JP7624867B2 - Photoresponsive deoxyribonucleoside triphosphates - Google Patents

Description

The present invention relates to photoresponsive deoxyribonucleoside triphosphates for use in a method for photocontrolled amplification of a target nucleic acid.

In genetic testing used in clinical diagnosis, extremely small amounts of target nucleic acid contained in clinical samples are amplified to increase signal strength, thereby achieving highly sensitive and highly reproducible measurements. Examples of amplification methods when the target nucleic acid is DNA include the polymerase chain reaction (PCR) method. In addition, examples of amplification methods when the target nucleic acid is RNA include the NASBA (Nucleic Acid Sequence-Based Amplification) method (Patent Documents 1 and 2, Non-Patent Document 1), the TMA (Transcription Mediated Amplification) method (Patent Document 3), and the TRC (Transcription-Reverse Transcription Concerted) method (Patent Document 4 and Non-Patent Document 2). RNA amplification methods can be easily automated because reactions can be performed at a constant, relatively low temperature (e.g., 41°C to 46°C). On the other hand, PCR methods, which require rapid temperature increases and decreases, have not been easily adapted to automation for the purpose of mass processing. However, by adding an enzyme with strand displacement activity, DNA amplification at a constant, relatively low temperature is now possible (Patent Document 5). However, in the above-mentioned reactions that allow amplification of target nucleic acids at a constant, relatively low temperature, the reaction may also proceed at room temperature.

The digital NASBA method (Patent Document 6) is one example of a technique for accurately quantifying minute amounts of target RNA contained in a sample. However, as mentioned above, the amplification reaction may proceed even at room temperature, that is, after the reaction solution is prepared, the amplification reaction may proceed until it is distributed to the microcompartments, making highly accurate quantification extremely difficult. Therefore, in order to detect extremely small amounts of target RNA present in a sample more efficiently and reproducibly, it was necessary to control the amplification reaction, particularly at room temperature.

Methods for controlling the amplification reaction of a target nucleic acid include a method of suppressing the amplification reaction by light irradiation using a substrate in which the primer or nucleic acid component dNTP (deoxyribonucleoside triphosphate) is protected with a photolabile protecting group (Non-Patent Documents 3 and 4). However, no photoresponsive dNTPs have been reported to date in which the hydroxyl group at the 3' position of the nucleic acid component dNTP is protected with a photolabile protecting group such as the 3-(dialkylamino)benzyl group (Non-Patent Document 5) or the 1-[3-(dialkylamino)phenyl]ethyl group (Non-Patent Document 6). No methods have been reported for photocontrolled amplification of a target nucleic acid in which such a dNTP is used as a raw material for the target nucleic acid and the amplification reaction is initiated by light irradiation.

Japanese Patent Application Publication No. 2-005864 Special Publication No. 4-503451 Special Publication No. 4-500759 JP 2000-014400 A JP 2015-116136 A US Publication No. 2014/0141502

de Baar, M. P. et al. , Journal of Clinical Microbiology, 39, 1895-1902 (2001) Ishiguro, T. et al. , Analytical Biochemistry, 314, 77-86 (2003) Jian, W. et al. , Proc. Natl. Acad. Sci. USA, 104, 42, 16462-16467 (2007) Weidong, W. et al. , Nucleic Acids Research, 35, 19, 6339-6349 (2007) Ding X. et al. , J. Org. Chem. , 83, 7459-7466 (2018) Ding X. et al. , J. Org. Chem. , 83, 10736-10742 (2018)

The present invention has been made based on the above circumstances, and aims to provide a photoresponsive deoxyribonucleoside triphosphate and its production precursor for use in a light-controlled amplification method in which the amplification reaction of a trace amount of target nucleic acid present in a sample is initiated by light irradiation.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that the above problems could be solved by using a photoresponsive deoxyribonucleoside triphosphate represented by general formula (1b), which can control the initiation of the amplification reaction of a target nucleic acid by light irradiation, and thus completed the present invention.

That is, the present invention includes the following embodiments [1] to [3].

[1] A compound represented by general formula (1).

(In the formula, ^R1 and ^R2 each independently represent an alkyl group having 1 to 8 carbon atoms. Alternatively, ^R1 and ^R2 may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded. ^R3 and ^R4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group. ^R5 represents a hydrogen atom or a group represented by the formula (7):
[-P(=O)(OH)-OP(=O)(OH)-OP(=O)(OH) ₂ (7)]
wherein Y represents a hydrogen atom or a hydroxyl group, and X represents a nucleic acid base represented by any one of the following formulas (2) to (5):

(In the formula, ^R6 represents a hydrogen atom or a methyl group, and when Y is a hydrogen atom, ^R6 represents a methyl group, and when Y is a hydroxyl group, ^R6 represents a hydrogen atom.)
[2] The compound represented by the general formula (1) according to [1], wherein Y is a hydrogen atom.

[3] The compound represented by the general formula (1) according to [2], wherein R ¹ and R ² are ethyl groups.

[4] A method for producing a deoxyribonucleoside triphosphate represented by formula (1b), comprising the steps of reacting a deoxyribonucleoside represented by formula (1a) with salicyl chlorophosphite in the presence of a base, followed by treatment with pyrophosphate to obtain a cyclic compound represented by formula (6), and reacting the cyclic compound represented by formula (6) with an oxidizing agent in the presence of water.

(In the formula, ^R1 and ^R2 each independently represent an alkyl group having 1 to 8 carbon atoms. Alternatively, ^R1 and ^R2 may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded. ^R3 and ^R4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group, and X represents a nucleic acid base of any of the following formulas (2), (3), (4a), or (5).)

(In the formula, R ¹ , R ² , R ³ , R ⁴ and X have the same meanings as defined above.)

(In the formula, R ¹ , R ² , R ³ , R ⁴ and X have the same meanings as defined above.)

By using the light-controlled amplification reagent for target nucleic acid containing the deoxyribonucleoside triphosphate (1b) of the present invention, the amplification reaction of a small amount of target nucleic acid present in a sample can be controlled by light irradiation. That is, in the amplification reaction of the target nucleic acid contained in a sample at a relatively low constant temperature, the start of the nucleic acid amplification reaction can be controlled by light, and highly accurate quantification of the target nucleic acid, which was previously difficult, can be performed. In addition, the deoxyribonucleoside (1a) of the present invention is useful as a manufacturing intermediate for deoxyribonucleoside triphosphate (1b).

The present invention will be described in detail below.

<1> Compound (1) of the present invention
The compound (1) of the present invention is represented by the following general formula (1).

(In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and X have the same meanings as defined above.)
In the present invention, the compound (1) includes its chemically acceptable salts and is represented by the general formula (1).

The alkyl group having 1 to 8 carbon atoms represented by R ¹ and R ² may be linear, branched or cyclic, and examples thereof include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, hexyl, heptyl and octyl. R ¹ and R ² may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded, and in this case, the carbon atom of the heterocycle may be replaced with at least one heteroatom selected from the group consisting of nitrogen and oxygen atoms. Examples of the heterocycle include pyrrolidine, piperidine and morpholine.

The alkyl group having 1 to 6 carbon atoms represented by ^R3 and ^R4 may be linear, branched, or cyclic, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, and a hexyl group.

Examples of the 3-(dialkylamino)benzyl group, which is the protecting group for the hydroxy group at the 3' position of compound (1) of the present invention, include 3-(dimethylamino)benzyl group, 3-(diethylamino)benzyl group, 3-(dipropylamino)benzyl group, 3-(dibutylamino)benzyl group, 3-(dioctylamino)benzyl group, 3-(ethylmethylamino)benzyl group, 3-(ethylpropylamino)benzyl group, 3-pyrrolidinobenzyl group, 3-piperidinobenzyl group, 1-[3-(dimethylamino)phenyl]ethyl group, 1-[3-(diethylamino)phenyl]ethyl group, [3-(dimethylamino)phenyl](phenyl)methyl group, [3-(diethylamino)phenyl](phenyl)methyl group, 1-[3-(dimethylamino)phenyl]-1-methylethyl group, and 1-[3-(diethylamino)phenyl]-1-methylethyl group.

In the compound (1) of the present invention, deprotection of the 3-(dialkylamino)benzyl group proceeds by irradiation with light having a wavelength of 200 nm or more and 400 nm or less. In terms of a fast photolysis rate, it is preferable to use a wavelength of 300 nm or more and 340 nm or less. The irradiance of the light is not particularly limited, but it is preferable to use light in the range of 0.1 mW/ ^cm2 to 3 W/ ^cm2 . The irradiation time of the light is not particularly limited, but an irradiation time of 30 minutes or less is preferable, and an irradiation time of 5 minutes or less is more preferable. As the 3-(dialkylamino)benzyl group, a 3-(diethylamino)benzyl group is preferable in terms of high photolysis.

Next, the method for producing compound (1) of the present invention will be described.

The deoxyribonucleoside (1a) of the present invention can be produced by referring to the methods disclosed in, for example, Proceedings of the National Academy of Sciences of the United States of America, 104, 16462-16467 (2007) and Nucleic Acids Research, 35, 6339-6349 (2007).

The method for producing the deoxyribonucleoside (1b) of the present invention is a method for producing the deoxyribonucleoside triphosphate (1b) of the present invention by triphosphorylating the deoxyribonucleoside (1a) of the present invention.

The deoxyribonucleoside (1a) of the present invention is reacted with salicyl chlorophosphite in the presence of a base, followed by treatment with pyrophosphate to obtain a cyclic compound (6) (Step-A). The compound is then reacted with an oxidizing agent in the presence of water (Step-B) to produce the deoxyribonucleoside triphosphate (1b) of the present invention. Triphosphorylation can be performed with reference to methods described in the literature (Acta Biochim. et Biophys. Acad. Sci. Hung., 16, 131-133 (1981); J. Org. Chem., 54, 631-635. (1989); Org. Lett., 13, 4156-4159 (2011); Org. Lett., 18, 580-583 (2016)).

The cyclic compound (6) obtained after step A may be isolated and purified, or may be directly subjected to the next step B without isolation and purification.

In the present invention, the cyclic compound (6) includes its chemically acceptable salts and is represented by the general formula (6).

The salicylic chlorophosphite used in the manufacturing method of the present invention is commercially available.

Examples of bases that can be used in the production method of the present invention include organic bases such as triethylamine, tributylamine, pyridine, and picoline.

Examples of pyrophosphates that can be used in the manufacturing method of the present invention include bis(tetrabutylammonium) dihydrogen pyrophosphate and bis(tributylammonium) pyrophosphate. These are commercially available.

An example of an oxidizing agent that can be used in the manufacturing method of the present invention is iodine.

The production method of the present invention may be carried out in a solvent as long as the solvent does not inhibit the reaction. Examples of solvents that can be used in the production method of the present invention include ether solvents such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane; hydrocarbon solvents such as hexane, pentane, and cyclohexane; aromatic hydrocarbon solvents such as benzene, toluene, and xylene; halogenated hydrocarbon solvents such as dichloromethane, chloroform, carbon tetrachloride, and 1,2-dichloroethane; acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and pyridine. Two or more of these solvents may be mixed and used. Among these solvents, it is preferable to use dimethylformamide because of its good yield.

The molar ratio of deoxyribonucleoside (1a) to chlorophosphite used in the production method of the present invention is preferably in the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

The molar ratio of deoxyribonucleoside (1a) to base used in the production method of the present invention is preferably in the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

The molar ratio of deoxyribonucleoside (1a) to pyrophosphate used in the production method of the present invention is preferably in the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

The molar ratio of deoxyribonucleoside (1a) to oxidizing agent used in the production method of the present invention is preferably in the range of 1:0.8 to 1:20, and more preferably 1:1 to 1:5 in terms of good yield.

The molar ratio of deoxyribonucleoside (1a) to water used in the manufacturing method of the present invention is not particularly limited, and it is sufficient to use an equal or greater amount of water relative to the deoxyribonucleoside (1a), and it is preferable to use water as a solvent.

The reaction temperature of the production method of the present invention can be appropriately selected from the range of -78°C to 150°C. Of these, a temperature in the range of 0°C to 120°C is preferable in terms of good yield.

The deoxyribonucleoside triphosphate (1b) obtained by the production method of the present invention can be purified from the reaction solution after the reaction is completed, if necessary. There are no particular limitations on the purification method, but the target product can be purified by a general method such as solvent extraction, silica gel column chromatography, preparative thin layer chromatography, preparative liquid chromatography, recrystallization, or sublimation.

The deoxyribonucleoside triphosphate (1b) of the present invention (hereinafter sometimes referred to as photoresponsive dNTP (1b)) can be used as a photocontrolled amplification reagent for target nucleic acids.

<2> Light-controlled amplification reagent The light-controlled amplification reagent for a target nucleic acid containing the light-responsive dNTP (1b) of the present invention can be used in an amplification reaction of a target nucleic acid contained in a sample at a constant relatively low temperature, thereby controlling the initiation of the nucleic acid amplification reaction by light.

The light-controlled amplification reagent is a reagent containing the following components (A) to (I). Note that the promoter sequence (G) below is added to the 5'-end of either (A) or (B). Other components that are more preferable for reasons other than those required for commonly used amplification reactions may be added.
(A) a first single-stranded oligonucleotide (first primer) having a sequence complementary to a portion of a target nucleic acid;
(B) a second single-stranded oligonucleotide (second primer) having a sequence homologous to a portion of the target nucleic acid;
(C) an enzyme having RNA-dependent DNA polymerase activity;
(D) photoresponsive dNTP (1b),
(E) an enzyme having ribonuclease H (RNase H) activity;
(F) an enzyme having DNA-dependent DNA polymerase activity;
(G) an enzyme having DNA-dependent RNA polymerase activity;
(H) Deoxyribonucleoside triphosphates (dNTPs: dATP, dTTP, dGTP, dCTP), provided that dNTPs having the same nucleic acid base as the photoresponsive dNTP (1b) described in (D) are not included, or may be included to the extent that the amplification reaction does not start before light irradiation.
(I) Ribonucleoside triphosphates (NTPs: ATP, UTP, GTP, CTP).

As described above, either (A) or (B) has the promoter sequence (G) added to its 5' end. An enhancer sequence consisting of several to several tens of nucleotides may be inserted between the added primer and the promoter sequence.

The enzymes (C), (E), and (F) may be different from each other, or some or all of them may be the same enzyme. For example, avian myeloblastoma virus (AMV) reverse transcriptase is an enzyme that encompasses all of the enzymatic activities of (C), (E), and (F), and is a particularly preferred embodiment as a light-controlled amplification reagent.

Examples of the enzyme (G) include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. The promoter sequence added to either (A) or (B) may be a sequence corresponding to the component (polymerase) used in (G).

The photoresponsive dNTP (1b) of (D) provides unmodified deoxynucleoside triphosphate (dNTP) that can be used as a substrate for DNA elongation upon irradiation with light. The photoresponsive dNTP (1b) used may be any one, two, three, or all of the photoresponsive dNTPs (1b) having four types of nucleic acid bases (adenine base (2), guanine base (3), thymine base (4), or cytosine base (5)) represented by formulas (2) to (5).

The photoresponsive dNTP (1b) (D) may be contained in the light-controlled amplification reagent of the present invention in the form of a free deoxyribonucleoside triphosphate (i.e., the form (H) described above), or in the form of a deoxyribonucleotide constituting the primers (A) and/or (B). Specifically, the primers (A) and/or (B) may be primers represented by the following general formula (0) in which the 3' end of the primer is modified with a 3-(dialkylamino)benzyl group.

In general formula (0), Z represents an oligodeoxyribonucleotide constituting the first or second primer, X represents a nucleic acid base selected from the group consisting of adenine base (2), guanine base (3), thymine base (4), and cytosine base (5), and Q represents a 3-(dialkylamino)benzyl group represented by general formula (8).

In the formula (8), R ¹ , R ² , R ³ and R ⁴ have the same meanings as defined above.

<3> Method for Amplifying Target Nucleic Acid The above-mentioned light-controlled amplification reagent can be used in a light-controlled amplification reaction of a target nucleic acid.

The target nucleic acid may be DNA or RNA having any base sequence, and may be any desired sequence, so long as it contains the above-mentioned (A) and (B) that are specific enough to be distinguished from other nucleic acids. There is no particular limit to the origin of the target nucleic acid, and it may be, for example, a polynucleotide synthesized by a nucleic acid amplification reaction such as the PCR method, a chemically synthesized oligonucleotide, a polynucleotide extracted from blood, tissue, cells, etc., or a polynucleotide extracted from food, soil, wastewater, etc.

When the target nucleic acid is DNA, a method such as PCR can be used to amplify the target nucleic acid.

When the target nucleic acid is RNA, the NASBA (Nucleic Acid Sequence-Based Amplification) method, the TMA (Transcription Mediated Amplification) method, the TRC (Transcription-Reverse Transcription Concerted) method, etc. can be used as a method for amplifying the target nucleic acid. These methods use a primer containing a promoter sequence, reverse transcriptase, and ribonuclease H (RNase H) for the target RNA to generate double-stranded DNA containing a promoter sequence, generate RNA containing a specific base sequence using RNA polymerase, and then perform a chain reaction using the generated RNA as a template for synthesizing double-stranded DNA containing the promoter sequence.

Although there is no particular limitation on the solvent used in the amplification reaction of the target nucleic acid, it is preferable to use a buffer solution. Examples of buffer solutions that can be used include Tris-HCl buffer solution, sodium acetate buffer solution, HEPES (2-[4-(2-HydroxyEthyl)-1-Piperazinyl]EthaneSulfonic acid)-KOH buffer solution, sodium phosphate buffer solution, and potassium phosphate buffer solution. An inorganic salt or a carboxylate salt may be added to the above-mentioned buffer solution at any concentration. Examples of the inorganic salt include sodium chloride, magnesium chloride, potassium chloride, sodium bromide, magnesium bromide, potassium bromide, sodium iodide, and potassium iodide, and examples of the carboxylate salt include magnesium acetate and manganese acetate. Two or more of these inorganic salts and carboxylate salts may be mixed and used. The concentration of the inorganic salt or the carboxylate salt added is preferably in the range of 0M to 5M. Furthermore, an organic solvent miscible with the buffer solution may be added at a ratio of 0% to 80%. Examples of the organic solvent include dimethyl sulfoxide (DMSO), methanol, ethanol, hexamethylphosphoric triamide, N,N-dimethylformamide, N,N-dimethylacetamide, 1,4-dioxane, tetrahydrofuran, ethylene glycol, and glycerin, and two or more of these organic solvents may be mixed and used.

The amplification reaction of the target nucleic acid is initiated by mixing the target nucleic acid with the light-controlled amplification reagent and irradiating the target nucleic acid with light. That is, when the light-responsive dNTP (1b) is contained as a free deoxyribonucleoside triphosphate, dNTPs having the same nucleic acid base as the light-responsive dNTP (1b) are not present in the amplification reaction system without light irradiation. Therefore, even if the target nucleic acid is added to the light-controlled amplification reagent in this state, the amplification reaction does not proceed. On the other hand, when the light-responsive dNTP (1b) is irradiated with light, the light-responsive protecting group is released, and unmodified dNTP, which is one of the substrates for DNA elongation, is present in the light-controlled amplification reagent. Therefore, the amplification reaction of the target nucleic acid proceeds by adding the target nucleic acid to the light-controlled amplification reagent in this state and setting the reagent to appropriate amplification reaction conditions (such as appropriate temperature).

In addition, when the photoresponsive dNTP (1b) is contained as a deoxyribonucleotide constituting the first and/or second primer (i.e., an embodiment of general formula (0)), the photodegradable protective group modified to the photoresponsive dNTP (1b) inhibits hybridization between the primer and the target nucleic acid without light irradiation, and the amplification reaction does not proceed even when the target nucleic acid is added. On the other hand, when the photoresponsive dNTP (1b) (general formula (0)) is irradiated with light, the photoresponsive protective group is released, and the primer becomes capable of hybridizing with the target nucleic acid. Therefore, the amplification reaction of the target nucleic acid proceeds by setting the light-controlled amplification reagent to which the target nucleic acid has been added under appropriate amplification reaction conditions (such as appropriate temperature).

The photoresponsive dNTP (1b) or the 3-(dialkylamino)benzyl group of the general formula (0) undergoes deprotection by irradiation with light having a wavelength of 200 nm or more and 400 nm or less, and an amplification reaction is initiated. In terms of a fast photolysis rate, it is preferable to use a wavelength of 300 nm or more and 340 nm or less. The irradiance of the light is not particularly limited, but it is preferable to use light in the range of 0.1 mW/ ^cm2 to 3 W/ ^cm2 . The irradiation time of the light is not particularly limited, but an irradiation time of 30 minutes or less is preferable, and an irradiation time of 5 minutes or less is more preferable.

The temperature at which the target nucleic acid is amplified may be set appropriately within the range of 0°C to 80°C, taking into consideration the composition of the solvent used. If the amplification reaction is carried out using the TRC method, it is preferable to carry out the reaction at a temperature in the range of 40°C to 50°C.

<4> Detection method In the above-mentioned method for amplifying a target nucleic acid, the amplified target nucleic acid can be detected by the following method. The target nucleic acid amplified with a light-controlled amplification reagent may be detected by adding a detection component before or after the amplification reaction and measuring the fluorescence intensity or chemiluminescence intensity derived from the component. A preferred embodiment of the detection component is an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before the formation.

An example of the probe is DNA bound to an intercalating fluorescent dye having a sequence complementary or homologous to a portion of the target nucleic acid. The sequence of the DNA portion must be a sequence present in the target nucleic acid that is complementary or homologous to a portion that can be sufficiently distinguished from nucleic acids other than the target nucleic acid. The length of the DNA portion is preferably 6 to 100 nucleotides, more preferably 10 to 30 nucleotides, for specific analysis of the target nucleic acid. It is preferable that the 3' end of the DNA portion has a sequence non-complementary to the target nucleic acid added thereto, or that the 3' end is chemically modified (e.g., aminated) so that extension from the 3' end by an enzyme having RNA-dependent DNA polymerase activity does not occur when the DNA portion forms a complementary bond with the amplified target nucleic acid.

When the aforementioned DNA portion forms a complementary bond with another nucleic acid, the intercalating fluorescent dye intercalates into the double-stranded portion, changing its fluorescent properties. For this purpose, for example, the intercalating fluorescent dye may be bound to DNA via a linker of an appropriate molecular length that does not prevent intercalation into the double-stranded portion. There are no particular limitations on such linkers, so long as they do not prevent the intercalating fluorescent dye from intercalating into the double-stranded portion. In particular, linker molecules selected from bifunctional hydrocarbons having functional groups at both ends are convenient and preferable for modifying oligonucleotides. A commercially available reagent set (for example, C6-Thiomodifier manufactured by Clontech) may also be used.

There are no particular limitations on the intercalating fluorescent dye, so long as its fluorescent properties change, for example, the emitted fluorescent wavelength changes, when it intercalates into the double strand. From the standpoint of ease of measurement, however, it is particularly preferred that the fluorescent intensity increases upon intercalation. Specifically, thiazole orange and oxazole yellow, which show particularly significant changes in fluorescent intensity, as well as their derivatives, are examples of preferred intercalating fluorescent dyes.

The position at which the intercalating fluorescent dye is bound to the DNA portion via a linker is not particularly limited, and may be the 5' end, 3' end, or center of the DNA portion, as long as it does not prevent the intercalating fluorescent dye from intercalating into the double strand and does not inhibit the complementary binding between the DNA portion and RNA.

Another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before formation is the molecular beacon. A molecular beacon is a DNA having a sequence complementary or homologous to a part of the target nucleic acid, and has a stem-loop structure with a fluorescent dye and a quencher at both ends. In the stem-loop structure, the fluorescence of the fluorescent dye is suppressed by the quencher, but when a region complementary to the target RNA present in the loop sequence hybridizes with the amplification product generated during the reaction, the stem portion is cleaved and emits fluorescence. The stem can be formed by forming a double strand of DNA within the molecule and introducing a pair of a fluorescent dye (such as Cy3) and a quencher (such as an azo compound) with high association ability into the DNA.

Another example of an oligonucleotide probe whose fluorescent properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before formation is the FRET (Fluorescence Resonance Energy Transfer) probe. In this case, two probes designed inside the first and second primers used for amplification in the sequence of the target nucleic acid are used. Of the two probes, one probe is modified with a donor fluorescent dye at the 3' end and the other probe is modified with an acceptor fluorescent dye at the 5' end. By designing the donor fluorescent dye and acceptor fluorescent dye to be close to each other upon hybridization, the target nucleic acid can be detected by the FRET phenomenon that occurs when the two probes hybridize with the amplification product generated during the reaction.

Yet another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before formation is the TaqMan probe. In this case, a single probe designed inside the first and second primers used for amplification in the sequence of the target nucleic acid is used. The 5' end of the TaqMan probe is labeled with a fluorescent dye (reporter dye), and the 3' end is labeled with a quencher. The fluorescence of the reporter dye is suppressed by the quencher, but if the probe is hybridized to the target RNA during the primer extension reaction by DNA polymerase, the probe is degraded by 5'→3' exonuclease activity, and the reporter dye is released, allowing detection by the fluorescence generated.

The present invention will now be described in more detail with reference to examples and reference examples, but the present invention is not limited to these. The reference examples do not constitute the present invention. The reagents and anhydrous solvents used in the reactions were commercially available products (Tokyo Chemical Industry Co., Ltd., Kanto Chemical Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., and Sigma-Aldrich Co., Ltd.) and were used without purification. Nuclear magnetic resonance (NMR) spectra were measured with a 400 MHz Bruker AVANCE III 400 and a 400 MHz Bruker AVANCE III HD. Chemical shift values are shown in ppm using tetramethylsilane (TMS) as an internal standard. The letter s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, quint represents a quintet, brs represents a broad singlet, and m represents a multiplet.

Reference Example 1
N ⁶ ,N ⁶ -bis(tert-butoxycarbonyl)-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine (2.88 g, 5.1 mmol) was dissolved in methylene chloride (20 mL), and sodium iodide (100 mg, 0.67 mmol), 1.5 M aqueous tetrabutylammonium hydroxide solution (6.78 mL, 10 mmol), 1.0 M aqueous sodium hydroxide solution (7.63 mL, 7.6 mmol) and distilled water (10 mL) were added and stirred at room temperature for 5 minutes. To the resulting mixture was added a solution of m-(diethylamino)benzyl chloride (3.02 g, 15 mmol) in methylene chloride (9.0 mL), and the mixture was reacted at room temperature for 15 hours under light-shielded conditions. The aqueous and organic phases were separated, and the aqueous phase was extracted twice with methylene chloride (50 mL). The organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=70:30) to give N ⁶ ,N ⁶ -bis(tert-butoxycarbonyl)-5′-O-tert-butyldimethylsilyl-3′-O-[3-(diethylamino)benzyl]-2′-deoxyadenosine (625 mg, 17%) as a yellow solid.

^1H NMR (400MHz, _CDCl3 ): δ8.84 (s, 1H), 8.42 (s, 1H), 7.19 (t, J=7.9Hz, 1H), 6.64-6.61 (m, 3H), 6. 55 ( m, 1H), 4.53 (d, J = 1.4Hz, 2H), 4.37 (m, 1H), 4.27 (m, 1H), 3.89 (dd, J = 11.1, 4.0 Hz, 1H), 3.78 (dd, J = 11.1, 3.2Hz, 1H), 3.36 (q, J = 7.1Hz, 4H), 2.70-2.59 (m, 2H ), 1.45 (s, 18H), 1.16 (t, J=7.1Hz, 6H), 0.88 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H).
Example 1
The N ⁶ ,N ⁶ -bis(tert-butoxycarbonyl)-5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxy Adenosine (625 mg, 0.87 mmol) was weighed out and placed in a light-shielding container, dissolved in methylene chloride (30 mL), activated (vacuum dried at 80°C for 3 hours) silica gel (6.0 g) was added, and the solvent was removed under reduced pressure. The residue obtained was heated at 80° C. under reduced pressure for 13 hours. The solid obtained was washed with methanol and chloroform, and the filtrate was concentrated to give 5′-O-tert-butyldimethylsilyl- 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (369 mg, 81%) was obtained as a yellow solid.

^1H NMR (400MHz, _CDCl3 ): δ8.32 (s, 1H), 8.14 (s, 1H), 7.17 (t, J=7.8Hz, 1H), 6.63-6.59 (m, 3H), 6. 47(t, J = 6.5Hz, 1H), 6.05 (brs, 2H), 4.51 (d, J = 3.7Hz, 2H), 4.34 (m, 1H), 4.24 (m, 1H) ,3. 88 (dd, J=11.0.4.2Hz, 1H), 3.76 (dd, J=11.0, 3.2Hz, 1H), 3.33 (q, J=7.0Hz, 4H) ， 2.64-2.61 (m, 2H), 1.13 (t, J=7.0Hz, 6H), 0.89 (s, 9H), 0.06 (s, 3H), 0.05 ( s, 3H).
Next, 5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (369 mg, 0.70 mmol) was dissolved in tetrahydrofuran (3.0 mL); The mixture was cooled to 0° C. A 1M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.70 mL, 0.70 mmol) was slowly added dropwise to the resulting mixture, and the temperature was then raised to room temperature. The mixture was allowed to react at room temperature for 2 hours, Methanol (3.0 mL) was added to quench the reaction. The reaction solution was concentrated, and the resulting residue was purified by flash silica gel column chromatography (ethyl acetate:methanol=90:10) to obtain 3'-O-[ As a result, 3-(diethylamino)benzyl]-2'-deoxyadenosine (Compound A, 202 mg, 70%) was obtained as a yellow solid.

^1H NMR (400MHz, _CDCl3 ): δ8.32 (s, 1H), 7.81 (s, 1H), 7.20 (t, J=8.1Hz, 1H), 6.65-6.62 (m, 3H), 6. 57 (m, 1H), 6.28 (m, 1H), 5.74 (brs, 2H), 4.54 (s, 2H) , 4.49 (m, 1H), 4.39 (s, 1H), 3.99 (m, 1H), 3.70 (m, 1H), 3.36 (q, J = 7.0Hz, 4H ), 3.01 (m, 1H), 2.44 (m, 1H), 1.17 (t, J=7.0Hz, 6H).
Example 2
Compound A (70.0 mg, 0.17 mmol) obtained in Example 1 was subjected to azeotropic distillation twice with dehydrated toluene (3.0 mL) and three times with dehydrated pyridine (3.0 mL) to remove water. To a solution of the solid in dehydrated pyridine (2.0 mL), chlorosalicyl phosphite (40.0 mg, 0.20 mmol) and 4-dioxane (1.0 mL) were added and the mixture was allowed to react at room temperature for 1.5 hours. Then, a solution (1.0 mL) of bis(tributylammonium) pyrophosphate (82.0 mg, 0.15 mmol) in DMF (N,N-dimethylformamide) and tributylamine (0.12 mL, 0.50 mmol) were added and the mixture was stirred at room temperature. After the reaction, a solution of iodine (32 mg, 0.13 mmol)/pyridine (9.0 mL)/distilled water (1.0 mL) was added until the reaction solution turned red. After confirming that the reaction solution did not fade even when stirred at room temperature for about 15 minutes, distilled water (2.0 mL) was added and reacted at room temperature for 12 hours. The reaction solution was washed with ethyl acetate (60 mL), The aqueous phase was concentrated. The resulting residue was purified by octadecyl-modified silica gel column chromatography (water:methanol=9:1), and the solvent was distilled off under reduced pressure. The resulting residue was diluted with distilled water (1.3 mL ), was suspended in methanol (0.5 mL), a solution of sodium perchlorate (850 mg) in acetone (10 mL) was added, and the mixture was centrifuged. After removing the supernatant, the solvent was distilled off under reduced pressure. This was carried out twice to obtain 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine triphosphate (Compound B, 13 mg, 12%) as a white solid.

^1H NMR ( _D2O , 400MHz): δ8.14 (m, 1H), 7.56 (m, 3H), 7.53 (m, 1H), 7.41 (m, 1H), 6.40 (m, 1H), 4.60 (m, 2H, obscured by the residual proton of D2O), 4.58 (m, 1H, obscured by the residual proton of _D2 O), 4.40 (m, 1H), 4.11 (m, 2H), 3.57 (m, 4H), 2.66 (m, 2H), 1.01 (m, 6H); ³¹ P NMR( _D2 O, 162MHz): δ-8.0 (m, 1P), -11.4 (d, 1P), -21.5 (m, 1P); HR-MS (ESI-TOF): molecular formula C ₂₁ H ₃₀ N ₆ O ₁₂ P ₃ ;[M-H+]-:651.1179(calculated:651.118).

Reference Example 2
2-Nitro-p-xylene glycol (5.00 g, 27.3 mmol) and imidazole (3.72 g, 54.6 mmol) were dissolved in DMF (15.0 mL) under an argon atmosphere and cooled to 0°C. To the resulting solution, a solution of t-butyldimethylsilyl chloride (4.25 g, 27.3 mmol) in DMF (10.0 mL) was added. The resulting mixture was warmed to room temperature and reacted for 1.5 hours, and then methanol (20 mL) was added and stirred at room temperature for 30 minutes to stop the reaction. The solvent in the reaction solution was distilled off under reduced pressure, and distilled water (200 mL) was added to the resulting residue, which was then extracted five times with ethyl acetate (200 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=4:1) to obtain 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzenemethanol (1.9 g, 23%) as a white yellow solid.

^1H NMR (400MHz, _CDCl3 ): δ8.06 (s, 1H), 7.66 (d, J = 8.0Hz, 1H), 7.60 (d, J = 8.0Hz, 1H), 4 .93(d, J=6.7Hz, 2H, CH ₂ -OH), 4.79 (s, 2H, CH ₂ -OTBS), 2.51 (t, J=6.7Hz, 1H, OH), 0.95 (s, 9H, TBS), 0.11 ( s, 6H, TBS).
Reference Example 3
4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzenemethanol (648 mg, 2.17 mmol) obtained in Reference Example 2, triphenylphosphine (1.71 g, 6.51 mmol), tetrachloride Carbon (2.16 g, 6.51 mmol) and diisopropylethylamine (929 mg, 7.16 mmol) were dissolved in THF (tetrahydrofuran) (7.3 mL) under an argon atmosphere and stirred at room temperature for 1 hour. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=15 :1) to give 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzyl bromide (747 mg, 83%) as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ8.00 (s, 1H), 7.55-7.51 (m, 2H), 4.82 (s, CH ₂ -Br), 4.79 (s, 2H, CH ₂ -OTBS), 0.95 (s, 9H, TBS), 0.11 (s, 6H, TBS).
Reference Example 4
5'-O-(4,4'-dimethoxytrityl)thymidine (2.72 g, 5.0 mmol) was dissolved in a mixed solvent (25 mL) of dichloromethane/dimethylacetamide (10:1), and triethylamine (607 mg, 6 After adding benzoyl chloride (773 mg, 5.5 mmol) and stirring at room temperature for 10 minutes, the mixture was reacted at room temperature for 15 hours. After the reaction, the mixture was diluted with ethyl acetate (50 mL) and washed with saturated saline (30 mL). Washed. The solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (toluene:ethyl acetate=3:1→1:1) to obtain N-benzoyl-5′-O-(4, A mixture of 3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine and 3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine was obtained (1.78 g, 55%, N-Bz form: O-Bz compound (6.5:1) was obtained as a colorless solid.

N-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine; ^1H NMR (400MHz, _CDCl3 ): δ 7.88 (dd, J = 7.2, 1.2 Hz, 2H), 7.73 (d, J = 0.84 Hz, 1H), 7.55 (t, J = 7.4 Hz, 1H), 7.37-7.42 (m, 4H), 7.25-7.30 (m, 5H), 7.19-7.23 (m, 2H), 6.82 (d, J = 8.0 Hz, 4H), 6.34 (t, J = 7.2Hz, 1H), 4.49 (quint, J = 2.5Hz, 1H), 3.97 (q, J = 2.5Hz, 1H), 3.74 (s, 6H), 3.42 (d d, J=10.6, 2.7Hz, 1H), 3.31 (dd, J=10.6, 2.7Hz, 1H), 2.23-2.34 (m, 2H), 1.41 (s, 3H).
3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine; ¹ H NMR (400 MHz, CDCl ₃ ): δ8.02 (t, J=6.9Hz, 2H), 7.66 (d, J=0.96Hz, 1H), 7.55 (t, J=7.4Hz, 1H), 7 .37-7.42 (m, 4H), 7.25-7.30 (m, 5H), 7.09-7.14 (m, 2H), 6.82 (d, J=8.0Hz, 4H), 6.50 (dd, J = 5.6Hz, 3.0Hz, 1H), 5.68 (d, J = 5.6Hz, 1H), 4.26 (d, J = 1.8H z, 1H), 3.73 (s, 6H), 3.53 (t, J=2.4Hz, 1H), 2.47-2.64 (m, 1H), 1.37 (s, 3H).
Reference Example 5
4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzyl bromide (377 mg, 1.05 mmol) obtained in Reference Example 3 was dissolved in methylene chloride (2.0 mL) and stirred at room temperature for 10 minutes, and then a mixture of N-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine and 3'-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine (521 mg, 0.81 mmol, N-Bz form: O-Bz form = 4:1) obtained in Reference Example 4, tetrabutylammonium iodide (236 mg, 0.64 mmol), 1M aqueous sodium hydroxide solution (1.62 mL, 1.62 mmol) and methylene chloride (6 mL) was added dropwise and reacted at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (20 mL), washed with saturated saline (20 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (toluene:ethyl acetate=30:1) to obtain a product (146 mg). The resulting product was then dissolved in a 2M ethanol/methylene chloride solution of ammonia (16 mL, ethanol:methylene chloride=2.5:1) and stirred at room temperature for 6.5 hours, after which the solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=1:1) to obtain 3'-O-[4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (46.7 mg, 7% over two steps) as a colorless solid.

^1H NMR (400MHz, _CDCl3 ): δ8.05 (brs, 1H), 7.98 (brs, 1H), 7.68 (d, J = 8.0Hz, 1H), 7.59 (d, J = 4.6Hz, 2H), 7.39 (d, J=8.6Hz, 2H), 7. 21-7.31 (m, 7H), 6.83 (dd, J = 8.9, 1.2Hz, 4H), 6.39 (dd, J = 8.4, 5.6Hz, 1H), 4 .85 (q, J=14.6Hz, 2H), 4.76 (br s, 2H), 4.33 (d, J = 5.6Hz, 1H), 4.22 (d, J = 2.4Hz, 1H), 3.78 (s, 6H), 3.52 (dd, J=10.6, 3.2Hz, 1H), 3.37(dd, J =10.6, 3.2Hz, 1H), 2.53-2.58 (m, 1H), 2.21-2.29 (m, 1H), 1.47 (d, J = 0.8Hz, 3H), 0.95 (s, 9H), 0.12 (s, 6H).
Reference Example 6
3'-O-[4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl) obtained in Reference Example 5 A 1M THF solution (0.23 mL, 0.23 mmol) of tetrabutylammonium fluoride was added to a solution of thymidine (157 mg, 0.19 mmol) in THF (3.8 mL), and the mixture was reacted at 0° C. for 2 hours. (20 mL) and washed with saturated saline (20 mL). The solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=1:1→1:4). After purification, 3'-O-[4-(hydroxymethyl)-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl)thymidine (124 mg, 92%) was obtained as a colorless oil. I got it at.

^1H NMR (400MHz, _CDCl3 ): δ8.10 (d, J=1.4Hz, 1H), 7.91 (brs, 1H), 7.70 (d, J=8.0Hz, 1H), 7.61 (dd, J=8 .6, 1.1Hz, 2H), 7.38(dd, J=7.0, 1. 5Hz, 2H), 7.31-7.20 (m, 7H), 6.82 (dd, J = 8.9, 3.4Hz, 4H), 6.39 (dd, J = 8.4, 5 .6Hz, 1H), 4.86 (q, J=14.9Hz, 2H), 4. 79 (s, 2H), 4.31 (d, J = 5.9Hz, 1H), 4.23 (d, J = 2.5Hz, 1H), 3.79 (s, 6H), 3.53 ( dd, J=10.6, 2.7Hz, 1H), 3.35(dd, J=1 0.6, 2.7Hz, 1H), 2.54 (dq, J = 13.6, 5.4Hz, 1H), 2.17-2.28 (m, 1H), 1.94 (t, J =6.0Hz, 1H), 1.49(d, J=0.96Hz, 3H).
Reference Example 7
3'-O-[4-(hydroxymethyl)-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl)thymidine (130 mg, 0.18 mmol) obtained in Reference Example 6 Succinic anhydride (22 mg, 0.21 mmol), triethylamine (36 mg, 0.36 mmol) and 4-dimethylaminopyridine (22 mg, 0.18 mmol) were added to a methylene chloride (3.6 mL) solution and reacted at room temperature for 18 hours. The mixture was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). The solvent was removed under reduced pressure to obtain the product (78.2 mg) as a colorless oil. A solution of the obtained product (21.6 mg) in dimethylformamide (3.6 mL) was diluted with 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (90 mg, 0.55 mmol). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (70 mg, 0.37 mmol) was added and reacted at room temperature for 18 hours. The mixture was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). did. The solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (chloroform:ethyl acetate=3:1) to give 3'-O-[[[[[4-(3,4 -dihydro-4-oxo-1,2,3-benzotriazin)-3-yloxy]carbonylethyl]carbonyl]oxymethyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl ) Thymidine (65 mg, 38% over two steps) was obtained as a colorless oil.

^1H NMR (400MHz, _CDCl3 ): δ8.36 (dd, J=8.0, 1.0Hz, 1H), 8.22 (dd, J=8.2, 0.48Hz, 1H), 8. 08 (dd, J=13.0, 1.6Hz, 1H), 8.00 (dt, J=7.4, 1.4Hz, 2H), 7.84 (dt, J=7.4, 1.1Hz, 1H), 7.74 (d, J=8.0, 2.5Hz, 1H), 7.63 (dd, J=8.0, 1. 7Hz, 1H), 7.59 (d, J=1.1Hz, 1H), 7.38 (dd, J=8.5, 1.5Hz, 2H), 7.30
-7.21 (m, 7H), 6.82 (dd, J=8.9, 2.0Hz, 4H), 6.39 (dd, J=8.4, 5.6Hz, 1H), 5. 29(s, 2H), 5 .24 (s, 1H), 4.86 (q, J=15.0Hz, 2H), 4.32 (d, J=5.9Hz, 1H), 4.22 (d, J=2.4Hz, 1H), 3.78( s, 6H), 3.52 (dd, J = 10.6, 2.7Hz, 1H), 3.36 (dd, J = 10.6, 2.7Hz, 1H), 3.13 (t, J =6.6Hz, 2 H), 2.91 (t, J=6.6Hz, 2H), 2.50-2.58 (m, 1H), 2.16-2.28 (m, 1H), 1.48 (d, J=1.0Hz, 3H).
Reference Example 8

Amino-SynBase CPG 1000/110 (LCAA) (LGC LINK Technologies) (400 mg, loading amount 105 μmol/g) was placed in a plastic centrifuge tube and set in a solid-phase synthesizer. A DMF solution of 3'-O-[[[[[4-(3,4-dihydro-4-oxo-1,2,3-benzotriazine)-3-yloxy]carbonylethyl]carbonyl]oxymethyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl)thymidine (61.6 mg, 64.5 μmol) and diisopropylethylamine (8.4 mg, 64.5 μmol) obtained in Reference Example 7 was added and reacted at room temperature for 3 days. After the reaction was completed, the solid phase was removed by suction filtration, washed with ethyl acetate, and dried to obtain a solid-phase-supported thymidine derivative (349.5 mg). The loading amount of the thymidine derivative in the solid phase was 100.5 μmol/g.

Hereinafter, the nucleic acid amplification method will be referred to as TRC (Transcription-Reverse transcription
The present invention will be described in more detail using examples in which a concealed (annotation concerted) method is used, but the present invention is not limited to these examples.

Example 3 Effect of dATP Concentration on TRC Reaction The effect of the dATP concentration added on the detection time in the TRC method was examined.

(1) A standard RNA (SEQ ID NO: 1) of hepatitis C virus was prepared by in vitro transcription from a plasmid into which the gene of the standard RNA of hepatitis C virus was inserted. The standard RNA was diluted with water for injection to ¹⁰ copies/2 μL, and this was used as an RNA sample.

(2) 12 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the RNA sample was added. The molecular beacon probe (SEQ ID NO: 2), which is a probe for detecting the standard RNA, contains a portion of the homologous strand of the standard RNA [from 108th to 123rd in SEQ ID NO: 1]. The first primer (SEQ ID NO: 3) is an oligonucleotide in which a T7 promoter sequence (SEQ ID NO: 6) has been added to the 5' end of a portion of the complementary strand of the standard RNA (specifically, from 125th to 145th in SEQ ID NO: 1: SEQ ID NO: 5).

Composition of reaction solution: Final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33mM each dCTP, dGTP, dTTP
2.0mM each ATP, CTP, GTP, UTP
3.3mM ITP
150 mM trehalose 50 nM molecular beacon probe (containing a portion of the homologous strand of standard RNA [from 108 to 123 of SEQ ID NO: 1]: SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO:3)
1.0 μM second primer (a portion of the homologous strand of standard RNA [from the 1st to the 16th of SEQ ID NO: 1]: SEQ ID NO: 4)
12.8 U AMV reverse transcriptase 166 U T7 RNA polymerase Various concentrations of dATP (0 to 3300 μM)
(3) The above reaction solution was incubated at 46° C. for 3 minutes, and then 6 μL of an initiator having the following composition was added.

Composition of initiator: Final concentration after addition of initiator (in 20 μL) was 18.4 mM magnesium chloride, 90.0 mM potassium chloride, 0.1% (w/v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
9.0% (v/v) DMSO
2.5% (w/v) glycerol (4) The PCR tube was then reacted at 46°C using a thermostatically controlled fluorescence spectrophotometer capable of direct measurement, and the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes. The time when the initiator was added was set to 0 minutes, and the time when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a specified time by the fluorescence intensity ratio of the background) exceeded 2.3 was defined as the detection time.

The results are shown in Table 1. It can be seen that if the dATP concentration in the reaction solution is 165 μM or higher, there is no effect on the amplification reaction.

Example 4 Effect of Photolabile Protective Group on TRC Reaction The effect of compound A synthesized in Example 1 on the TRC reaction was examined.

(1) Using Example 1, 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (Compound A) was synthesized, in which the 3'-terminal hydroxyl group of dATP was modified with a 3-(diethylamino)benzyl group.

(2) A solution containing 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (compound A) synthesized in (1) was dissolved in DMSO to a certain concentration.

(3) 8 μL of the reaction solution having the composition described below was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the RNA sample prepared in Example 3 (1) was added.

Composition of reaction solution: Final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33mM each dATP, dCTP, dGTP, dTTP
2.0mM each ATP, CTP, GTP, UTP
3.3mM ITP
150 mM Trehalose 50 nM Molecular Beacon Probe (SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO:3)
1.0 μM second primer (SEQ ID NO:4)
12.8 U AMV reverse transcriptase 166 U T7 RNA polymerase (4) The above reaction solution was incubated at 46° C. for 3 minutes, and then 10 μL of an initiator having the following composition was added.

Composition of initiator: Final concentration after addition of initiator (in 20 μL) was 18.4 mM magnesium chloride, 90.0 mM potassium chloride, 0.1% (w/v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
2.5% (w/v) glycerol 10% (v/v) DMSO (containing 0 to 200 μM as a photolabile protecting group, (2)
(Prepared with
(5) Measurement was performed in the same manner as in Example 3(4) to determine the detection time.

The results are shown in Table 2. It can be seen that when a diethylaminobenzyl group is used as a photodegradable protecting group, there is no effect on the TRC reaction at least up to a concentration of 200 μM.

Reference Example 9: Photocontrol of TRC reaction using a photolabile protective group-modified primer (1) Using the solid-phase-supported thymidine derivative obtained in Reference Example 8, a first primer (hereinafter also referred to simply as a "photolabile protective group-modified primer") consisting of the nucleotide sequence set forth in SEQ ID NO: 7, in which the hydroxyl group at the 3' position of the thymidine at the 3' end (the 49th position) was modified with a photolabile protective group, was synthesized (consigned to Gene Design, Inc.). Of the first primer consisting of the sequence set forth in SEQ ID NO: 7, the 28 bases on the 5' end side are the T7 promoter sequence (SEQ ID NO: 6). The synthesized product was purified using HPLC, and the molecular weight was confirmed using an electrospray ionization mass spectrometer (ESI-MS), and then it was diluted to 100 μM with TE buffer and stored at -20°C.

(2) 8 μL of the reaction solution with the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

Composition of the reaction solution: Final concentration after addition of the initiator described below (in 20 μL) was 0.1% (w/v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
66mM Tris-HCl buffer (pH 8.36)
2.5% (w/v) glycerol 0.33 mM each dATP, dCTP, dGTP, dTTP
2.0mM each ATP, CTP, GTP, UTP
3.3mM ITP
94.5 mM Trehalose 50 nM Molecular Beacon Probe (SEQ ID NO: 2)
1.0 μM second primer (SEQ ID NO:4)
(3) The photolabile protecting group-modified primer solution synthesized in (1) or the first primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 7 (not modified with a photolabile protecting group), prepared to 10 μM using water for injection, was added in an amount of 2 μL to the above reaction solution without UV light irradiation (UV light irradiation time 0 minutes) (final concentration after addition of an initiator described below: 1 μM).

(4) The 10 μM photodegradable protective group-modified primer solution prepared in (3) was irradiated with ultraviolet light (wavelength 365 nm, intensity 1 mW/cm ² ) for 1 minute or 10 minutes using an ultraviolet light irradiation device (Omnicure LX405S UV LED spot curing device, manufactured by Sanei Tech Co., Ltd.) and 2 μL of the irradiated primer solution was added to the above reaction solution.

(5) Standard RNA (SEQ ID NO: 1) was diluted with TE buffer (containing 0.01% (w/v) cholic acid) to ¹⁰ copies/2 μL, and then 2 μL each was added to the reaction solutions containing the primer solutions prepared in (3) and (4) above.

(6) After the standard RNA was added, the reaction solution was incubated at 46°C for 3 minutes, and then 8 μL of the initiator having the composition described in Example 3(3) that had been similarly incubated at 46°C for 3 minutes was added.

(7) Measurements were performed in the same manner as in Example 3 (4) to determine the detection time.

The results are shown in Table 3. From the results with 0 minutes of UV irradiation, it can be seen that when the photolabile protective group-modified primer synthesized in (1) is used as the first primer, the detection time is delayed by about 1 minute compared to when a first primer not modified with a photolabile protective group is used. This shows that the TRC reaction can be inhibited by modifying the deoxyribonucleotide at the 3' end of the primer with a photolabile protective group. On the other hand, even when the photolabile protective group-modified primer synthesized in (1) is used as the first primer, the detection time is equivalent to that when a first primer not modified is used when the primer is irradiated with UV light for 1 minute or 10 minutes to decompose the protective group. This shows that the TRC reaction can be controlled by light by modifying the deoxyribonucleotide at the 3' end of the primer with a photolabile protective group.

Reference Example 10: Effect of photocontrol of TRC reaction on TRC reactivity In Reference Example 9, only the first primer was irradiated with UV light, but in this experiment, the reaction solution including the first primer was irradiated with UV light to examine the effect of the irradiation on the TRC reaction.

(1) 8 μL of the reaction solution having the composition described in Reference Example 9 (2) was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

(2) 2 μL each of the 10 μM photolabile protecting group modified primer (SEQ ID NO: 7) solution or the first primer (unmodified with a photolabile protecting group) (SEQ ID NO: 7) solution prepared in Reference Example 9 (3) was added to the above reaction solution (final concentration after addition of the initiator described below: 1 μM).

(3) Standard RNA (SEQ ID NO: 1) was diluted with TE buffer (containing 0.01% (w/v) cholic acid) to ¹⁰ copies/2 μL, and then 2 μL of the diluted standard RNA was added to the reaction solution containing the primer solution prepared in (2) above.

(4) After the addition of the standard RNA, the reaction solution was incubated at 46°C for 3 minutes, and then 8 μL of initiator having the composition described in Example 3(3) that had been similarly incubated at 46°C for 3 minutes was added, followed by irradiation with ultraviolet light (wavelength 365 nm, intensity 1 mW/ ^cm2 ) for 1 minute.

(5) Measurements were performed in the same manner as in Example 3 (4) to determine the detection time.

The results are shown in Table 4. Even when the entire reaction solution was irradiated with UV light, the detection time was the same as in Reference Example 9. This shows that irradiating the entire reaction solution with UV light does not affect the TRC reaction.

In Reference Examples 9 and 10, a 2-nitrobenzyl group (o-nitrobenzyl group) is used as the photodegradable protecting group, but it is easily conceivable that the TRC reaction can be controlled by light even if a 3-(dialkylamino)benzyl group (an embodiment of compound (1) of the present invention) is used as the photodegradable protecting group instead of a 2-nitrobenzyl group.

Claims (4)

A compound represented by general formula (1):
(In the formula, ^R1 and ^R2 each independently represent an alkyl group having 1 to 8 carbon atoms. Alternatively, ^R1 and ^R2 may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded. ^R3 and ^R4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group. ^R5 represents a hydrogen atom or a group represented by the formula (7):
It represents a group represented by [-P(=O)(OH)-O-P(=O)(OH)-O-P(=O)(OH) ₂ (7)], Y represents a hydrogen atom or a hydroxyl group, and X represents any of the nucleic acid bases represented by the following formulas (2) to (5).
(In the formula, ^R6 represents a hydrogen atom or a methyl group, and when Y is a hydrogen atom, R6 represents a methyl group, and when Y is a hydroxyl group, ^R6 represents a hydrogen atom.) A compound represented by general formula (1) according to claim 1, in which Y is a hydrogen atom. The compound represented by the general formula (1) according to claim 2, wherein R ¹ and R ² are ethyl groups. A method for producing a deoxyribonucleoside triphosphate represented by formula (1b), comprising the steps of: reacting a deoxyribonucleoside represented by formula (1a) with salicyl chlorophosphite in the presence of a base, followed by treatment with a pyrophosphate to obtain a cyclic compound represented by formula (6); and reacting the cyclic compound represented by formula (6) with an oxidizing agent in the presence of water.
(In the formula, ^R1 and ^R2 each independently represent an alkyl group having 1 to 8 carbon atoms. Alternatively, ^R1 and ^R2 may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded. ^R3 and ^R4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group, and X represents a nucleic acid base of any of the following formulas (2), (3), (4a), or (5).)
(In the formula, R ¹ , R ² , R ³ , R ⁴ and X have the same meanings as defined above.)
(In the formula, R ¹ , R ² , R ³ , R ⁴ and X have the same meanings as defined above.)