JP2008256494A - Labeling agent and method of detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a labeling agent capable of detecting an object nucleic acid sample with high sensitivity, and to provide a method of detecting nucleic acid using the same. <P>SOLUTION: This labeling agent wherein a linker connecting nucleotide to an electrochemical emission portion is allowed to have an unsaturated hydrocarbon and a cyclic compound is provided. By allowing the linker for bonding the electrochemical emission portion to a substrate to have a stiff structure, bending is not generated, and intramolecular association caused by electrostatic adsorption between the substrate and the electrochemical emission portion is suppressed, to thereby prevent a reaction between the substrate in nucleic acid synthesis from being impeded. In addition, steric hindrance with oxygen is also mitigated, and a modification amount of the labeling agent to a generated nucleic acid sample is increased, to thereby enable highly sensitive detection. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a labeling agent and a nucleic acid detection method for detecting a nucleic acid sample with high sensitivity by performing a nucleic acid extension reaction using a specific nucleic acid present in a biological sample.

  As a technique for detecting a specific nucleic acid present in a sample, a technique for labeling using a nucleic acid extension reaction has been proposed. This is a technique for labeling a nucleic acid sample by performing a nucleic acid extension reaction using a substrate to which a labeling agent is bound, and incorporating the labeling agent into the generated nucleic acid. A typical example of a labeling agent incorporated into a nucleic acid is a radioisotope. This radioisotope is excellent in sensitivity, but due to the danger inherent in radioactivity, its handling is complicated and its use is limited. For this reason, fluorescent dyes and enzymes are used as labeling agents with low risk (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3).

  However, the above-described fluorescent labeling agent requires excitation light at the time of detection, and fluorescence from other than the labeling agent is generated. Therefore, background noise is increased, and there is a limit to increasing the sensitivity. Also, when an enzyme is used, the enzyme is deactivated during the reaction, and the sensitivity is lowered.

Therefore, a nucleic acid detection method in which electrochemiluminescence is labeled on a substrate has been proposed (Patent Document 4). Electrochemiluminescence does not require excitation light like fluorescent dyes, and light emission occurs when a voltage is applied in the presence of a reducing agent. Therefore, detection with low background noise and high sensitivity can be expected.
JP-A-6-234787 JP 2003-34696 A Special table 2001-519354 gazette JP 2002-34561 A

  As described above, since the labeling agent in which the electrochemiluminescence site is modified on the substrate has low background noise, it can be expected for highly sensitive detection. However, in this labeling agent, the electrochemiluminescent site is charged to a positive charge, and the substrate containing phosphoric acid is charged to a negative charge. Therefore, in the reaction solution, the substrate and the electrochemiluminescent site are electrostatically charged. Adsorbs and forms an intramolecular association. Therefore, when performing nucleic acid amplification, the electrochemiluminescent site that has been electrostatically adsorbed acts as a steric hindrance and not only inhibits the reaction site of the substrate, but also inhibits the enzyme reaction, reducing the amount of modification and detecting with high sensitivity. Had the problem of not being able to.

  The present invention has been made to solve the above problems, and provides a labeling agent having a structure that improves the amount of labeling agent bound to a nucleic acid sample generated by nucleic acid synthesis, and a nucleic acid detection method using the same. For the purpose.

  In order to solve the above problems, the labeling agent of the present invention is a labeling agent represented by the following general formula (Formula 2).

（但し、式中、Ｐはリン酸残基、Ｎはヌクレオシド、Ｌは飽和炭化水素、不飽和炭化水素、アミド、環式化合物の組み合わせから構成されるリンカー部位、リンカー部位、Ｅは電気化学発光部位を示す化合物である。）
また、本発明の核酸検出方法は、標識剤を含む基質が、酵素反応を利用した核酸伸長反応によって核酸合成される核酸合成工程と、合成された核酸サンプルを抽出する核酸抽出工程と、抽出した前記核酸サンプルを電極に固定させる固定化工程と、前記電極に固定させた前記核酸サンプルに含まれる前記標識剤由来の電気化学発光を検出する、検出工程とを含む、核酸検出方法において、前記標識剤が上記（化２）の一般式で表されるものである。

(Wherein, P is a phosphate residue, N is a nucleoside, L is a linker moiety composed of a combination of a saturated hydrocarbon, an unsaturated hydrocarbon, an amide, and a cyclic compound, a linker moiety, and E is electrochemiluminescence. It is a compound showing a site.)
Further, the nucleic acid detection method of the present invention extracts a nucleic acid synthesis step in which a substrate containing a labeling agent synthesizes nucleic acid by a nucleic acid extension reaction utilizing an enzymatic reaction, a nucleic acid extraction step in which a synthesized nucleic acid sample is extracted, In the nucleic acid detection method, comprising: an immobilization step of immobilizing the nucleic acid sample on an electrode; and a detection step of detecting electrochemiluminescence derived from the labeling agent contained in the nucleic acid sample immobilized on the electrode. The agent is represented by the general formula (Formula 2).

  Furthermore, the nucleic acid detection method of the present invention includes a nucleic acid synthesis step in which a substrate containing a labeling agent is synthesized by a nucleic acid extension reaction utilizing an enzymatic reaction, a nucleic acid extraction step of extracting a synthesized nucleic acid sample, and the detection A single-stranded capture probe having a sequence complementary to the base sequence of the nucleic acid to be prepared is prepared, the immobilization step of immobilizing the capture probe on a solid phase, and the nucleic acid sample and the capture probe are hybridized. A nucleic acid sample capturing step of allowing soybean to form a double strand and capturing the nucleic acid sample on the solid phase; and a detection step of detecting electrochemiluminescence derived from the labeling agent contained in the nucleic acid sample. In the nucleic acid detection method, the labeling agent has a general formula represented by the above (Chemical Formula 2).

  According to the labeling agent of the present invention, the linker that binds the electrochemiluminescence site and the substrate has a rigid structure, so that bending does not occur and intramolecular association between the substrate and the electrochemiluminescence site is suppressed by electrostatic adsorption. And does not inhibit the reaction between the substrates in nucleic acid synthesis. Also, steric hindrance with the enzyme is alleviated, the amount of labeling agent modification to the generated nucleic acid sample is increased, and highly sensitive detection is possible.

  Below, the labeling agent of this invention and its usage method are demonstrated in detail.

(Embodiment 1)
Hereinafter, the labeling agent in Embodiment 1 will be described. The labeling agent in the present embodiment is represented by the following general formula (Formula 3).

（但し、式中、Ｐはリン酸残基、Ｎはヌクレオシド、Ｌは飽和炭化水素、不飽和炭化水素、アミド、環式化合物の組み合わせから構成されるリンカー部位、Ｅは電気化学発光部位を示す化合物である。）
このように、リンカー部位に不飽和炭化水素及び環式化合物を持たせたことにより、リンカー部位が剛直な構造となるため、屈曲しなくなり、基質と電気化学発光部位との静電吸着による分子内会合が抑制され、電気化学発光部位の立体障害による反応阻害がなくなる。

(Wherein, P is a phosphate residue, N is a nucleoside, L is a linker moiety composed of a combination of a saturated hydrocarbon, an unsaturated hydrocarbon, an amide, and a cyclic compound, and E is an electrochemiluminescence site. Compound.)
As described above, since the unsaturated hydrocarbon and the cyclic compound are provided in the linker portion, the linker portion has a rigid structure, so that the linker portion does not bend and the intramolecular due to electrostatic adsorption between the substrate and the electrochemiluminescent portion. Association is suppressed, and reaction inhibition due to steric hindrance at the electrochemiluminescence site is eliminated.

  Further, the linker is an integer of 4 to 50. This is because when the linker length is 3 or less, when the labeling agent of the present invention is introduced into the nucleic acid, the distance between the substrate and the light emitting site is close, so that the electrochemiluminescent site becomes sterically hindered and inhibits the nucleic acid reaction. The linker itself becomes a target of steric hindrance and inhibits the nucleic acid reaction.

  Further, the site where the nucleoside and the linker are bonded is not particularly limited. However, in the case of adenine and guanine, it is desirable that the linker is bonded to the linker at the C7 or C8 site, more preferably at the C8 site. When the nucleoside is cytosine, it is desirable to bind to the linker at the C5 or C6 site, more preferably at the C5 site. When the nucleoside is thymine and uracil, it is desirable that the linker is bonded to the linker at the C5 or C6 site, more preferably at the C6 site. This is because the nucleic acid sample does not inhibit the hydrogen bonding necessary to form a double-stranded nucleic acid.

  E, which is an electrochemiluminescent site, is not particularly limited as long as it is an electrochemiluminescent substance. For example, a metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, fluoranthene, Examples include chrysene, phenanthrene, perylene, binaphthyl, and octatetraene.

  Furthermore, examples of the metal complex having a heterocyclic compound in the aforementioned ligand include a heterocyclic compound containing oxygen, nitrogen, etc., for example, a metal complex having a pyridine moiety, a pyran moiety, etc. A metal complex having a pyridine moiety as a ligand is preferred.

  Furthermore, examples of the metal complex having the above-mentioned pyridine moiety as a ligand include a metal bipyridine complex and a metal phenanthroline complex.

  Furthermore, in the metal complex having a heterocyclic compound as the ligand, as the central metal, for example, ruthenium, osnium, zinc, cobalt, platinum, chromium, molybdenum, tungsten, technetium, rhenium, rhodium, iridium, palladium, Examples thereof include copper, indium, lanthanum, praseodymium, neodymium, and samarium.

  In particular, a complex in which the central metal is ruthenium or osmium has good electrochemiluminescence properties, and examples of the material having such good electrochemiluminescence properties include ruthenium bipyridine complexes, ruthenium phenanthroline complexes, and osmium. Bipyridine complex, osnium phenanthroline complex, etc. can be mentioned.

  Specific examples of the labeling agent of the first embodiment described above are shown in the following (Chemical Formula 4) to (Chemical Formula 8).

（実施の形態２）
以下に、実施の形態１で説明した標識剤を用いた核酸検出方法について詳細に説明する。なお、ここでは、例としてＤＮＡポリメラーゼを用いた核酸増幅について記述するが、これに限定されない。

(Embodiment 2)
The nucleic acid detection method using the labeling agent described in Embodiment 1 will be described in detail below. Here, nucleic acid amplification using DNA polymerase is described as an example, but the present invention is not limited to this.

  In addition, the nucleic acid sample in the following embodiment is, for example, blood, leukocyte, serum, urine, feces, semen, saliva, cultured cells, tissue cells such as various organ cells, and any sample containing other genes , Cells in the sample are destroyed to release double-stranded nucleic acids. In addition, the nucleic acid sample in the present embodiment may be a nucleic acid fragment that is cut with a restriction enzyme and purified by separation by electrophoresis or the like.

(Step 1)
First, the above-described nucleic acid sample, an arbitrary primer, a buffer adjusted to an arbitrary pH, a polymerase, dNTP, and a labeling agent of the present invention are added to a sample tube.

(Step 2)
Next, the sample described above is heated to 98 ° C. for several minutes to denature the nucleic acid sample into single strands. Thereafter, the primer and the nucleic acid sample are hybridized at an arbitrary temperature, and the nucleic acid sample is amplified from the 3 ′ end with a polymerase. After amplification, the nucleic acid sample is again denatured into single strands by applying heat at 98 degrees for several minutes. By repeating this operation several times, the labeling agent-bound nucleic acid sample can be amplified. This operation varies depending on the nucleic acid used and the purpose. Further, as shown in (Embodiment 1), the labeling agent has a structure in which the linker site is rigid. Therefore, the labeling agent is incorporated into the enzyme without inhibiting the nucleic acid elongation reaction between the substrates, and is added to the nucleic acid sample. Increased amount of labeling agent introduced.

(Step 3)
The labeling agent-bound nucleic acid sample amplified by the operation described above is extracted. In the extraction method, only the labeling agent-bound nucleic acid sample is precipitated with ethanol, acetonitrile or the like, centrifuged, and then the supernatant is removed. This labeling agent-binding nucleic acid sample can be obtained by repeating this operation a few times and finally substituting it with sterilized water or the like. Other methods include means for purification by HPLC or fractionation by gel filtration. Moreover, it can extract rapidly using a commercially available DNA extraction kit.

(Step 4)
Next, the labeling agent-bound nucleic acid sample is immobilized on the electrode. The electrode used in the present invention is not particularly limited, and examples thereof include noble metals such as gold, platinum, platinum black, palladium, and rhodium, carbides such as graphite, glassy carbon, pyrolytic graphite, carbon paste, and carbon fiber. And oxides such as titanium oxide, tin oxide, manganese oxide and lead oxide, and semiconductors such as Si, Ge, ZnO, CdS, TiO and GaAs.

  A labeling agent-bound nucleic acid sample solution is dropped onto this electrode and fixed. The immobilization method is not particularly limited, a method of drying on the electrode, a method of applying a cationic or anionic resin on the electrode and electrostatically adsorbing it, or making the electrode have a positive or negative charge And a method of electrical suction.

(Step 5)
As a result, electrochemiluminescence derived from a plurality of labeling agents contained in the labeling agent-binding nucleic acid sample immobilized on the electrode is measured with a photomultiplier or the like.

  The presence of the target nucleic acid can be detected with high sensitivity by using the labeling agent of the present invention improved so that the efficiency of introduction into a nucleic acid sample is improved.

  In addition, although the PCR technique has been described here, as another technique, a terminal transferase enzyme is used, and when the 3 ′ end of a single-stranded or double-stranded nucleic acid is extended, the present labeling agent is introduced, Random primer method that introduces this labeling agent by DNA polymerase using oligonucleotide consisting of any combination of bases as a primer, this labeling agent is introduced when DNA sample is randomly cleaved by DNase I and repaired by DNA polymerase In addition, the present invention can be applied to the nick translation method in which the present labeling agent is introduced when cDNA is synthesized from RNA using reverse transcriptase and a gene sample is amplified using the cDNA as a template.

(Embodiment 3)
Hereinafter, embodiments of the nucleic acid detection method of the present invention will be described in detail. Here, nucleic acid amplification using DNA polymerase is described as an example, but the present invention is not limited to this.

(Step 1)
E. coli cultured in a liquid medium is collected with a centrifuge, and then a proteolytic solution is added and homogenized. Thereafter, phenol / chloroform extraction is performed to extract DNA and RNA. RNA can be extracted easily and with high yield by using a commercially available RNA extraction kit.

(Step 2)
Next, the RNA of the nucleic acid sample described above, reverse transcriptase, any primer, buffer adjusted to any pH, polymerase (preferably derived from thermophile), dNTP, and the labeling agent of the present invention are added to the sample tube. . Labeling agent-bound cDNA can be obtained by incubating at an arbitrary temperature for an arbitrary time.

  As shown in (Embodiment 1), since the labeling agent has a rigid linker site, the labeling agent is incorporated into the reverse transcriptase without inhibiting the nucleic acid reaction with the substrate, and labeled on the nucleic acid sample. The amount of agent introduced increases.

(Step 3)
Labeling agent-bound cDNA is extracted. Since details are described in (Embodiment 2), they are omitted here.

(Step 4)
Next, a capture probe having a nucleic acid sequence complementary to the labeling agent-bound cDNA is prepared. As the capture probe, a single-stranded nucleic acid obtained by chemical synthesis or a nucleic acid obtained by cleaving a nucleic acid extracted from a biological sample with a restriction enzyme and separating it by electrophoresis or the like can be used. In the case of nucleic acid extracted from a biological sample, it is preferable to dissociate into single-stranded nucleic acid by heat treatment or alkali treatment.

(Step 5)
Thereafter, the capture probe obtained as described above is immobilized on a solid phase. The solid phase used in the present invention is not particularly limited, and examples thereof include noble metals such as gold, platinum, platinum black, palladium, and rhodium, and carbides such as graphite, glassy carbon, pyrolytic graphite, carbon paste, and carbon fiber. And oxides such as titanium oxide, tin oxide, manganese oxide, and lead oxide, and semiconductors such as Si, Ge, ZnO, CdS, TiO, and GaAs. The above substances can be used as electrodes. In that case, these electrodes may be coated with a conductive polymer, and by coating in this way, a more stable capture probe-immobilized electrode can be prepared.

  A known method is used as a method for immobilizing the capture probe on the solid phase. For example, when the solid phase is a gold electrode, for example, a thiol group is introduced at the 5′- or 3′-end (preferably 5′-end) of the capture probe to be immobilized. The capture probe is fixed to the gold electrode via a covalent bond. Methods for introducing thiol groups into this capture probe are described in the literature (M. Maeda et al., Chem. Lett., 1805-1808 (1994) and BA Connolly, Nucleic Acids Res., 13, 4484 (1985). ) Are listed.

  That is, the capture probe having a thiol group obtained by the above method is dropped on a gold electrode and left at a low temperature for several hours, whereby the capture probe is fixed to the electrode and a capture probe is produced.

  Another example of the solid phase is a magnetic particle generally called a magnetic bead. In the case of magnetic beads, the capture probe can be immobilized by avidin-biotin binding technique. First, avidin is coated on the surface of magnetic beads. On the other hand, biotin is bound to the end of the capture probe. By adding this capture probe to the magnetic beads, an antigen-antibody reaction occurs, and the capture probe can be bound to the magnetic beads. Since this method is well known, details are omitted.

(Step 6)
A solution containing the labeling agent-bound cDNA is added to the capture probe immobilized on the solid phase. As a result, the capture probe and the labeling agent-bound cDNA are hybridized, and the labeling agent-bound cDNA is immobilized on a solid phase. Since this hybridization method is well known, the description thereof is omitted here.

(Step 7)
A double-stranded DNA is formed by the capture probe and the labeling agent-bound cDNA, and then washed with a phosphate buffer or the like to remove the unreacted labeling agent-bound cDNA.

(Step 8)
After washing, the presence of the target nucleic acid can be detected with high sensitivity by measuring the electrochemiluminescence derived from the labeling agent-bound nucleic acid sample immobilized on the solid phase with a photomultiplier or the like. This is because the labeling agent of the present invention improved so as to improve the introduction efficiency into the nucleic acid sample was used during reverse transcription.

  Here, a technique for directly detecting a labeling agent-bound nucleic acid sample obtained by reverse transcription has been described. However, as another technique, a single-stranded or double-stranded nucleic acid using a PCR method or a terminal transferase enzyme is used. When extending the 3 ′ end of DNA, a method for introducing the labeling agent, a random primer method in which the oligonucleotide is composed of any combination of bases as a primer, and the labeling agent is introduced by DNA polymerase, and a gene sample is DNase Using nick translation method that introduces this labeling agent when randomly cleaved by I and repaired by DNA polymerase, or using reverse transcriptase, cDNA is synthesized from RNA, and gene sample is amplified using the cDNA as a template The method to introduce this labeling agent can be developed when That.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

(1) Synthesis of guanine-modified ruthenium complex A labeling agent represented by the following (Chemical 9) was obtained as follows.

まず、ＴＨＦ６０．０ｍLに溶解させた４，４’−ジメチル−２，２’ビピリジン２．５０ｇ（１３．５ｍｍｏｌ）溶液を窒素雰囲気の容器に注入した後、リチウムジイソプロピルアミド２Ｍ溶液１６．９ｍＬ（２７．０ｍｍｏｌ）を滴下し、３０分撹拌した。一方、同様に窒素気流中で乾燥させた容器に、１，３−ジブロモプロパン４．２ｍＬ（４１．１ｍｍｏｌ）とＴＨＦ１０ｍＬとを加え、撹拌させた。この容器に、先程の反応液を３０分かけて滴下させて２．５時間反応させた。反応溶液は２Ｎの塩酸で中和し、ＴＨＦを留去した後、クロロホルムで抽出した。溶媒を留去して得た粗生成物をシリカゲルカラムで精製し、生成物Ｃを得た（収率４７％）。

First, a solution of 2.50 g (13.5 mmol) of 4,4′-dimethyl-2,2′bipyridine dissolved in 60.0 mL of THF was poured into a container in a nitrogen atmosphere, and then 16.9 mL (27 of lithium diisopropylamide 2M solution) (27 0.0 mmol) was added dropwise and stirred for 30 minutes. On the other hand, 4.2 mL (41.1 mmol) of 1,3-dibromopropane and 10 mL of THF were added to a container that was similarly dried in a nitrogen stream and stirred. The previous reaction solution was dropped into this vessel over 30 minutes and allowed to react for 2.5 hours. The reaction solution was neutralized with 2N hydrochloric acid, and THF was distilled off, followed by extraction with chloroform. The crude product obtained by distilling off the solvent was purified with a silica gel column to obtain the product C (yield 47%).

  To a container in a nitrogen atmosphere, 1.0 g (3.28 mmol) of the product C, 0.67 g (3.61 mmol) of potassium phthalimide, and 30.0 mL of dimethylformamide (dehydrated) were added and refluxed in an oil bath for 18 hours. After the reaction, the mixture was extracted with chloroform and washed with distilled water with 50 mL of 0.2N sodium hydroxide. The solvent was distilled off and recrystallization was performed from ethyl acetate and hexane to obtain the product D (yield 61.5%).

  Ruthenium (III) chloride (2.98 g, 0.01 mol) and 2,2′-bipyridine (3.44 g, 0.022 mol) were refluxed in dimethylformamide (80.0 mL) for 6 hours, and then the solvent was distilled off. Left. Then, acetone was added and the black deposit obtained by cooling for 8 hours was extract | collected, 170 mL of ethanol aqueous solution (ethanol: water = 1: 1) was added, and it heated and refluxed for 1 hour. After filtration, 20 g of lithium chloride was added, ethanol was distilled off, and the mixture was further cooled at 4 ° C. for 8 hours. The deposited black substance was collected by suction filtration to obtain a product E (yield 68.2%).

  To the container purged with nitrogen, 0.50 g (1.35 mmol) of the product D, 0.78 g (1.61 mmol) of the product E, and 50 mL of ethanol were added. After refluxing in a nitrogen atmosphere for 9 hours, the solvent was distilled off, dissolved in distilled water, and precipitated with a 1.0 M aqueous perchloric acid solution. This precipitate was collected and recrystallized from methanol to obtain a product F (yield 81.6%).

  Further, 1.0 g (1.02 mmol) of the product F and 70.0 mL of methanol were refluxed for 1 hour. After cooling to room temperature, 0.21 mL (4.21 mmol) of hydrazine monohydrate was added and refluxed again for 13 hours. After the reaction, 15 mL of distilled water was added and methanol was distilled off.

  Next, 5.0 mL of concentrated hydrochloric acid was added, and the reaction solution obtained by refluxing for 2 hours was cooled at 4 ° C. for 8 hours, and impurities were removed by natural filtration.

  After neutralizing this with sodium hydrogen carbonate, water was distilled off and inorganic substances were removed with acetonitrile. The crude product obtained by distilling off the solvent was purified with a silica gel column to obtain the product G (yield 71.4%).

  0.28 g (0.33 mmol) of product G was dissolved in acetonitrile, and 96.8 mg (0.96 mmol) of triethylamine was added. Next, a solution obtained by dissolving 0.32 g (3.3 mmol) of succinic anhydride in 5 mL of acetonitrile was added dropwise to the solution of the product G and stirred at room temperature for 2 hours. Acetonitrile was distilled off and purified by a silica gel column to obtain the product H (yield 63.4%).

  0.20 g (0.22 mmol) of the product H was dissolved in 10 mL of acetonitrile, 0.43 g (4.26 mmol) of triethylamine, 0.30 g (2.13 mmol) of 1,4-cyclohexanebis (methylamine), 0.23 g of dicyclohexylcarbodiimide. 13 g (0.63 mmol) was added and stirred at room temperature for 48 hours.

  After the reaction, the white precipitate was removed, concentrated with an evaporator, and then purified with a silica gel column to obtain the product I shown below (Chemical Formula 10) (yield 57.4%).

下記の表１は、前述のようにして得た生成物Ｉに示す物質の^１Ｈ-ＮＭＲ結果である。

Table 1 below shows ¹ H-NMR results of the substance shown in the product I obtained as described above.

¹ H-NMR (300 MHz, DMSO d-6)
σ:
1.40 (8H, m)
1.55 (2H, m)
1.62-1.67 (3H, m)
2.02 to 2.06 (3H, m)
2.56 (3H, s)
2.61 (2H, q)
2.75 (2H, t)
2.92 (2H, d)
3.06 (2H, q)
7.36 (2H, d)
7.42 (2H, d)
7.56 (6H, m)
7.78 (4H, m)
8.12 (2H, t)
8.76 (4H, t)
8.82 (6H, m)
Next, 30.0 mg (52.3 μmol) of 2′-deoxyguanosine-5′-triphosphate was dissolved in 9.0 mL of carbonate buffer, and 93.2 mg (0.52 mmol) of N-bromosuccinimide was added thereto. And stirred at room temperature for 1 hour. Thereafter, 13.8 mg (13.1 μmol) of Product I was added dropwise and stirred at 50 ° C. for 6 hours. The reaction solution was purified by HPLC to obtain the compound represented by the above (Chemical 9) (yield 26.8%).

  Hereinafter, other examples of the present invention will be shown, but the present invention is not limited thereto.

(1) Nucleic Acid Amplification First, an oral swab sample was purified using a Qiaamp DNA Mini Kit. The nucleic acid sample used here is chromosome 6 and its base sequence is as follows.

5'-TAACATTCAGAAGTGTTCTCAGTGATAGTTCTGTTCTCAGATTTGCTTTCTCTCCTTATACCTCCTGCATGCAGCGGTGGATGCATGG
Moreover, the sequences of the primers are 5'-CACTGGAACACATCTGAATGTTA-3 '(primer 1) and 5'-CAGAGGCTCTAACTGCTCTAACTG-3' (primer 2).

  To 2 μL of this nucleic acid sample, 5 × L of 10 × EX Taq buffer, 5 μL of 25 mM magnesium chloride solution, 4.2 μL of 2.5 mM dNTP solution, 2.5 μL of 10 μM primer, 30.2 μL of distilled water, 0.5 μL of EX Taq (5 U / μL) And 0.6 microliters of labeling agents (2.5 mM) synthesize | combined in Example 1 were added to the sample tube. This sample was heated as described below to amplify the nucleic acid.

  First, using a thermal cycler, heating at 98 ° C. for 5 minutes to denature to single strand, followed by primer binding for 30 seconds at 50 ° C., extension at 72 ° C. for 3 minutes, and denaturation again at 98 ° C. for 10 seconds 30 cycles Repeated times.

  After amplification, a labeling agent-bound nucleic acid sample was extracted with a PCR product purification kit (MinElute PCR Purification Kit) manufactured by Qiagen.

(2) Immobilization of capture probe on solid phase Magnetic beads were used for the solid phase. The magnetic beads used were CM01N / 5896 streptavidin magnetic beads manufactured by Bangs Laboratories (particle size: 0.35 μm). As the capture probe, a probe having the sequence of the primer 2 and modified with biotin via a phosphate group at the 5 ′ end was used.

  First, 1 mg of magnetic beads were sampled and washed with TTL buffer (500 mM Tris-HCl (pH 8.0): Tween 20: 2 M lithium chloride: ultrapure water = adjusted to a volume ratio of 2: 10: 5: 3). , 20 μL of TTL buffer. Thereafter, 5 μL of 100 nM supplemental probe was added and shaken at room temperature for 15 minutes.

  The solution was decanted, and the remaining magnetic beads were washed with 0.15 M aqueous sodium hydroxide solution, and then the volume ratio of TT buffer (500 mM Tris-HCl (pH 8.0): Tween 20: ultrapure water = 1: 2: 1). Washed to prepare).

After washing, the solution was replaced with TTE buffer and left at 80 ° C. for 10 minutes to remove unstable bonds. Thereby, a magnetic bead having a capture probe immobilized thereon was obtained.

Furthermore, in Example 2, as a comparison target, a capture probe having a sequence that is non-complementary to the nucleic acid sample (hereinafter referred to as “control probe”) is used, and the same treatment as that of the nucleic acid probe is performed. It was. Here, as a control probe, a probe modified with biotin via a phosphate group at the 5 ′ end having the sequence of 30-mer Poly-A, 5′-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 ′ was used.

(3) Hybridization First, 10 μL of the magnetic beads on which the capture probe was immobilized were transferred to a microtube and replaced with 4XSSC buffer.

  Next, the labeling agent-binding nucleic acid sample described above was heated at 98 ° C. for 5 minutes, and then cooled with ice water. 10 μL of this nucleic acid sample was collected, added to the microtube, and shaken at 60 ° C. After 1 hour, the solution was decanted and washed with 2XSSC to obtain magnetic beads α on which double-stranded nucleic acids were formed.

  The magnetic beads having the control probe immobilized thereon were also subjected to the same treatment as described above to obtain magnetic beads β in which no double-stranded nucleic acid was formed.

(3) Electrochemical measurement After the above steps, 5 μL each of magnetic beads α on which double-stranded nucleic acids were formed and magnetic beads β on which double-stranded nucleic acids were not formed were dropped onto the electrodes.

  The electrode used here was a gold electrode. This gold electrode was prepared by forming a gold substrate with a thickness of 10 nm on a glass substrate using a sputtering apparatus (SH-350, ULVAC, Inc.) and forming an electrode pattern by a photolithography process. In addition, a permanent magnet sheet is attached under the electrode so that the magnetic beads are concentrated only on the working electrode.

  After standing for 5 minutes, 75 μL of an electrolyte solution in which 0.1 M PBS and 0.1 M triethylamine were mixed was dropped onto the electrode x aggregated with the magnetic beads α and the electrode y aggregated with the magnetic beads β. . Thereafter, a voltage was applied to the electrode where each magnetic bead was gathered, and the electrochemiluminescence generated at this time was measured. The voltage was applied at 1.3 V, and electrochemical measurement was performed for 5 seconds. The electrochemiluminescence amount was measured using a photomultiplier tube (H7360-01 manufactured by Hamamatsu Photonics), and the maximum luminescence amount was measured.

  In addition, as a comparison target, a conventional labeling agent having a flexible linker site is prepared, and the above-described steps are performed in the same manner, and the magnetic bead electrode x2 in which the double-stranded nucleic acid is formed and the double-stranded nucleic acid is not formed. A magnetic bead electrode y2 was obtained. In addition, since the synthesis | combination of the conventional labeling agent is well-known, it abbreviate | omits.

  FIG. 1 shows the maximum amount of electrochemiluminescence detected in the magnetic bead electrode x in which double-stranded nucleic acid was formed and the magnetic bead electrode y in which double-stranded nucleic acid was not formed in Example 2. .

  FIG. 2 shows the maximum electrochemical detected in the magnetic bead electrode x2 in which the double-stranded nucleic acid was formed and the magnetic bead electrode y2 in which the double-stranded nucleic acid was not formed, using the conventional labeling agent in Example 2. It shows the amount of luminescence.

  As is clear from FIG. 1, the amount of light emitted from the electrode x is significantly higher than the amount of light emitted from the electrode y. Further, compared with FIG. 2, the labeling agent of the present invention was used. The technique shows that the amount of light introduced from the nucleic acid product is larger than that of the conventional labeling agent, so that the amount introduced into the nucleic acid sample is large. Thus, by using the method of the present invention, a double-stranded nucleic acid, that is, a target gene sample can be detected with high sensitivity.

  The labeling agent and the method for using the same according to the present invention can detect a nucleic acid having a specific sequence with high sensitivity, and can be applied to uses such as genetic diagnosis, infectious disease diagnosis, and genomic drug discovery.

The figure which measured the maximum amount of electrochemiluminescence in Example 2 of this invention The figure which measured the maximum electrochemiluminescence amount of the conventional labeling agent in Example 2 of this invention

Claims (14)

A labeling agent represented by the following general formula (Formula 1).

(Wherein, P is a phosphate residue, N is a nucleoside, L is a linker moiety composed of a combination of a saturated hydrocarbon, an unsaturated hydrocarbon, an amide, and a cyclic compound, and E is an electrochemiluminescence site. Compound.) The labeling agent according to claim 1,
M is an integer of 4 to 50.
A labeling agent characterized by the above. The labeling agent according to claim 1,
The electrochemiluminescence site E is a metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, fluoranthene, chrysene, phenanthrene, perylene, binaphthyl, or octatetraene,
A labeling agent characterized by the above. The labeling agent according to claim 3,
The metal complex having a heterocyclic compound in the ligand is a metal complex having a pyridine moiety in the ligand.
A labeling agent characterized by the above. The labeling agent according to claim 4,
The metal complex having a pyridine moiety in the ligand is either a metal bipyridine complex or a metal phenanthroline complex.
A labeling agent characterized by the above. The labeling agent according to claim 5,
The central metal of the metal complex having a heterocyclic compound as the ligand is ruthenium, osnium, rhenium, or iridium.
A labeling agent characterized by the above. A nucleic acid synthesis step for nucleic acid synthesis by a nucleic acid extension reaction using a substrate containing a labeling agent, a nucleic acid extraction step for extracting the synthesized nucleic acid sample, an immobilization step for fixing the extracted nucleic acid sample to an electrode, A detection step of detecting electrochemiluminescence derived from the labeling agent contained in the nucleic acid sample immobilized on an electrode, and a nucleic acid detection method comprising:
As the labeling agent, one represented by the general formula of (Chemical Formula 1) is used.
A method for detecting a nucleic acid. A nucleic acid synthesis step for nucleic acid synthesis by a nucleic acid extension reaction using a substrate containing a labeling agent, a nucleic acid extraction step for extracting a synthesized nucleic acid sample, and a sequence complementary to the base sequence of the nucleic acid to be detected A single-stranded capture probe, and immobilizing the capture probe on a solid phase, and hybridizing the nucleic acid sample and the capture probe to form a double strand, thereby forming the nucleic acid In a nucleic acid detection method comprising a nucleic acid sample capturing step of capturing a sample on the solid phase, and a detection step of detecting electrochemiluminescence derived from the labeling agent contained in the nucleic acid sample,
As the labeling agent, one represented by the general formula of (Chemical Formula 1) is used.
A method for detecting a nucleic acid. In the nucleic acid detection method according to claim 7 and 8,
M is an integer of 4 to 50.
A method for detecting a nucleic acid. In the nucleic acid detection method according to claim 7 and 8,
The electrochemiluminescence site E is a metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, fluoranthene, chrysene, phenanthrene, perylene, binaphthyl, or octatetraene,
A method for detecting a nucleic acid. The method for detecting a nucleic acid according to claim 10,
The metal complex having a heterocyclic compound in the ligand is a metal complex having a pyridine moiety in the ligand.
A method for detecting a nucleic acid. The method of detecting a nucleic acid according to claim 11,
The metal complex having a pyridine moiety in the ligand is either a metal bipyridine complex or a metal phenanthroline complex.
A method for detecting a nucleic acid. The nucleic acid detection method according to claim 12,
The central metal of the metal complex having a heterocyclic compound as the ligand is ruthenium, osnium, rhenium, or iridium.
A method for detecting a nucleic acid. In the nucleic acid detection method according to claim 7 and 8,
A nucleic acid detection method, wherein the nucleic acid synthesis includes any one of a PCR reaction, a reverse transcription reaction, a ligase chain reaction, a TMA method, a terminal transferase reaction, a random prime method, and a nick translation method.

Images