JPH0675519B2 - Nucleic acid probe, method for producing the same, and detection method using the nucleic acid probe - Google Patents

Description

TECHNICAL FIELD The present invention relates to a nucleic acid probe having a ruthenium bis (bipyridyl) ²⁺ ion coordinately bonded, a method for producing the same, and a method for detecting DNA using the nucleic acid probe. .

[Conventional technology]

With the remarkable progress of molecular biology, it is becoming more and more important to determine the existence and position of a specific nucleotide sequence in a cell or virus gene.

In order to determine the presence and position of this specific base sequence, a single-stranded nucleic acid having a target sequence is prepared in advance and labeled with some method (labeled nucleic acid; called nucleic acid probe) It has been performed to bind to a homologous sequence portion by utilizing the complementarity (hybridization) of the.

And this technology has been indispensable for obtaining a target gene in the field of genetic engineering, and recently, also in the fields of medicine and agriculture, genetic diseases, pathogenic bacteria, and viruses have been developed. It has been widely used for analysis and so on, and it is expected to be one of the innovative technologies along with monoclonal antibodies.

By the way, nucleic acid probes include deoxyribonucleic acid probe (DNA probe) and ribonucleic acid probe (RNA probe).
However, since the former is mainly used, the DNA probe will be described below. Conventional DNA probes are ³ H and ³² P
A nucleic acid is labeled with a radioisotope (hereinafter abbreviated as RI) as described above, and a method of detecting by autoradiography is adopted, which is an excellent method widely used at the laboratory level.

However, this method has an extremely large drawback in that RI must be handled, especially when considering its wide use as described above. That is, there are problems such as restrictions on the place of use, qualification of the user, and the life of the DNA probe.

Therefore, recently, attention has been paid to the development of DNA probes that do not use RI, and a typical example is a biotinylated probe.

This is designed by designing a color reaction by an enzyme labeled with biotin and utilizing a specific binding force between biotin and avidin on avidin to which the enzyme is bound in advance.

[Problems to be Solved by the Invention]

The biotinylated probe is stable and sensitive, and is an excellent probe, but the drawback is that avidin and alkaline phosphatase used in the color reaction are proteins, which makes them expensive and difficult to store. , And biotin originally present in the cell interferes with the detection.

Various studies have been made on these drawbacks and attempts have been made to avoid them, but the preparation of probes, detection, storage of reagents, etc. are not entirely satisfactory.

The present invention, without the use of RI or enzyme as in the above conventional,
DNA probe applying the photocatalytic action of ruthenium complex,
Manufacturing method and ruthenium tris (bipyridyl) ²⁺
It is an object of the present invention to provide a method for detecting ions.

[Means for Solving the Problems]

The nucleic acid probe of the present invention for achieving the above object is characterized in that a ruthenium bis (bipyridyl) ²⁺ ion is coordinate-bonded to the base of a nucleic acid.

Further, the method for producing a nucleic acid probe of the present invention is characterized in that the one-stranded nucleic acid having a base sequence is dissolved in water-organic mixed solvent, and ruthenium bis (bipyridyl) ²⁺ salt is added to the solution and then refluxed. To do.

Furthermore, the present invention also relates to a method for detecting the presence or absence of a base sequence of interest using the nucleic acid probe. The method for detecting coenzymes is that the analyzed DNA sequence in a single-stranded form is hybridized with a nucleic acid probe for coenzyme, and the product is reacted with an excess of bipyridyl to produce ruthenium tris (bipyridyl) ^2+. The ions are irradiated with light in the presence of a color reagent and a reducing agent to generate ruthenium tris (bipyridyl) ³⁺ ions through excited ruthenium tris (bipyridyl) ²⁺ ions and reduce the color reagent.
Ruthenium tris (bipyridyl) produced by coloring
^{The 3+} ion is converted to ruthenium tris (bipyridyl) ³⁺ ion by the reducing agent. The method for detecting ruthenium bis (bipyridyl) ²⁺ ion, which is the base of the DNA probe of the present invention, comprises ruthenium tris (bipyridyl) ²⁺ ion irradiated with light in the presence of a color reagent and a reducing agent to produce ruthenium tris (bipyridyl). ) ³⁺ ions are generated and the coloring reagent is reduced and colored, and the generated ruthenium tris (bipyridyl) ³⁺ ions are returned to ruthenium tris (bipyridyl) ²⁺ ions by the reducing agent. It is a thing.

The present invention is based on the following facts found by the present inventors.

(1) Ruthenium tris (bipyridyl) ²⁺ ion [Ru (b
ipy) ₂ ²⁺ ] with DNA is refluxed in a water-organic mixed solvent such as ethanol to give ruthenium bis (bipyridyl).
A DNA-probe of the invention is obtained in which the ²⁺ ion is coordinated to a purine or pyrimidine residue of DNA.

(2) This DNA-probe
y) and refluxed in ethanol, Ru (bipy) ₂ ²⁺
Dissociates from the DNA and ruthenium tris (bipyridyl) ²⁺
[Ru (bipy) ₃ ²⁺ ] is formed.

(3) This Ru (bipy) ₃ ²⁺ , under light irradiation, catalytically reduces a color reagent, for example, a tetrazonium salt, by a reaction cycle represented by the following formula (1) to cause color.

Here, TEA is a reducing agent of Ru (bipy) ₃ ³⁺ , for example, triethanolamine, and NBT is nitroblue tetrazonium represented by the following formula.

What is important in this cycle is the photocatalytic reaction of Ru (bipy) ₃ ²⁺ of (3), and the reduction of NBT ²⁺ is enhanced. That is, Ru (bipy) ₃ ²⁺ is excited by light irradiation to become [Ru (bipy) ₃ ²⁺ ] ^* and reduce NBT ²⁺ to NBT, but at the same time Ru (bipy) ₃ ³⁺ produced by TEA Ru (bipy) ₃ again
^{It is} to return to ²⁺ and reduce NBT ²⁺ again by light irradiation. This reaction cycle is repeated many times by irradiation with light, and even a trace amount of Ru (bipy) ₃ ²⁺ can be sensitively colored.

Next, a method for producing the DNA probe of the present invention and a detection method using this probe will be described.

First, about 50 mer one-stranded DNA having the target nucleotide sequence is dissolved in an ethanolic solvent such as water / ethanol (1/1) solvent, and Ru (bipy) ₂ ²⁺ Cl ₂ is added to the number of moles of DNA. 1
Add ~ 2 times amount.

Then, the mixture is refluxed at 100 ° C. for 1 hour to obtain a DNA probe. This probe can be stored frozen even in a solution state, but can be stored more stably if it is stored frozen without solvent.

Hybridization of DNA is performed by known methods, for example, T. Maniatis et al., Molecular Cloning, Cold Spring Ha.
It is performed according to the method of rbor Laboratory, 1982. That is, the DNA to be analyzed is denatured with alkali to form a single strand, and immobilized on a nitrocellulose membrane.

Then, pre-hybridization and hybridization are performed by a predetermined method, and washing is performed.

After the hybridized membrane is dried, it is immersed in ethanol containing excess bipyridyl and refluxed for 1 hour. As a result, Ru (bipy) ₂ ²⁺ is dissociated from the DNA sample hybridized with the DNA probe, and Ru (bipy) ₂
py) ₃ ²⁺ is generated. Finally, this ethanol is collected, NBT ²⁺ as a coloring reagent and TEA as a reducing agent are added, and it is irradiated with a xenon lamp for about 1 hour to give a purple color.

In the above, the case where NBT ²⁺ is used as the color reagent has been described, but the present invention is not limited to this, and any compound can be used as long as it is a compound that is colored by reduction. can do.

For example, the following tetrazolium salts MTT, TNBT, dimethyl viologen, etc. may be mentioned.

3- (4 ', 5'-dimethylthiazol-2-yl) -2,4-diphenyltetrazolium 2,2 ', 5', 5'-Tetra (p-nitrophenyl) -3,3 '-(3-dimethoxy-4-diphenylene) ditetrazolium Also, the reducing agent for Ru (bipy) ₃ ³⁺ is TEA. Without limitation, Ru (bip
y) ₃ ³⁺ can be reduced, as long as it does not reduce the coloring reagent, it may be used of any type. For example,
Includes ethylenediaminetetraacetic acid and cysteine.

Since the absorption of Ru (bipy) ₃ ²⁺ at 455 nm, the irradiation light is preferably and effectively adjusted to this.

In addition, since the color reagent tetrazolim salt also has photosensitivity,
It is desirable to use a filter that cuts 430 to 440 nm on the short wavelength side.

The DNA probe has been described above, but the RNA probe is exactly the same. However, as a practical matter, RNA is difficult to synthesize, so only natural ones are used.

Examples of the present invention will be described below.

〔Example〕

DNA probe with 69 mer synthetic oligonucleotide,
It was confirmed that this method could be used for selection with a plasmid into which a 42-mer oligonucleotide having the partial sequence was inserted.

(1) Preparation of DNA probe Single-stranded DNA having a nucleotide sequence represented by the following formula (2)
Was prepared with a DNA synthesizer (Nihon ZEON: Genet A-III) and purified by high performance liquid chromatography. ^{5 ′} GAT CCA ATG GTT CTC GAG GCC ATG AAG ATG GAG AC
C GAG ATC AAC GCT CCC ACC GAC GGC AAG GTC GAG ^{3 '}
(2) Transfer 12 μg (5 × 10 ^-10 M) of this DNA to an Eppendorf tube (hereinafter abbreviated as E tube) and put it in Ru (bipy) ₂ Cl ₂ 1
A water / ethanol solution containing 3.5 × 10 ⁻¹⁰ M was prepared.
Next, make a thin glass tube on the E tube and put it on the oil bath.
Refluxed at 104 ° C for 1 hour.

(2) Hybridization Partial sequence of the above formula (2) and its homologous sequence with Hind at the end
An oligonucleotide represented by the following formula (3) having a III site was similarly synthesized with a DNA synthesizer.

AGC TTG CTC GTT CTC GAG GCC ATG AAG ATG GAG ACC GA
G ATC AC GAG CAA GAG CTC CGG TAC TTC TAC CTC TGG C
TC TAG TCC A (3) Next, using T4 ligase, the plasmid was inserted into the plasmid pBR322, transformed into Escherichia coli JM109 and cultured to isolate the plasmid, which was designated as pBP42.

Separately, a plasmid pBR322 was prepared separately. this
pBR322 is a control and does not have the partial sequence of the above formula (2).

5.5 μg of pBP42 and 8.5 μg of pBR322 were collected,
Add 100 μl of alkaline denaturing solution (0.5N NaOH, 1.5M NaCl), leave it for 30 minutes to denature, then 0.5M Tris, 3M NaCl 0.1.
8 ml was added to neutralize. 1 μl to 10 μl of this solution was sampled and a nitrocellulose membrane (Schleicher & Schn
ell BA85) film pieces were dropped on 1 × 0.5 cm ² and heated at 80 ° C. for 2 hours to be fixed.

Pre-hybridization and hybridization were performed as follows according to the method described by T. Maniatis et al.

Pre-hybridization solution (6 x SSC, 5 x Denhardt's solution) 3 ml, 10% SDS solution 30 μl, 10 mg / ml salmon sperm DNA solution 75 μ
The above-mentioned membrane piece was dipped in 1 and sealed in a polyethylene bag, which was informed at 42 ° C for 3 hours. Next, this solution is discarded and hybridization solution (6 x SSC, 0.01M EDTA, 5 x Denhardt's solution) 3 ml, 10% SDS solution 30 μl, 10 mg / ml salmon sperm DNA solution 30
μl and 1 ml of the above-prepared DNA probe were added, and the mixture was similarly sealed in a polyethylene bag and notified at 42 ° C. overnight. After hybridization, the membrane is washed 2x for washing
Shake with 100 ml of SCC and 1% SDS for 5 minutes, then 0.16% SSC, 1
Shake with 100 ml of SDS solution at 50 ℃ for 15 minutes, 2 × SSC, 0.1% SD
Shake with 100 ml of S liquid.

(3) Color reaction E
The mixture was placed in a tube, 100 μl of 1 mM bipy ethanol solution and 0.9 μl of ethanol were added, and a glass thin tube was set up in an E tube and refluxed on an oil bath at 104 ° C. for 1 hour. This solution was transferred to another E tube, the solvent was removed with a centrifugal concentrator, and then NBT ²⁺ ethanol solution 50 mM 20 μl,
Ethanol solution of triethanolamine 10 mM, 100 μl
And shaken well.

Take 100 μl of this solution in a Durham tube with an outer diameter of 6 mm, plug it
A 500W xenon lamp was fitted with a shortcut filter (Toshiba color glass filter Y45) and irradiated for 1 hour. When observed during irradiation, the liquid from pBP42 emitted a bluish fluorescence, and the liquid from pBR322 had no bluish color, and a clear difference could be observed. After irradiation, the solution was diluted with ethanol to 2 ml and the infrared absorption spectrum was measured. The results shown in FIG. 1 were obtained. The blue precipitation of pBR42 shown by the curve a is remarkably observed, and the difference from the pBR322 of the curve b is clear.

〔The invention's effect〕

As described above, the DNA probe of the present invention is a single-stranded DNA
Since Ru (bipy) ₂ ²⁺ is simply refluxed, the production is extremely easy.

The color reaction is sensitive because it utilizes the photocatalysis of Ru (bipy) ₃ ²⁺ .

Since the DNA probe and the reagent used for coloring do not contain RI or enzyme and are ordinary analytical reagents, they are inexpensive and stable.

The present invention is a new method in which photocatalysis is applied to analysis. However, this principle can be applied to analysis other than DNA, and detection methods other than color reaction are also possible, which opens a wide range of applications. .

[Brief description of drawings]

Figure 1 shows the plasmid (pBP4) isolated by culturing the transformed oligonucleotide in E. coli.
FIG. 3 is a diagram showing an infrared absorption spectrum of a liquid obtained by adding a color reagent and a reducing agent to an eluent (curve a) and a control liquid (curve b) from the nitrocellulose membrane identified in 2), and irradiating with light.

Claims (3)

[Claims] 1. A nucleic acid probe having a ruthenium bis (bipyridyl) ²⁺ ion coordinately bonded to the base of a nucleic acid. 2. A single-stranded nucleic acid having a base sequence of interest is treated with water.
A method for producing a nucleic acid probe having a ruthenium bis (bipyridyl) ²⁺ ion coordinate-bonded, which comprises dissolving in an organic mixed solvent, adding a ruthenium bis (bipyridyl) ²⁺ salt thereto, and then refluxing. 3. A method for detecting the presence or absence of a target nucleotide sequence, which comprises a single-stranded DNA sequence to be analyzed, in which a ruthenium bis (bipyridyl) ²⁺ ion is coordinate-bonded. Excited ruthenium tris (bipyridyl) ²⁺ by irradiating the produced ruthenium tris (bipyridyl) ²⁺ ion with light in the presence of a coloring reagent and a reducing agent by reacting the product with excess bipyridyl. Ruthenium tris (bipyridyl) via ion
^The above-mentioned ruthenium tris (bipyridyl) ³⁺ ion is reduced to the ruthenium tris (bipyridyl) ²⁺ ion by the reducing agent while generating ³⁺ ion and reducing and coloring the coloring reagent. A method for detecting the presence or absence of a base sequence.