynylene group having 2 to 4 carbon atoms, R4 represents an alkylene group having 1 to 4 carbon atoms, and A1 represents a hydrogen atom, a phosphate group, or a phosphodiester bond. And B1 represents a hydrogen atom, a phosphate group, a nucleotide or an oligonucleotide.)
An oligonucleotide comprising either or both of: