JP2005080535A - Method for detecting reaction between nucleic acid base and helicase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily detecting a reaction between a nucleic acid base and a helicase in a short time and in high accuracy. <P>SOLUTION: The method comprises the following steps: (1) A solution of a fluorescence-labeled nucleic acid base is prepared using PCR (polymerase chain reaction). (2) A mixed solution is prepared by mixing together the above solution and a helicase and made to react at a temperature in a living body, e.g. at 37°C; alternatively, the mixed solution may be prepared by adding an enzyme acting on the reaction product of the nucleic acid base and helicase. (3) The reacted mixed solution is subjected to FCS(fluorescence correlation spectroscopy) and based on the measurement, the presence/absence of the reaction is detected. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for detecting a reaction between a nucleobase and a helicase.

Patent Document 1 (Japanese Patent Publication No. 2002-540800) discloses that a nucleobase and a helicase are mixed and reacted, and the reaction between the nucleobase and the helicase is measured by radiation, surface plasmon, or nuclear magnetic resonance. It is disclosed to go and detect.
Patent Document 2 (Japanese Patent Laid-Open No. 2000-197483) discloses that a nucleobase and a helicase are mixed and reacted, and a reaction between the nucleobase and the helicase is detected by performing an antibody reaction experiment.
Patent Document 3 (Japanese Patent Publication No. 2000-508521) discloses that a reaction between a nucleobase and a helicase is detected using a nucleobase immobilized on a support or a helicase and agarose beads immobilized on a support. doing.
Patent Document 4 (Japanese Patent Application Laid-Open No. 2000-166600) discloses a method for screening a compound using suppression of the expression level of a helicase gene as an index.

  Patent Document 5 (Japanese Patent Application Laid-Open No. 2003-88369) discloses a method for detecting DNA endonuclease activity using fluorescence correlation spectroscopy (FCS), and the magnitude of molecular weight and translational diffusion time. The relationship is disclosed.

Patent Document 6 (Japanese Patent Publication No. 2002-543414) describes a method for characterizing fluorescent molecules or other particles in a sample, as well as fluorescence intensity multiple distribution analysis (FIMDA) and fluorescence autoconvoluted intensity distribution analysis. (FACID) discloses that translational diffusion time can be obtained.
Special Table 2002-540800 Japanese Unexamined Patent Publication No. 2000-197483 Special Table 2000-508521 JP 2000-166600 A JP 2003-88369 A Special Table 2002-543414

  Among the above-mentioned conventional techniques, each detection method described in JP 2000-197483 A, JP 2000-508521 A and JP 2001-321199 is a measurement of radiation when detecting a helicase reaction. , Surface plasmon measurement or nuclear magnetic resonance measurement, antibody reaction, nucleobase or helicase immobilized on a support, agarose beads, etc. There is a problem that it takes time until. In addition, it is difficult to detect the reaction of a helicase without an antibody or a helicase that cannot be immobilized on a substrate.

  Japanese Unexamined Patent Publication Nos. 2000-197483, 2000-508521, 2001-321199 and 2000-166600 describe the use of fluorescence correlation spectroscopy (FCS), fluorescence intensity for helicase analysis. There is no disclosure of using multiple distribution analysis (FIMDA) and fluorescence autoconvoluted intensity distribution analysis (FACID).

  Japanese Patent Application Laid-Open No. 2003-88369 and Japanese Patent Application Publication No. 2002-543414 do not disclose a method for detecting the reaction between DNA and helicase.

  An object of the present invention is to provide a method for detecting a reaction between a nucleobase and a helicase easily and accurately in a short time.

  The feature of the present invention that achieves the above object is that the size, brightness, or number of substances having a fluorescent label in a mixed solution by fluorescent labeling a nucleobase or helicase, mixing a nucleobase and a helicase, and using a fluorescence analysis method Is to seek.

  According to this feature, the size, brightness, or number of substances having fluorescent labels can be determined by mixing each solution and fluorescence analysis, so measurement of radiation, measurement of surface plasmons or measurement of nuclear magnetic resonance, Without complicated operations such as fixation to the support, presence or absence of reaction such as binding or action of nucleobase and helicase, helicase reaction activity, change in reaction product size, change in brightness, number As a result, it is possible to easily obtain a detection result such as a change in the time and accuracy with a short time.

  In addition, since a solid support is not used, helicase that cannot be immobilized can be reacted and analyzed. Moreover, since an antibody reaction is not used, it is possible to react and analyze even a helicase without an antibody.

  In addition, mixing each solution, fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity distribution analysis method (FIDA), multi-item fluorescence intensity distribution analysis method (FIMDA) or fluorescence polarization analysis method ( By using FIDA-polarization), it is possible to detect a change in the size, brightness, and number of reaction products in a floating system with very good sensitivity of the order of nM.

  Alternatively, double-stranded DNA fluorescently labeled as a nucleobase may be used, and DNA helicase II may be used as a helicase. In this case, if the translational diffusion time of the reaction product is significantly shorter than that of the unreacted fluorescently labeled double-stranded DNA, the reaction between the double-stranded DNA and DNA helicase II can be detected.

  Exonuclease I may be further mixed in a mixed solution of double-stranded DNA and DNA helicase II. In this case, the reaction between the double-stranded DNA and DNA helicase II could be detected if the translational diffusion time of the reaction product was an order of magnitude less than that of the unreacted fluorescently labeled double-stranded DNA. Become.

  Further, an enzyme may be further mixed in a mixed solution of nucleobase and helicase. This enzyme preferably reacts with a reaction product of a nucleobase and a helicase. With this enzyme, a substance with a fluorescent label undergoes a two-step reaction, and then the size, brightness, or number of the substance can be determined. Appearing, these results can be obtained more accurately.

Another feature of the present invention is that a fluorescently labeled nucleobase, a helicase, and a sample are mixed, and the size, brightness, or number of reaction products in the mixed solution is obtained by fluorescence analysis, It is to compare the size, brightness, or number of the reaction product with and helicase, or the size, brightness, or number of unreacted nucleobases. If the translational diffusion time of the reaction product is significantly shorter than that of unreacted fluorescently labeled double-stranded DNA, or if there is no significant difference from the translational diffusion time of fluorescently labeled single-stranded DNA, then double-stranded Since the reaction between DNA and DNA helicase II has been carried out, it can be said that there is no helicase inhibitor in the sample.

  According to this feature, the size, brightness, or number of substances having fluorescent labels can be determined by mixing each solution and fluorescence analysis, so measurement of radiation, measurement of surface plasmons or measurement of nuclear magnetic resonance, The presence or absence of the helicase inhibitor can be easily and accurately obtained in a short time without performing a complicated operation such as fixing to a support.

  According to the present invention, the size, brightness or number of a substance having a fluorescent label can be determined by mixing each solution and analyzing fluorescence, so that measurement of radiation, measurement of surface plasmon or measurement of nuclear magnetic resonance, Without complicated operations such as fixation to the support, presence or absence of reaction such as binding or action of nucleobase and helicase, helicase reaction activity, change in reaction product size, change in brightness, number As a result, it is possible to easily obtain a detection result such as a change in the time and accuracy with a short time.

  In addition, mixing each solution, fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity distribution analysis method (FIDA), multi-item fluorescence intensity distribution analysis method (FIMDA) or fluorescence polarization analysis method ( By using FIDA-polarization), it is possible to detect a change in the size, brightness, and number of reaction products in a floating system with very good sensitivity of the order of nM.

  In this embodiment, the reaction between the nucleobase and the helicase is analyzed by the following procedure. FIG. 1 shows an example of the procedure.

(1) Nucleobase fluorescent labeling and preparation Using the polymerase chain reaction (PCR), a fluorescently labeled nucleobase (hereinafter referred to as a fluorescently labeled nucleobase) is prepared and approximately 10 nM of fluorescently labeled nucleic acid. Make a base solution.

(2) Reaction experiment of fluorescent nucleobase and helicase A solution of fluorescently labeled nucleobase and helicase are mixed to prepare a mixed solution, which is reacted at in-vivo temperature conditions, for example, 37 ° C. Alternatively, a mixed solution may be prepared by adding an enzyme that acts on a reaction product of a nucleobase and a helicase.

(3) FCS analysis The reacted mixed solution is transferred to a glass bottom plate for FCS measurement, for example, a microplate, and FCS measurement is performed. The presence or absence of reaction is detected based on the measured value in FCS measurement.

  In the FCS measurement, the fluctuation of the fluorescent molecule in the minute region is measured, and the translational diffusion time (Diffusion Time) is obtained based on the obtained value. Since the magnitude of the translational diffusion time indicates the magnitude of the molecular weight, an increase or decrease in the molecular weight can be found by comparing the translational diffusion times before and after the reaction. An increase in molecular weight indicates a binding reaction between biomolecules, a decrease in molecular weight indicates a degradation reaction of biomolecules, and a maintenance of molecular weight indicates that there is no binding or degradation to biomolecules. Therefore, the reaction between the fluorescently labeled nucleobase and the helicase can be detected by detecting an increase in the translational diffusion time of the fluorescently labeled substance before and after the reaction between the fluorescently labeled nucleobase and the helicase.

  In the above procedure, fluorescence correlation spectroscopy (FCS) was used to determine the translational diffusion time of the reaction product in (3), but instead of fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation (FCS) was used. Spectroscopy (fluorescence cross correlation spectroscopy), fluorescence intensity distribution analysis (Fluorescence Intensity Distribution Analysis), multi-item fluorescence intensity distribution analysis (Fluorescence Intensity Multiple Distribution Analysis) or fluorescence polarization analysis (FIDA-polarization) may be used. . From these analysis methods, data related to the size, number, and brightness of the nucleobases after the reaction are obtained. From these data, changes in the size, number and brightness of the nucleobases before and after the reaction can be obtained. For example, if there is no significant difference in the nucleobase size before and after the reaction as a result of FCS measurement, but there is a change in the brightness of fluorescence per molecule, the fluorescence intensity distribution analysis method (FIDA) is performed. Thus, it can be known whether or not there is a reaction.

[Example]
(Reaction experiment of fluorescently labeled double-stranded DNA with DNA helicase II)
In this embodiment, the following is used. In particular, fluorescently labeled double-stranded DNA is used as a nucleobase, DNA helicase II is used as a helicase, and exonuclease I is used as an enzyme that acts on the reaction product of a nucleobase and a helicase.

Human DNA;
Non-labeled primer (F) 19mer;
EvoBlue (633nm) label primer (R) 20mer (may be TAMRA labeling);
TITANIUM TaQ DNA polymerase;
QIAquick PCR Purification Kit;
Exonuclease I (EPICENTRE X40501K) 20U / μl;
DNA Helicase II E coli: Wako 200 μg / ml; and
Helicase Buffer (25 mM Tris HCl (pH 7.4), 20 mM NaCl, 3 mM MgCl ₂ , 2 mM β-ME and 2 mM ATP, water (H ₂ O))
(1) Preparation of double-stranded DNA solution Polymerase chain reaction (PCR) is performed using the reagents shown in Table 1 and fluorescently labeled double-stranded DNA (hereinafter referred to as fluorescent-labeled double-stranded DNA). Got. The length of fluorescently labeled double-stranded DNA is 160 bp. PCR temperature control was 94 ° C for 2 minutes, followed by 30 cycles of 94 ° C for 30 seconds and 74 ° C for 30 seconds, and stored at 4 ° C after 74 ° C for 5 minutes.

In order to remove unreacted primers and single-stranded DNA from the solution, 3 to 5 μl of exonuclease I was added to 25 μl of the PCR solution. At the same time, MgCl ₂ was added to a final concentration of 10 mM (because Mg ++ is necessary for the reaction), and the reaction was carried out at 37 ° C. for about 1 hour. Thereafter, unreacted primers, nucleotides, enzyme (Enzyme), salt, etc. were removed using QIAquick PCR Purification Kit. The fluorescently labeled double-stranded DNA was purified and electrophoresed with 3% agarose / TAE to confirm the band.

  Fluorescently labeled double-stranded DNA obtained by purification is measured by fluorescence correlation spectroscopy (FCS), diluted with Helicase Buffer so that the number of particles (n) is about 10, and a solution of fluorescently labeled double-stranded DNA (About 10nM). When each experiment was carried out using a fluorescently labeled double-stranded DNA solution with a particle number (n) of about 10, the number of particles in the confocal volume was 0.3 to 2 during FCS measurement, and FCS measurement could be performed with high accuracy and fluorescence. The reaction between labeled double-stranded DNA and helicase can be detected with high accuracy.

(2) Reaction experiment and FCS measurement Fig. 1 shows the procedure of the reaction experiment. The FCS measurement was performed three times after each reaction under the condition that a laser beam having a wavelength of 633 nm and an output of 400 μW was irradiated for 15 seconds at a time. MF20 (manufactured by Olympus Optical Co., Ltd.) was used for FCS measurement (reaction experiment 1: fluorescently labeled double-stranded DNA only)
The reagents shown in Table 2 were mixed and reacted at 37 ° C. for 15 minutes. The reacted mixture was transferred to a glass bottom plate for FCS measurement, for example, a microplate, and FCS measurement was performed to determine the translational diffusion time.

(Reaction experiment 2: Reaction of fluorescently labeled double-stranded DNA with DNA helicase II)
The reagents shown in Table 3 were mixed and reacted at 37 ° C. for 15 minutes. The reacted mixture was transferred to a glass bottom plate for FCS measurement, for example, a microplate, and FCS measurement was performed to determine the translational diffusion time.

(Reaction experiment 3: reaction of fluorescently labeled double-stranded DNA, DNA helicase II and exonuclease I)
The reagents shown in Table 4 were mixed and reacted at 37 ° C. for 15 minutes. The reacted mixture was transferred to a glass bottom plate for FCS measurement, for example, a microplate, and FCS measurement was performed to determine the translational diffusion time.

(Reaction experiment 4: Reaction of fluorescently labeled double-stranded DNA with exonuclease I)
The reagents shown in Table 5 were mixed and reacted at 37 ° C. for 15 minutes. The reacted mixture was transferred to a glass bottom plate for FCS measurement, for example, a microplate, and FCS measurement was performed to determine the translational diffusion time.

Table 6 shows the results of FCS measurement in Reaction Experiments 1 to 4. The results of FCS measurement using only the fluorescent dye are also shown.

The graph of the translational diffusion time of the product in reaction experiment 1-4 in FIG. 2 is shown.
In Reaction Experiment 1, since neither DNA helicase II nor exonuclease I is mixed, the fluorescence-labeled double-stranded DNA does not change. Therefore, the translational diffusion time in Reaction Experiment 1 is that of unreacted fluorescently labeled double-stranded DNA.

  In Reaction Experiment 2, the translational diffusion time of the reaction product is significantly smaller than the translational diffusion time of unreacted fluorescently labeled double-stranded DNA. This indicates that the double-stranded DNA has become single-stranded by DNA helicase II. Therefore, the translational diffusion time in Reaction Experiment 2 is that of fluorescently labeled single-stranded DNA.

  In Reaction Experiment 3, the translational diffusion time of the reaction product was significantly shorter than that of the fluorescently labeled single-stranded DNA. This indicates that the double-stranded DNA was made into a single strand by DNA helicase II, and the single-stranded DNA was specifically decomposed by exonuclease I into a short base sequence. Therefore, the translational diffusion time of Reaction Experiment 3 is that of the short base sequence A that is fluorescently labeled.

  The translational diffusion time in Reaction Experiment 4 was not significantly different from that of unreacted fluorescently labeled double-stranded DNA. This indicates that exonuclease I does not react with double stranded DNA.

  Therefore, when detecting whether or not DNA helicase II is present in the sample, the fluorescently labeled double-stranded DNA and the sample are mixed, the reaction product is subjected to FCS measurement, and the translational diffusion time is obtained. The value may be compared with the translational diffusion time of unreacted fluorescently labeled double stranded DNA. If the translational diffusion time of the reaction product is significantly shorter than that of unreacted fluorescently labeled double-stranded DNA, it can be said that DNA helicase II is present in the sample.

  In addition, when detecting whether or not DNA helicase II is present in the sample, exonuclease I may be further mixed in a mixed solution of the fluorescently labeled double-stranded DNA and the sample. If the translational diffusion time of the reaction product is reduced by an order of magnitude, it can be said that DNA helicase II is present in the sample.

  When detecting whether a helicase inhibitor is present in a sample, fluorescently labeled double-stranded DNA, DNA helicase II and the sample are mixed, and the reaction product is subjected to FCS measurement to determine the translational diffusion time. The obtained value may be compared with the translational diffusion time of unreacted fluorescently labeled double-stranded DNA or the translational diffusion time of fluorescently labeled single-stranded DNA. If the translational diffusion time of the reaction product is significantly less than that of unreacted fluorescently labeled double-stranded DNA, or if there is no significant difference from the translational diffusion time of fluorescently labeled single-stranded DNA, then double-stranded Since the reaction between DNA and DNA helicase II has been carried out, it can be said that there is no helicase inhibitor in the sample.

  According to this example, the reaction between the fluorescence-labeled double-stranded DNA and DNA helicase II can be detected with high accuracy by mixing each solution and reacting them, and measuring the reaction product by FCS. In addition to the double-stranded DNA, the presence or absence of a reaction can also be detected by using a nucleobase such as a genome, single-stranded DNA, or RNA and a helicase other than DNA helicase II.

  In addition, mixing each solution, fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity distribution analysis method (FIDA), multi-item fluorescence intensity distribution analysis method (FIMDA) or fluorescence polarization analysis method ( By using FIDA-polarization, it is possible to detect changes in the size, brightness, and number of reaction products in a floating system with very good sensitivity on the order of nM.

  In addition, the detection result can be easily obtained in a short time without performing complicated operations such as measurement of radiation, measurement of surface plasmon or nuclear magnetic resonance, and fixation to a support. In addition, since a solid support is not used, helicase that cannot be immobilized can be reacted and analyzed. Moreover, since an antibody reaction is not used, it is possible to react and analyze even a helicase without an antibody.

  In this example, the double-stranded DNA is fluorescently labeled, but the double-stranded DNA may not be fluorescently labeled but may be fluorescently labeled with DNA helicase II to conduct a reaction experiment.

  In addition, the present invention can be applied to screening of a large amount of sample by using an automatic dispenser and a plate stacker.

The figure which shows the example of the protein analysis procedure in this embodiment. The graph which shows the translational diffusion time of each reaction product in the reaction experiments 1-4.

Claims (5)

Fluorescently labeling a nucleobase or helicase;
Mixing the nucleobase and the helicase to form a mixed solution;
A method for detecting a reaction between a nucleobase and a helicase, comprising a step of obtaining a size, brightness, or number of a substance having a fluorescent label in a mixed solution by a fluorescence analysis method.   The fluorescence analysis methods include fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), fluorescence intensity distribution analysis method (FIDA), multi-item fluorescence intensity distribution analysis method (FIMDA), or fluorescence polarization analysis method (FIDA- The method for detecting a reaction between a nucleobase and a helicase according to claim 1, wherein The step of fluorescent labeling comprises the step of fluorescently labeling a nucleobase;
2. The method for detecting a reaction between a nucleobase and a helicase according to claim 1, wherein the nucleobase is a double-stranded DNA and the helicase is a DNA helicase II. The step of preparing the mixed solution includes:
The method for detecting a reaction between a nucleobase and a helicase according to claim 1, comprising a step of mixing an enzyme that reacts with a reaction product of the nucleobase and the helicase. Mixing a fluorescently labeled nucleobase, a helicase and a sample to form a mixed solution;
Determining the size, brightness or number of reaction products in the mixed solution by fluorescence analysis;
The obtained size, brightness or number of the substance and the size, brightness or number of the reaction product of nucleobase and helicase, or the size, brightness or number of unreacted nucleobase And a method for detecting a helicase inhibitor.

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