JPH11286616A - Nucleic acid staining agent, method for detecting double-stranded nucleic acid, and reagent for detecting target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nucleic acid staining agent which undergoes a marked change in fluorescent characteristics, especially, in fluorescent intensity depen dent of the presence or absence of a double-stranded nucleic acid by selecting a 3,8-diazo-6-phenylphenanthridine derivative. SOLUTION: A derivative (ethidium bromide derivative) of the formula is used. In the formula, R1 is an alkyl; R2 is a halide ion; R3 and R4 are each phenyl, anilino, hydroxyphenyl, an alkylanilino, a dialkylanilino, a dihydroxyalkylanilino, a 2-(N-alkylanilino)ethanol, naphthyl, or naphthol. The ethidium bromide derivative may be used as such or in the form dissolved or dispersed in a suitable solvent or dispersion medium. The solvent and the dispersion medium are exemplified by water, acetonitrile, dimethyl sulfoxide, and various buffer solutions such as a phosphate buffer solution, and an acetate buffer solution. The solution or the dispersion has an ethidium bromide derivative concentration of, desirably, 1×10<-8> to 1×10<-5> M.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to viruses, microorganisms,
Nucleic acid stains useful for detecting and identifying specific nucleotide sequences of nucleic acids of animals, plants, humans, etc. using optical means, or detecting the presence or absence of mutations in various nucleotide sequences, gene diagnosis using the same, and cloning of useful genes The present invention relates to a double-stranded nucleic acid detection method and a detection reagent for searching for unknown genes and the like.

[0002]

2. Description of the Related Art With the development of nucleic acid analysis technology, many mutant genes have been found, and it has become clear that gene mutations cause protein mutations, thereby causing various diseases. At present, these diseases are mainly diagnosed and detected by enzyme-based assays and immunological methods using antibodies.However, from the viewpoint of early detection and early treatment, it is important to detect mutations in genes as early as possible. The importance of possible genetic diagnosis has been pointed out. Genetic diagnosis is also often used to identify infected bacteria.

[0003] In the field of detection or identification of the gene of the causative bacterium in bacterial infections, attempts have been made to use a DNA-DNA hybridization method or a DNA-RNA hybridization method. This method is problematic in a sample by focusing on a specific portion of a nucleic acid of a bacterium and measuring whether or not the base sequence of that portion is present in the test nucleic acid sample of interest by a hybridization method. This is a method for determining whether or not bacteria exist.

As a typical procedure of the hybridization method, a test nucleic acid is denatured and bound to an insoluble carrier, which is then incubated with a probe having a base sequence complementary to a target nucleic acid labeled with an enzyme or radioisotope. To form a hybrid, then wash the insoluble carrier to wash away the probe not bound to the target sequence,
This is an operation for quantifying the amount of a target nucleic acid by an enzyme reaction, radioisotope, or the like.

However, in the hybridization method using the insoluble carrier as described above, it is necessary to remove the unreacted probe (B / F separation), and the operation is extremely complicated. Further, since the probe is adsorbed on the insoluble carrier, a signal derived from the probe adsorbed on the insoluble carrier is mixed into the measurement result at the stage of measuring the labeling substance of the hybrid on the insoluble carrier, and the target in the sample is removed. There is a problem that an error occurs in the measurement result of the presence or absence of the nucleic acid and the amount thereof, and it is difficult to make a correct determination.

Therefore, as a method of avoiding these problems, a hybridization method in a solution, so-called,
The development of a homogeneous method is desired. The biggest challenge with this homogeneous system is that it is necessary to distinguish between probes that bind to the target nucleic acid and those that do not.

As a method for detecting a hybrid without performing B / F separation, a method utilizing a fluorescence depolarization method has been proposed (JP-A-2-75958, JP-A-2-295496). . This method uses a fluorescent dye-labeled probe to determine the presence or absence of a target nucleic acid based on the difference between the fluorescence polarization of a single-stranded nucleic acid and the fluorescence polarization of a hybrid. Is made to be difficult to move due to the double-stranded structure, and the fact that the fluorescence anisotropy is increased is used. However, in this method, if impurities such as proteins are contained in the sample, the probe is non-specifically adsorbed to them and causes an increase in the background of hybrid detection. Requires a complicated operation of completely removing the.

A method for detecting a hybrid using the transfer of photoexcitation energy has been proposed (Proc. Nat.
l. Acad. Sci. USA, 85, 8790-8794). This method
It consists of an energy donor compound and an energy acceptor compound. When the probe hybridizes to the target nucleic acid, the excitation energy is transferred from the energy donor to the acceptor due to the presence of both compounds in close proximity, resulting in energy donation. A decrease in the excitation lifetime and fluorescence intensity of the body or an increase in the fluorescence intensity of the receptor is observed. By utilizing these phenomena, the presence or absence of hybridization can be measured as a solution, and it is an epoch-making method that can eliminate a series of complicated operations, but the sensitivity is several orders of magnitude lower than existing methods, At present, it has not been put to practical use.

Further, a method for detecting a hybrid using an intercalating fluorescent dye has been proposed (JP-A-8-21110). This method is a method of detecting a hybrid by measuring a change in fluorescence characteristics when a dye is intercalated into the hybrid. JP-A-9-406611 discloses that a pyrylium salt or a salt thereof can be used as an intercalating dye.

[0010]

However, these dyes have a problem that the change in fluorescence intensity with respect to the presence or absence of double-stranded nucleic acid is small. An object of the present invention is to provide a nucleic acid stain having a large change in fluorescence characteristics with respect to the presence or absence of a double-stranded nucleic acid, particularly a large change in fluorescence intensity, and a method and a reagent for detecting a double-stranded nucleic acid using the same. It is.

[0011]

The present inventors have made intensive studies to provide such a nucleic acid stain, and as a result, have found that 3,8-
The present inventors have found that a diazo-6-phenylphenanthlysine derivative changes its fluorescence intensity and fluorescence wavelength by interacting with a nucleic acid, and arrived at the present invention.

That is, the first invention comprises a 3,8-diazo-6-phenylphenanthridine derivative represented by the following general formula (1) (hereinafter referred to as an ethidium bromide derivative). The gist of the invention is a nucleic acid stain.

[0013]

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(Wherein R ₁ represents an alkyl group;
R ₂ represents a halogen ion; R ₃ and R ₄ represent phenyl, anilino, hydroxyphenyl, alkylanilino, dialkylanilino, dihydroxyalkylanilino, 2- (N-alkylanilino) ethanol A naphthyl group or a naphthol group. )

[0015] The second invention is characterized in that a change in fluorescence characteristics caused by the interaction of the ethidium bromide derivative represented by the general formula (1) with a double-stranded nucleic acid is measured. The gist is a method for detecting a nucleic acid. Further, a third invention is a target nucleic acid comprising a probe consisting of a single-stranded nucleotide having a nucleic acid sequence complementary to a target nucleic acid, and an ethidium bromide derivative represented by the general formula (1). Of the present invention.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The ethidium bromide derivative used in the present invention is represented by the above general formula (1). R ₁ in the above general formula
Examples thereof include an alkyl group such as a methyl group, an ethyl group and a propyl group.Examples of R ₂ include a bromine and a halogen ion such as chlorine.As R ₃ and R ₄ , a phenyl group which is an electron donating group, Alkylanilino group such as anilino group, hydroxyphenyl group, methylanilino group, ethylanilino group, dimethylanilino group, dialkylanilino group such as diethylanilino group, dimethanolanilino group,
Examples include a dihydroxyalkylanilino group such as a diethanolanilino group, a 2- (N-alkylanilino) ethanol group such as a 2- (N-ethylanilino) ethanol group, a naphthyl group, and a naphthol group.

As the nucleic acid stain of the present invention, the above-mentioned ethidium bromide derivative may be used as it is, or may be dissolved or dispersed in an appropriate solvent or dispersant. Examples of such a solvent and a dispersant include various buffers such as water, acetonitrile, dimethyl sulfoxide, a phosphate buffer, and an acetate buffer. At this time, the concentration of the ethidium bromide derivative is preferably 1 × 10 ⁻⁸ to 1 × 10 ⁻⁵ M.

The nucleic acid stain of the present invention may be in a form in which the above ethidium bromide derivative is bound to a probe for detecting a target nucleic acid. As a probe,
It is preferable to have a nucleic acid sequence completely complementary to a specific base sequence in the target nucleic acid, but there is no problem even if some bases are not complementary sequences as long as they do not interfere with binding to the target nucleic acid. . The specific base sequence is a nucleic acid sequence consisting of 10 to 30 bases in the target nucleic acid, and this sequence is preferably a sequence that can be distinguished from other nucleic acids.

The ethidium bromide derivative can be bound to the probe by directly binding the ethidium bromide derivative to the probe or via a linker having an appropriate molecular length. The linker is not particularly limited as long as it does not hinder the formation of the hybrid and the interaction between the ethidium bromide derivative and the double-stranded nucleic acid. It is preferred to use a difunctional hydrocarbon having groups.

The binding site between the ethidium bromide derivative and the probe is not particularly limited as long as it does not interfere with the formation of the hybrid and the interaction between the ethidium bromide derivative and the double-stranded nucleic acid. It may be any of the 'end, 3' end or the central part. However, when performing gene amplification such as PCR in the presence of the probe, it is preferable to attach an ethidium bromide derivative to the 5 ′ end of the probe so as not to inhibit the elongation reaction of the nucleic acid by the DNA synthase. .

Next, the method for detecting a double-stranded nucleic acid of the present invention will be described. The method for detecting a double-stranded nucleic acid of the present invention uses the above ethidium bromide derivative as a nucleic acid stain,
The measurement is performed by measuring a change in the fluorescent property caused by the ethidium bromide derivative interacting with the double-stranded nucleic acid. The interaction between the ethidium bromide derivative and the double-stranded nucleic acid is considered to be a state in which the ethidium bromide derivative is intercalated into the double-stranded nucleic acid. It is not limited to curation.

In the present invention, the ethidium bromide derivative before interacting with the double-stranded nucleic acid is surrounded by highly hydrophilic water molecules, but when interacting with the double-stranded nucleic acid, the compound becomes hydrophobic. Since the situation is surrounded by nucleic acid bases having high affinity, the fluorescence characteristics change. For this reason, by measuring the change in the fluorescence characteristic by, for example, a change in the fluorescence intensity at a specific wavelength or a change in the maximum maximum wavelength,
Double-stranded nucleic acids can be detected and quantified.

The detection method of the present invention can be used to detect a hybrid of a probe and a target nucleic acid in a hybridization method using a probe. In this case, the ethidium bromide derivative can be bound to the probe in advance and detected by hybridizing with the target nucleic acid, or the probe without the ethidium bromide derivative bound thereto can be first hybridized with the target nucleic acid, and Can be detected by adding an ethidium bromide derivative to the mixture. Further, the detection method of the present invention can be applied to hybridization in a state where a probe or a sample is fixed and in a solution.

The amount of the ethidium bromide derivative to be added to the double-stranded nucleic acid is preferably an excess of the ethidium bromide derivative with respect to the double-stranded nucleic acid, preferably 1 × 10 ⁻⁸
It is preferable to add so as to be 1 × 10 ⁻⁵ M.

The method for measuring the change in the fluorescence characteristics is based on the fluorescence spectrum of the ethidium bromide derivative measured in the absence of the double-stranded nucleic acid and the fluorescence spectrum of the fluorescent substance measured in the presence of the double-stranded nucleic acid. The change in the fluorescence characteristic may be measured by comparing the fluorescence maximum wavelengths or by comparing the fluorescence intensities of both spectra at the fluorescence maximum wavelengths of both spectra.

The reagent for detecting a target nucleic acid according to the present invention comprises a probe and an ethidium bromide derivative, and the ethidium bromide derivative may or may not be bound to the probe.

[0027]

The present invention will be specifically described below with reference to examples. Example 1 1 g of ethidium bromide represented by the following structural formula (2)
(2.5 mmol) was dissolved in 50 ml of acetone, and 2.8 ml of 6M hydrochloric acid (15 mmol), 10 ml of water and 340 mg (5.0 mmol) of sodium nitrite were added thereto, and the mixture was stirred at 0 ° C. for 2 hours. . Thereafter, 826 mg (5.
(0 mmol) of 2- (N-ethylanilino) ethanol and stirred at 0 ° C. for 20 hours. After adding sodium hydrogen carbonate to make the solution neutral, the precipitate was collected by filtration and washed with water. The precipitate was dissolved in methylene chloride and purified with a silica gel column to obtain an ethidium bromide derivative represented by the following structural formula (3) (hereinafter, referred to as EB2NEE) (yield 0.78 g).

[0028]

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[0029]

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Using the obtained EB2NEE as a nucleic acid stain, double-stranded nucleic acids in the solution were measured. As the double-stranded nucleic acid, a commercially available double-stranded nucleic acid (Calf thymus DNA, manufactured by Boehringer) was used. First, EB2NEE was dissolved in dimethyl sulfoxide to a concentration of 100 mM, and then diluted 100-fold with a 5 mM TrisHCl buffer (pH 7.5) containing 50 mM NaCl, and the excitation wavelength was changed to 350 n.
m was measured using a fluorescence spectrometer FP-777.
(Made by JASCO Corporation) (dotted line in FIG. 1). Next, the base pair concentration of the double-stranded nucleic acid was 25% of the EB2NEE concentration.
The double-stranded nucleic acid was added so as to be twice as large, and the fluorescence spectrum was measured at the same excitation wavelength (solid line in FIG. 1). FIG.
EB2NEEE in the presence and absence of double-stranded nucleic acid
It is a diagram showing a fluorescence spectrum of, the fluorescence intensity on the vertical axis,
The horizontal axis indicates the fluorescence wavelength. As can be seen from FIG. 1, the maximum fluorescence wavelength of EB2NEE in the absence of double-stranded nucleic acid is 425 nm, while the maximum fluorescence wavelength in the presence of double-stranded nucleic acid is 398 nm. The fluorescence wavelength shifted by 27 nm by interacting with. In addition, the fluorescence intensity at 398 nm when the double-stranded nucleic acid was present was about 50 times that when the double-stranded nucleic acid was not present. This indicates that double-stranded nucleic acid can be detected by measuring the fluorescence intensity at a fluorescence wavelength of 398 nm.

Example 2 A 20-mer oligonucleotide having a base sequence partially complementary to a single-stranded M13mp18 DNA (manufactured by Takara Shuzo Co., Ltd.) represented by SEQ ID NO: 1 was synthesized by Applied Biosystems Inc. 39
1 DNA was synthesized using an automatic synthesizer. Excision from CPG carrier, deprotection, high performance liquid chromatography (HP
Purification by LC) was performed by a method specified by Applied Biosystems. A thiol linker was bound to the 5 'end of this oligonucleotide using a 5' thiol modifier C6 manufactured by Glen Research.

The resulting oligonucleotide (260 nm
Was dissolved in 50 ml of 100 mM TrisHCl buffer (pH 7.5), and 7.5 ml of a 1 M silver nitrate solution was added thereto. After stirring, the mixture was allowed to stand at room temperature for 40 minutes. 10m of 1M dithiothreitol (DTT) solution
After adding and stirring, the mixture was left at room temperature for 30 minutes. The precipitate was removed by centrifugation, and the probe was purified by high performance liquid chromatography. 20 ml of 0.01 M DTT was added thereto, and the mixture was stirred and replaced with argon. (Solution 1)

1 g (1.3 mmol) of EB2NEE and 0.1 g
08 g (1.4 mmol) of potassium hydroxide was dissolved in 10 ml of glycol monomethyl ether.
g (1.4 mmol) of 1,3-diiodopropane are added,
The mixture was refluxed for 5 hours. To this, 10 ml of water was added, and after shaking well, the aqueous layer was removed. Hexane was added thereto, and the precipitated solid was collected by filtration (yield 0.58 g).
15 mg of the precipitated solid was dissolved in dimethylformamide 2
00 ml, 1 M phosphate buffer (pH 10.0) 300 m
l and 500 ml of water were added, followed by purging with argon.
(Solution 2)

The solution 1 was mixed with the solution 2 so that the volume became 2: 1 and left at room temperature for 2 hours, and then subjected to gel filtration using Sephadex G-25 (manufactured by Pharmacia) to obtain the obtained product. Is dried and purified by high performance liquid chromatography to obtain EB2N represented by the following structural formula (4).
An EE-bound probe was obtained. The buffer used in the high performance liquid chromatography operation was 0.1 M
Is TrisHCl (pH 7.5) / 50% by weight acetonitrile. The buffer used for the gel filtration operation of Sephadex G-25 is 0.1M TrisHCl (pH 7.5) / 5% by weight acetonitrile.

[0035]

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Example 3 M13mp18 DNA was detected using the EB2NEE binding probe prepared in Example 2. M13mp18DN
A 200 pmol (manufactured by Takara Shuzo Co., Ltd.) and 200 pmol of BE2NEE binding probe were added to 5 mM Tris containing 50 mM NaCl.
It was dissolved in 1 ml of HCl buffer (pH 7.5), heated to 90 ° C., allowed to cool, and gradually returned to room temperature (about 20 ° C.) to hybridize the probe to M13mp18 DNA. The fluorescence spectrum of the hybrid at an excitation wavelength of 350 nm was measured. Also, M13mp18 DNA
The fluorescence spectrum was measured in the same manner except that no was added. As a result, the fluorescence maximum wavelength was 425 nm in the solution containing only the probe, whereas the fluorescence maximum wavelength was 399 nm in the case of the hybrid, and the fluorescence intensity in the case of the hybrid was the fluorescence intensity in the case of only the probe. It was about 5 times the strength. From these results, EB was found in the sample.
It can be seen that it is possible to detect a specific nucleic acid by adding a 2NEE binding probe.

Example 4 EB2NEE binding probe 200 pm prepared in Example 2
ol, 0.1 of M13mp18 DNA (Takara Shuzo),
0.2, 0.5, 1.0, 2.0, 3.0, 5.0,
7.5, 10.0, 15.0 or 20.0 equivalents are added,
Excitation wavelength 350 nm, fluorescence wavelength 3
The fluorescence intensity at 98 nm was measured. The result is shown in FIG. FIG. 2 shows that M13 using the EB2NEE binding probe
It is a figure which shows the result at the time of quantifying mp18 DNA, and a vertical axis | shaft shows fluorescence intensity and a horizontal axis shows probe quantity. FIG.
This indicates that the fluorescence intensity increases in proportion to the amount of the target nucleic acid in the sample. Therefore, it is understood that the specific nucleic acid in the sample can be quantified by adding the EB2NEE binding probe to the sample and measuring the fluorescence intensity.

[0038]

The nucleic acid stain of the present invention interacts with a double-stranded nucleic acid to change its fluorescence intensity and fluorescence wavelength, so that it can be used for detection and quantification of a target nucleic acid in a homogeneous system. . Further, according to the method and reagent for detecting a double-stranded nucleic acid of the present invention, the presence / absence and amount of a target nucleic acid can be determined without performing an operation of removing a labeled probe.

[Brief description of the drawings]

FIG. 1. EB2N in the presence and absence of double-stranded nucleic acid
It is a figure which shows the fluorescence spectrum of EE.

FIG. 2 is a view showing the results of quantifying a target nucleic acid using a probe labeled with EB2NEE.

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTTTTCCCAG TCCACGCGTT
20

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/533 G01N 33/533 33/566 33/566

Claims (3)

[Claims] 1. A nucleic acid staining agent comprising a 3,8-diazo-6-phenylphenanthridine derivative represented by the following general formula (1). Embedded image (In the formula, R ₁ represents an alkyl group, R ₂ represents a halogen ion, and R ₃ and R ₄ represent a phenyl group, anilino group, hydroxyphenyl group, alkylanilino group, dialkylanilino group, dihydroxyalkyl Anilino group,
2- (N-alkylanilino) represents an ethanol group, a naphthyl group, or a naphthol group. ) 2. The method according to claim 1, wherein a change in fluorescence characteristics caused by the interaction of the 3,8-diazo-6-phenylphenanthridine derivative represented by the general formula (1) with a double-stranded nucleic acid is measured. For detecting double-stranded nucleic acids. 3. A probe comprising a single-stranded nucleotide having a nucleic acid sequence complementary to a target nucleic acid and a 3,8-diazo-6-phenylphenanthridine derivative represented by the general formula (1). A reagent for detecting a target nucleic acid, comprising: