WO2007105623A1

WO2007105623A1 - Oligonucleotide-immobilized solid support

Info

Publication number: WO2007105623A1
Application number: PCT/JP2007/054645
Authority: WO
Inventors: Mitsuo Sekine; Kohji Seio; Akihiro Ohkubo; Kunihiko Tanaka
Original assignee: Tokyo Institute Of Technology
Priority date: 2006-03-10
Filing date: 2007-03-09
Publication date: 2007-09-20
Also published as: JP2009153381A

Abstract

[PROBLEMS] To provide a solid support to which an oligonucleotide having a leaving group at the 5'-terminus is bound and which has a capability of forming a double bond. [MEANS FOR SOLVING PROBLEMS] Disclosed is an oligonucleotide-immobilized solid support which comprises a solid support and an oligonucleotide derivative represented by the general formula (1) bound to the solid support: wherein R1's may be different from or same as one another and independently represent a hydrogen or an alkoxy group; R2's may be different from or same as one another and independently represent a hydrogen or an alkoxy group; B's may be different from or same as one another and independently represent a natural or non-natural nucleotide; X represents a leaving group; and n is an integer of 2 to 50.

Description

Specification

Oligonucleotide-immobilized solid support

Technical field

[0001] The present invention relates to an oligonucleotide-immobilized solid phase carrier.

The oligonucleotide-immobilized solid phase carrier of the present invention is useful for improving the sensitivity of long-chain DNA synthesis and SNPs analysis.

Background art

[0002] In genetic engineering, the cleavage and ligation of nucleic acid molecules such as DNA is one of the most important basic techniques. Therefore, with the development of genetic engineering, efficient technology for binding DNA is required. Conventionally, the ligation of double-stranded nucleic acids using restriction enzymes has been the main method for linking two types of nucleic acids. In recent years, chemical ligation, a new DNA binding technology, has been reported (Non-patent Document 1). Chemical ligation is the complementary D-complementation of an oligonucleotide having a highly nucleophilic functional group such as phosphate at the 3 ′ end of a nucleic acid and an oligonucleotide having a leaving group such as a iodine at the 5 ′ end. This is a technology that uses NA as a template to synthesize a single oligonucleotide by forming a covalent bond.

[0003] On the other hand, attempts have been actively made to use genome sequences in various fields such as medicine and pharmaceuticals using genome sequences of various organisms including humans. Among them, genetic polymorphism, which is a single nucleotide substitution in the genome sequence, is being analyzed from the viewpoint of investigating the relationship between genes and diseases or drug sensitivity. Such analysis has revealed the relationship between individual genetic polymorphisms and diseases and the sensitivity of drugs, and tests called genetic diagnosis have been conducted.

[0004] As a technique for performing the genetic diagnosis as described above, a DNA chip, a DNA microarray, and the like are known. DNA chips and DNA microarrays are made by aligning a large number of DNA molecules on a substrate such as a glass slide, silicon, or plastic, and are useful for analyzing gene polymorphisms.

On the other hand, DNA is grown on microporous glass (CPG), which is the most suitable material for DNA synthesis. After synthesizing the lobe, a probe-on-carrier method has been developed that can be used to detect SNPs for each CPG without separating the probe molecule from the CPG carrier force. This technique not only improves the efficiency of DNA probe strand extension, but also reduces the cost of the DNA chip and allows the probe to be arranged three-dimensionally, so it can be expected to improve detection sensitivity. However, it is desired to further improve the SN p _s detection efficiency.

[0005] Oligonucleotides having a leaving group at the 5 'end are useful for genetic diagnosis, and many methods for their synthesis have been reported (for example, Non-Patent Document 2 and Non-Patent Document 3). In addition, the chemical ligation technique as described above is useful for genetic diagnosis to examine gene polymorphism, but it binds an oligonucleotide having a leaving group at the 5 ′ end and forms a duplex. A solid support having a function has not been reported yet.

Such a solid phase carrier is useful for genetic diagnosis as described above, and development is desired.

[0006] Non-Special Reference 1: Yanzheng Xu and Eric T, Kooi, Journal of the American hemical Soc iety, 2000, 122, 9040-9041

Non-Patent Document 2: Mathias K. Herrlein, Jeffrey S. Nelson and Robert L. Letsinger, Journal of the American Chemical Society, 1995, 117, 10151-10152

Non-Patent Document 3: Yanzheng Xu and Eric T, Kool, Tetrahedron Letters, 1997, 38, 5595

-5598

Disclosure of the invention

Problems to be solved by the invention

[0007] When a solid phase carrier to which an oligonucleotide having a leaving group at the 5 'end as described above is bound by a general DNA synthesis method, the leaving group present at the 5' end is eliminated. Or the oligonucleotide itself will be detached.

Accordingly, an object of the present invention is to provide a solid phase carrier which binds an oligonucleotide having a leaving group at the 5 ′ end and has a double bond forming ability.

Means for solving the problem

[0008] In order to achieve the above object, the present inventors diligently studied, and as a result, have found that the above object can be achieved by a solid phase synthesis method using chemical ligation. The present invention has been made based on the above knowledge, and provides an oligonucleotide-immobilized solid phase carrier bound to an oligonucleotide derivative-forced solid phase carrier represented by the general formula (1). .

[Chemical 1]

[In the above formula, R ¹ represents hydrogen or alkoxy group, which may be the same or different, and R ² represents hydrogen, which may be the same or different, or Represents an alkoxy group, B represents a natural or non-natural nucleobase, which may be the same or different, X represents a leaving group, and n represents an integer of 2 to 50.)

[0011] The present invention also provides a method for producing an oligonucleotide-immobilized solid phase carrier, wherein the oligonucleotide derivative represented by the general formula (1) is bound to a solid phase carrier,

As a reaction accelerator in the solid phase synthesis of oligonucleotides by the phosphoramidide method. 1-hydroxybenzotriazole is used, and a method for producing an oligonucleotide-immobilized solid phase carrier is provided.

[Chemical 2]

B

(In the above formula, R ¹ represents hydrogen or an alkoxy group, which may be the same or different, and R ² represents hydrogen, which may be the same or different, or Represents an alkoxy group, B represents a natural or non-natural nucleobase, which may be the same or different, X represents a leaving group, and n represents an integer of 2 to 50.)

[0014] The present invention also relates to the oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a gene with a leaving group bonded to the 5 'end side and fixed to the 3' end side. Complementary oligonucleotide-immobilized solid phase carrier formed by binding a phase carrier and a complementary base sequence portion complementary to a base sequence portion adjacent to a predetermined base sequence portion in the gene A complementary oligonucleotide having a highly nucleophilic functional group at the 3 'end of the oligonucleotide

An oligonucleotide solid-phase synthesis method is provided, which comprises a step of reacting.

[0015] The present invention also provides a method for binding a leaving group to the 5 'end side of an oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a normal gene, and Complementary oligonucleotide-immobilized solid phase carrier formed by binding a solid phase carrier and an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the predetermined base sequence portion in the normal gene A specific nucleotide sequence in a normal gene and a sample gene corresponding to the sample gene, which has a step of reacting with a complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end of A method for confirming the difference from the base sequence portion is provided.

The present invention also includes a step of chemically synthesizing an oligonucleotide having a predetermined base sequence by coupling base-part unprotected nucleotide phosphoramidides in a predetermined order. Ligated oligonucleotide solid phase is a method of chemically synthesizing a solid phase carrier, which is a nucleotide phosphoramidide having a leaving group at the 5 ′ end as a 5 ′ terminal base unprotected nucleotide phosphoramidide And a method using a nucleotide phosphoramidite having a solid phase carrier bound to the 3 ′ end as a 3′-terminal base-unprotected nucleotide phosphoramidide.

The invention's effect

[0016] The oligonucleotide-immobilized solid phase carrier of the present invention can improve the sensitivity of long-chain DNA synthesis and SNPs analysis.

The method for confirming the difference between a predetermined base sequence portion in a normal gene and a corresponding base sequence portion in a sample gene of the present invention has improved sensitivity.

The method for chemically synthesizing an oligonucleotide-immobilized solid phase carrier of the present invention can synthesize long-chain DNA and an oligonucleotide-immobilized solid phase carrier that can improve the sensitivity of SNPs analysis. .

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Hereinafter, the oligonucleotide-immobilized solid phase carrier of the present invention will be described. The oligonucleotide-immobilized solid phase carrier of the present invention is obtained by binding an oligonucleotide derivative represented by the general formula (1) to a solid phase carrier.

[Chemical 3]

In the general formula (1), R ¹ and R ² may be the same or different and each represents hydrogen or an alkoxy group. As the alkoxy group, an alkoxy group having from 5 to 5 carbon atoms is preferable, for example, a methoxy group, an ethoxy group, a propoxy group, a 1 ptyloxy group, a 1 pentyloxy group, and the like. In addition, an alkoxy group in which a part or all of the side chain thereof is cyclized, such as a branched alkoxy group such as a group, an isopropyloxy group, a cyclopropyloxy group, a cycloptynoxy group, and a cyclopentyloxy group is also included.

B is a natural or non-natural nucleic acid salt which may be the same or different. Represents a group. Specifically, natural bases such as adenine, cytosine, guanine, thymine, and urashinore are artificial bases such as 7-dazaadenine, 7-dazaadenine, 3-dazaadenine, 6-thioguanine, 2 thiouracil, 2 thiotimine, Various substituents (alkyl, alkenyl, alkynyl, halogen, nitro group, acyl group, hydroxyl group, etc.) introduced at the 7-position, 7_deazaadenine, and various substituents at the 8-position (alkyl, alkenyl, alkynyl, halogen, Nitro group, acyl group, hydroxyl group, etc.) introduced adenine, 8_position, various substituents (alkyl, halogen, nitro group, acyl group, hydroxyl group, etc.) introduced 7_ diazaadenine, 7th and 8th position Introduced various substituents (alkyl, alkenyl, alkynyl, halogen, nitro group, acylol group, hydroxyl group, etc.) Denine, 7-Dazaguanine, 7-Daza-1-8-azaguanine, 3-Dazaguanine, various substituents (alkyl, alkenyl, alkynyl, halogen, nitro group, acyl group, hydroxyl group, etc.) were introduced at position 7 —Dazaguanine, guanine with various substituents (alkyl, alkenyl, alkynyl, halogen, nitro, acyl, hydroxyl group) introduced at the 8-position, various substituents (alkyl, alkenyl, alkynyl, halogen, nitro at the 8-position Group, acyl group, hydroxyl group, etc.) introduced 7-deazaguanine, 7- and 8-position various substituents (alkyl, alkenyl, alkynyl, halogen, nitro group, acyl group, hydroxyl group, etc.) introduced 7- Diazaguanine, various functional groups at the 5-position (alkyl, alkenyl, alkynyl, halogen, nitro group, acyl group, hydroxyl group, etc.) Introduced cytosine, pseudoisocytosine, various functional groups (alkyl, alkenyl, alkynyl, asinole group, hydroxyl group, etc.) introduced at the 1-position, various functional groups (alkyl, alkenyl, ananolinyl at the 5-position) Such as uracil, pseudouracil, etc., into which various functional groups (alkyl, alkenyl, alkynyl, acyl group, hydroxyl group, etc.) are introduced at the 1-position. Can be mentioned.

X represents a leaving group. Examples of the leaving group include a halogen atom, a tosyl group, a mesinole group, a trifluoromethanesulfonyl group, and a phosphate group. Examples of the halogen atom include a chlorine atom, a bromine atom, and an iodine atom.

n represents an integer of 2 to 50, preferably an integer of 5 to 20. Less than n force, chemical ligation is not performed efficiently, while if it is larger than 50, base discrimination ability decreases. However, the accuracy of chemical ligation is reduced.

[0020] The oligonucleotide-immobilized solid phase carrier of the present invention is bound to a solid phase carrier. As the solid phase carrier, those conventionally used for producing DNA chips and DNA microarrays can be used without particular limitation. For example, slide glass, porous glass, polystyrene beads, plastic, Examples thereof include gold particles, gold plates, silver particles and silver plates, glass such as microporous glass and porous glass, magnetic beads having polystyrene, metal and ferrite as the core and the surface covered with glycine methacrylate. The shape of the carrier may be any shape such as a plate (substrate) or a bead. For example, a thermoplastic resin or a thermosetting resin can be used as the plastic. Examples of the thermoplastic resin include linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, and fluorine-containing resins. When microporous glass is used, a DNA chip that can be used in the probe-on-carrier method can be obtained.

In the oligonucleotide-immobilized solid phase carrier of the present invention, the hydroxyl group at the 3-position of the sugar moiety of the residue on the 3 ′ end side is bonded to the carrier. That is, the oligonucleotide-immobilized solid phase carrier of the present invention is represented by the general formula (2).

[0021] [Chemical 4]

D

[0022] In the general formula (2),

R ² , B, X and n are the same as described in the general formula (1), and D represents a solid support.

The method for producing the oligonucleotide-immobilized solid phase carrier of the present invention is not particularly limited, but after producing the oligonucleotide derivative represented by the general formula (1), it may be immobilized or adsorbed on the carrier. Each nucleotide unit may be bound. In the latter case, the production can be performed in solid phase synthesis using 1-hydroxybenzotriazole as a reaction accelerator.

[0023] That is, according to the present invention, there is provided a method for producing an oligonucleotide-immobilized solid phase carrier in which an oligonucleotide derivative represented by the general formula (1) is bound to a solid phase carrier, As a reaction accelerator in solid phase synthesis of oligonucleotides by the dye method 1-hydroxybenzotriazole is used, and a method for producing an oligonucleotide-immobilized solid phase carrier is provided.

In the solid-phase synthesis method, the first step is to use a material in which a base on the 3 'end side is fixed to a solid-phase support as a material, and to this, a phosphoroamidide for binding oligonucleotides that contact P At this time, 1-hydroxybenzotriazole is used as a reaction accelerator. As 1-hydroxybenzotriazole, even a compound having a substituent can be used without particular limitation. For example, 6-nitro-1-hydroxybenzotriazole can be used. The amount used is not particularly limited, but is about 0.5 to 40 equivalents, preferably 5 to 35 equivalents, more preferably about 10 to 30 equivalents with respect to phosphoroamidide. In addition, this reaction is preferable from the viewpoint that an oligonucleotide derivative having a leaving group can be synthesized with high purity since an oligonucleotide derivative can be produced without requiring a protecting group in the base moiety.

The extension reaction by solid-phase synthesis reaction includes steps of detrityl lysis with trichloroacetic acid, etc., washing with dichloromethane, oxidation with iodine, etc., washing with pyridine and the like. In order to improve the reaction yield, the coupling and subsequent washing steps are carried out several times, preferably twice.

Next, the oligonucleotide solid phase synthesis method of the present invention will be described.

In the oligonucleotide solid phase synthesis method of the present invention, a leaving group is bonded to the 5 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a gene, and the 3 ′ end side Complementary oligonucleotide-immobilized solid phase carrier (hereinafter, also simply referred to as “complementary oligonucleotide-immobilized solid phase carrier”), which is formed by binding a solid phase carrier to a specific nucleotide sequence in the above gene Complementary oligonucleotide (hereinafter simply referred to as “complementary oligonucleotide”) having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the portion (Also called).

As the complementary oligonucleotide-immobilized solid phase carrier, the above-described oligonucleotide-immobilized solid phase carrier of the present invention is used.

[0025] In the oligonucleotide solid-phase synthesis method of the present invention, first, the oligo to be synthesized is synthesized. A gene having a nucleotide sequence complementary to the nucleotide chain is prepared. Next, a predetermined (arbitrary site) base sequence portion of this gene is selected, and a leaving group is bound to the 5 ′ end side of the oligonucleotide having a complementary base sequence portion complementary to this base sequence portion. Prepare a complementary oligonucleotide-immobilized solid phase carrier having a solid phase carrier bound to the 3 ′ end. Next, the complementary oligonucleotide-immobilized solid phase carrier is reacted with the complementary oligonucleotide.

[0026] A functional group having high nucleophilicity is bonded to the 3 'terminal side of the complementary oligonucleotide.

Examples of the functional group having high nucleophilicity include a thiophosphate group, a phosphate group, an amino group, a carboxy group, and a thiol group. By this reaction, the 5 ′ end side of the complementary oligonucleotide-immobilized solid phase carrier and the 3 ′ end side of the complementary oligonucleotide are linked by a nucleophilic reaction, and the chain length of the oligonucleotide is increased. The reaction between the complementary oligonucleotide-immobilized solid phase carrier and the complementary oligonucleotide can be carried out in an appropriate buffer, and the reaction temperature and reaction time are 5 to 60 ° C, respectively: ~ 48 hours is sufficient. This reaction is a reaction generally called chemical ligation, and the reaction conditions can be appropriately selected depending on the number of bases of the oligonucleotide and the type of base pair. In addition, it is preferable to perform ammonia treatment after completion | finish of reaction. The treatment with ammonia is preferably a treatment for a short time, preferably 6 to 18 hours. By this ammonia treatment, the protecting group of the phosphate group is deprotected.

In the oligonucleotide solid-phase synthesis method of the present invention, a new second complementary oligonucleotide is further linked by attaching a leaving group to the 5 ′ end side of the complementary oligonucleotide. Can be made. The second complementary oligonucleotide should also be complementary to the adjacent sequence of the gene that is complementary to the complementary oligonucleotide. As described above, by sequentially using new complementary oligonucleotides, it is possible to extend the chain length of the oligonucleotides, and it is possible to synthesize oligonucleotides of several hundred bases by chemical ligation.

[0028] Next, a method for confirming the difference from the corresponding nucleotide sequence in the sample gene of the present invention will be described. The method for confirming the difference with the corresponding base sequence portion in the sample gene of the present invention is a complementary base sequence portion complementary to the predetermined base sequence portion in the normal gene. A complementary oligonucleotide-immobilized solid phase carrier having a leaving group bonded to the 5 ′ end side of an oligonucleotide having a base and a solid phase carrier bonded to the 3 ′ end side, and a predetermined in the normal gene A step of reacting a complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the base sequence portion. It is characterized by that.

[0029] In this method, a functional group having high nucleophilicity is bonded to the 3 'end of the complementary oligonucleotide. The functional group having high nucleophilicity is as described above. By this reaction, the 5 ′ end side of the complementary oligonucleotide-immobilized solid phase carrier and the 3 ′ end side of the oligonucleotide are linked by a nucleophilic reaction, and the oligonucleotide chain length is increased. The reaction between the complementary oligonucleotide-immobilized solid phase carrier and the complementary oligonucleotide can be carried out in an appropriate buffer, and the reaction temperature and reaction time are 5 to 60 ° C and 1 to 5 respectively. 48 hours is sufficient. This reaction is a reaction generally called chemical ligation, and the reaction conditions can be appropriately selected depending on the number of bases of the oligonucleotide and the type of base pair.

In this method, when the complementary oligonucleotide is complementary to a base sequence portion adjacent to a predetermined base sequence portion of a normal gene, chemical ligation occurs and the complementary oligonucleotide-immobilized solid phase carrier And the above complementary oligonucleotides are linked. Therefore, it is possible to confirm that chemical ligation has occurred by binding a labeling substance to the 5 ′ end of the complementary oligonucleotide. Any labeling substance can be used without any particular limitation. For example, radioactive consent elements, fluorescent substances, enzymes, and the like can be used. That is, a labeled compound is bound to a complementary oligonucleotide or contains a radioisotope, and chemical ligation can be detected using this label. If the base sequence part adjacent to the normal base part of the normal gene and the complementary oligonucleotide used are not completely complementary, chemical ligation will not occur and ligation cannot be confirmed by the labeling substance. It is possible to detect the presence of a normal gene and base substitution, deletion or addition.

[0030] The nucleotide solid phase synthesis method described above and the corresponding nucleotide sequence in the sample gene In the method for confirming the difference, the above-described oligonucleotide solid phase carrier of the present invention can be used.

[0031] Next, a method for chemically synthesizing the oligonucleotide-immobilized solid phase carrier of the present invention will be described. The method of chemically synthesizing an oligonucleotide-immobilized solid phase carrier of the present invention comprises chemically synthesizing an oligonucleotide consisting of a predetermined base sequence by coupling base unprotected nucleotide phosphoramidides in a predetermined order. A method of chemically synthesizing an oligonucleotide-immobilized solid phase carrier comprising an oligonucleotide bound to a solid phase carrier, comprising 5′-terminal base-unprotected nucleotide phosphoramidite, Use a nucleotide phosphoramidite with a leaving group at the 5 'end, as a nucleotide unprotected nucleotide phosphoramidide at the 3' end, and a nucleotide phosphoramidite with a solid phase carrier bound to the 3 'end. Use a dido.

[0032] The base-unprotected nucleotide phosphoramidide used in this method can be produced, for example, as in the examples described later. In addition, as the base-unprotected nucleotide phosphoramidide, it is commercially available.

As the nucleotide phosphoramidite having a leaving group at the 5 ′ end, a commercially available phosphoramidite unit (3′—O— (2-cyanoethyl) represented by the formula (7) can be used. For example, 1 N, N′-diisopropyl phosphoramidite) -5′-odothymidine) can be synthesized as follows.

Thymidine (1.94 g, 8 mmol) was dissolved in anhydrous dimethylformamide (20 ml), followed by methyl triphenoxyphosphonium iodide (4.35 g, 9.6 mmol). After stirring at room temperature for 10 minutes, methanol (10 ml) was added. Methanol (30 ml) was added again to the mixture obtained by evaporating the solvent under reduced pressure to obtain the desired 5′-odothymidine as crystals. (1.61 g, 57%) 5, _odothymidine (1.61 g, 4.6 mmol) was dissolved in anhydrous acetonitrile, azeotroped with anhydrous acetonitrile (X3), dissolved in anhydrous salt-methylene, followed by Then, isopropylethylamine (1.11 ml, 6.8 mmol) was added, and finally 2-cyanodiisopropyl phosphoramidite redite (1.11 ml, 5.0 mmol) was added and stirred at room temperature for 4.5 hours. Water was added to stop the reaction. The reaction solution was extracted 5 times with methylene chloride Z5% aqueous sodium bicarbonate. The organic layer is dried over anhydrous sodium sulfate and the solvent is distilled off under reduced pressure. Purification by chromatography (N60 spherical neutral silica gel) with hexane / chloroform 50% → 80% with addition of 1% triethylamine gave the compound of formula (7). (2.16 g, 3.9 mmol)

Other phosphoramidite units can also be manufactured as described in the examples described later.

[0033] Further, in this method, a nucleotide phosphoramidite having a solid phase carrier bound to the 3 'end is used as the 3' end base-unprotected nucleotide phosphoramidide. Three

'Nucleotide phosphoramidides having a solid phase carrier bound to the terminal can be produced, for example, by binding a solid phase carrier into which 16-hydroxyhexadecanoic acid has been introduced and a base part unprotected nucleotide phosphoramidide. it can. The conditions at this time are not particularly limited, but can be carried out under the conditions described in the examples described later.

[0034] In the method of chemically synthesizing the oligonucleotide-immobilized solid phase carrier of the present invention, the oligonucleotide-immobilized solid phase carrier is obtained by coupling base-unprotected nucleotide phosphoramidides in a predetermined order. Is obtained. For the reaction, a conventional method for producing an oligonucleotide using phosphoramidide is used. It is preferable to use 1-hydroxybenzotriazole as a reaction accelerator during the reaction. By including such a reaction accelerator, a side reaction to the base amino group can be suppressed, and hydroxyl group-selective phosphorylation can be performed.

Example

Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples. Using five types of phosphoramidide units represented by the following formulas (3) to (7), using an automated synthesizer of Applied Biosynthesis Inc, trade name “DNA / RNA Synthesizer 392”, SEQ ID NO: 1 A probe (oligonucleotide-immobilized solid phase carrier) having a base sequence of 1 (TCCGGTCATTTTTT) was synthesized (R ³ in formulas (3) to (6) is a dimethoxytrityl group). In addition, the 5 'end T has a odode. Automatic synthesis of DNA oligomers The machine synthesis is a microporous glass with 16-hydroxyhexadecanoic acid at the end. (CPG) A solid phase carrier (10 mg, 10 / i mol / g) was used. Each chain extension cycle was as shown in Table 1 below, and benzoimidazolium triflate (BIT) and 6-nitro-1-hydroxybenzotriazole (nHoBt) were used in the condensation reaction. The obtained probe has a leaving group at the 5 'end and a solid support bound to the 3' end.

[Chemical 5]

[Chemical 6]

[8S00]

91 · CZ9S0l / .00Z OAV

[6soo]

LY CZ9S0l / .00Z OAV

[6] [0 00]

81 · CZ9S0l / .00Z OAV

The phosphoramidide units represented by the formulas (3) to (5) were synthesized by the following method.

5 '-0- (dimethoxy) trityl mono-N-phenoxyacetyl mono-2'-deoxyadenosine 3,-(2-cyanoethyl N, N-diisopropyl phosphoramidite (215 mg, 0.24 mmol) in 2M ammonia- After dissolving the resulting solution in methanol solution (2.5 ml), the resulting solution was stirred for 1 hour, and then the solvent was distilled off under reduced pressure to obtain a crude product, which was purified by silica gel chromatography (1% triethylamine). ), Elution with 50 to 100% chloroform in hexane, and then with a gradient of 0 to 3% methanol in chloroform and distilling off the solvent, 5'—O— Dimethoxy) trityl-2'-deoxyadenosine 3 '-(2-cyanoethyl N, N -Diisopropyl phosphoramidite) (phosphoramidite unit represented by the formula (3)) was obtained as a white solid. (177 mg, 98%)

[0042] In addition, 5, 1-O- (dimethoxy) trityl N-phenoxyacetyl 2, deoxyadenosine 3,-(2-cyanethyl N, N-diisopropyl phosphoramidite (420 mg, 0.44 mmol) 2M The resulting solution was dissolved in ammonia-methanol solution (4.5 ml), and the resulting solution was stirred for 2 hours, and then the solvent was distilled off under reduced pressure to obtain a crude product, which was obtained by silica gel chromatography (1% Elution with hexane in a gradient of 50-: 100% black mouth form, followed by 0-3% methanol on the black mouth form, and the solvent was distilled off to remove 5 '-0- ( Dimethoxy) trityl-2'-deoxyguanosine 3 '-(2-cyanoethyl N, N-diisopropyl phosphoramidite) (phosphoramidite unit represented by formula (4)) was obtained as a white solid. (298 mg, 88%)

[0043] In addition, 5'-0- (dimethoxy) tritinole N-acetinole 2'-deoxycytidine 3 '-(2-cyanethyl N, N-diisopropyl phosphoramidite) (215 mg, 0.28 mmol) was added to 2M ammonia monomethanol. Dissolved in solution (2.5 ml). After stirring the obtained solution for 2 hours, the solvent was distilled off under reduced pressure to obtain a crude product. The resulting crude product was purified by silica gel chromatography (1% triethylamine) and eluted with a hexane gradient of 50-: 100% black mouth form and then a black mouth form with a 0-3% methanol gradient. The solvent was distilled off, and 5′-O— (dimethoxy) trityl-2′-deoxycytidine 3′-3′one (2-cyanethyl N, N-diisopropyl phosphoramidite) (phosphoramido represented by the formula (5) Dyed unit) was obtained as a white solid. (190 mg, 94%)

The phosphoramidite units represented by formulas (6) and (7) were purchased from Darren Research.

[0044] [Table 1] Sequence operation Reagent time (min)

1 Wash CH ₃ CN 0.2

2 Deprotection 3% Cl ₃ CCOOH / CH ₂ C1 ₂ 1.5

3 Wash CH ₃ CN 0.4

Condensation 0.1 M phosphoramidide + 0.2 M nHOBt + BIT in 1.0

CH ₃ CN-N-methyl-2-pyrrolidone (15: 1, v / v)

5 Condensed 0.1 M phosphoramidide + 0.2 M nHOBt + BIT in 1.0

CH ₃ CN-N-methyl-2-pyrrolidone (15: 1, v / v)

6 Wash CH ₃ CN 0.2 Oxidation 0.1 MI ₂ in pyridine-H ₂ 0-THF (v / v / v) 0.5

8 Wash CH ₃ CN 0.4

[0045] After the chain extension, ammonia treatment was performed for 10 minutes to remove the cyanoethyl group of the phosphate portion, and the target oligonucleotide-immobilized solid phase carrier was obtained. In Table 1, steps 4 and 5 mean that the same procedure is repeated twice.

[0046] Example 2

The oligonucleotide-immobilized solid phase carrier having the base sequences of SEQ ID NOs: 2 to 4 shown below was synthesized in the same manner as in Example 1. All sequences have a 5 'terminal side and a 3' terminal side with CPG.

SEQ ID NO: 2 (TCTGGTTCATTTTTT)

酉己歹 IJ number: 3 (TCCGGTTCATTTTTT)

酉己歹 IJ number: 4 (TCCAGTTCATTTTTT)

In addition, an oligonucleotide having the base sequence of SEQ ID NO: 5 shown below was synthesized in the same manner as in Example 1. In addition, fluorescein is bonded to the 5 ′ end and a thiophosphate group is bonded to the 3 ′ end.

SEQ ID NO: 5 (ATGGGCC)

Separately, an oligonucleotide having the base sequence of SEQ ID NO: 6 was synthesized in the same manner as in Example 1. SEQ ID NO: 6 (ATGAACCAGAGGCCCAT)

An oligonucleotide having a base sequence of SEQ ID NO: 6 (0.25 nmol), an oligonucleotide-immobilized solid phase carrier having a base sequence of SEQ ID NO: 2 (10 / ig), and a base sequence of SEQ ID NO: 5 Oligonucleotide (0.25 nmol) was immersed in an oligonucleotide solution (0.5 mL, 30 mM phosphate buffer (pH 7.2), 1 M NaCl, 50 μM DTT), and stirred at 40 ° C. for 14 hours. After completion of the stirring, the solid support was washed with water and dried. After drying, the fluorescence brightness of the solid phase carrier was measured at a wavelength of 470 to 490 nm using a fluorescence microscope (manufactured by Olympus Corporation, BX51WI).

[0048] Using the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 3 instead of the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2, the same operation was carried out, and the fluorescence intensity was changed. Was measured.

When using an oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2, the fluorescence intensity is about as compared to when using an oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 3. It was 10 times. The base sequence of SEQ ID NO: 2 is complementary to a part of the base sequence of SEQ ID NO: 6, and the base sequence of SEQ ID NO: 3 is different by only one base. Therefore, in the case of the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2, a chemical ligation reaction occurs, and when the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 3 is used. The chemical ligation reaction did not occur. Therefore, it was found that a single base mismatch (A-C mismatch) can be detected by this example.

Further, it was found that the oligonucleotide-immobilized solid phase carrier of the present invention has a double-strand forming ability.

[0049] Example 2

The fluorescence intensity was measured in the same manner using the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 4 instead of the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2. .

When using an oligonucleotide-immobilized solid phase carrier having a base arrangement of SEQ ID NO: 2 When using an oligonucleotide-immobilized solid phase carrier having a base arrangement of SEQ ID NO: 4 Compared with, the fluorescence brightness was about 10 times. The base sequence of SEQ ID NO: 2 is complementary to a part of the base sequence of SEQ ID NO: 6, and the base sequence of SEQ ID NO: 3 differs by only two bases. Therefore, in the case of the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 2, a chemical ligation reaction occurs, and when the oligonucleotide-immobilized solid phase carrier having the nucleotide sequence of SEQ ID NO: 4 is used. The chemical ligation reaction did not occur. Therefore, it was found that a mismatch of two bases (A-C mismatch, C-A mismatch) can be detected by this example.

In conventional SNP detection, the ratio of match to single-base mismatch is about 1.9 times. Considering this, it can be seen that the method using the oligonucleotide-immobilized solid phase carrier of the present invention has an accuracy of 5 times or more that of the conventional method.

Claims

The scope of the claims

An oligonucleotide-immobilized solid phase carrier in which an oligonucleotide derivative represented by the general formula (1) is bound to a solid phase carrier.

[Chemical 1]

(In the above formula, R ¹ represents the same or different hydrogen or alkoxy group, and R ² represents the same or different hydrogen or alkoxy group, respectively. B represents a natural or non-natural nucleobase, which may be the same or different, X represents a leaving group, and n represents an integer of 2 to 50.)

An oligonucleotide-immobilized solid phase carrier represented by the general formula (2).

[Chemical 2]

(In the above formula, R ¹ may be the same or different and each represents hydrogen or an alkoxy group; R ² may be the same or different; each represents hydrogen or an alkoxy group; B represents a natural or non-natural nucleobase, which may be the same or different, X represents a leaving group, D represents a solid support, and n is an integer from 2 to 50 Represents.)

The oligonucleotide-immobilized solid phase carrier according to claim 1 or 2, wherein the solid phase carrier is a slide glass, porous glass, polystyrene beads, plastic, gold particles, a gold plate, silver particles, and a silver plate. An oligonucleotide derivative represented by the general formula (1):

A method for producing an oligonucleotide-immobilized solid phase carrier, wherein 1-hydroxybenzotriazole is used as a reaction accelerator in solid phase synthesis of an oligonucleotide by a phosphoramidide method.

[Chemical 3]

B

An oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a gene A complementary oligonucleotide-immobilized solid phase carrier comprising a leaving group bound to the 5 ′ end side of the nucleotide and a solid phase carrier bound to the 3 ′ end side;

A complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the predetermined base sequence portion in the gene; The

An oligonucleotide solid phase synthesis method comprising a step of reacting.

6. The oligonucleotide solid phase synthesis method according to claim 5, wherein the solid phase carrier is a slide glass, porous glass, polystyrene bead, plastic, gold particle, gold plate, silver particle and silver plate.

[7] The oligonucleotide solid-phase synthesis method according to [5] or [6], wherein the leaving group is a halogen, a tosinore group, a mesinole group, a trifluoromethanesulfonyl group or a phosphate group.

[8] A leaving group is bound to the 5 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to a predetermined base sequence portion in a normal gene, and a solid phase carrier is bound to the 3 ′ end side. A complementary oligonucleotide-immobilized solid phase carrier,

A complementary oligonucleotide having a highly nucleophilic functional group on the 3 ′ end side of an oligonucleotide having a complementary base sequence portion complementary to the base sequence portion adjacent to the predetermined base sequence portion in the normal gene; The

A step of reacting,

A method for confirming the difference between a predetermined nucleotide sequence in a normal gene and a corresponding nucleotide sequence in a sample gene.

[9] The method according to claim 8, wherein the solid phase carrier is a glass slide, a porous glass, a polystyrene bead, a plastic, a gold particle, a gold plate, a silver particle and a silver plate.

[10] The method according to claim 8 or 9, wherein the leaving group is a halogen, a tosinore group, a mesinole group, a trifluoromethanesulfonyl group or a phosphoric acid group.

[11] The labeling compound is bound to the complementary oligonucleotide or contains a radioisotope, and the reaction is detected using the label.

The method according to item 1.

[12] The oligonucleotide immobilization according to claim 1, as a complementary oligonucleotide solid phase carrier 12. The method according to claim 8, wherein a solidified carrier is used.

[13] By coupling base-unprotected nucleotide phosphoramidides in a predetermined order, the oligonucleotide is bound to the solid phase carrier, including a step of chemically synthesizing the oligonucleotide having a predetermined base sequence. A method of chemically synthesizing an oligonucleotide-immobilized solid phase carrier comprising:

As the 5′-terminal base-unprotected nucleotide phosphoramidide, a nucleotide phosphoramidide having a leaving group at the 5′-end is used.

A method of using a nucleotide phosphoramidite having a solid phase carrier bound to the 3 ′ end as the nucleotide-side unprotected nucleotide phosphoramidide on the 3 ′ end side.

[14] The method according to claim 13, wherein 1-hydroxybenzotriazole is used as a reaction accelerator.