WO2020140829A1 - 一种可检测多种dna糖基化酶活性的方法 - Google Patents

一种可检测多种dna糖基化酶活性的方法 Download PDF

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WO2020140829A1
WO2020140829A1 PCT/CN2019/128834 CN2019128834W WO2020140829A1 WO 2020140829 A1 WO2020140829 A1 WO 2020140829A1 CN 2019128834 W CN2019128834 W CN 2019128834W WO 2020140829 A1 WO2020140829 A1 WO 2020140829A1
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dna
detection system
dna glycosylase
double
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French (fr)
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傅新元
闫娟
毛卓
郭剑南
杨盛莲
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上海仕谱生物科技有限公司
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical field of biological analysis, and particularly relates to a method for detecting the activities of various DNA glycosylases and its application.
  • the BER (base excision repair) pathway is the main way to repair endogenous DNA base damage caused by oxidation, alkylation and deamination.
  • DNA glycosylase is an important link in the BER pathway, including: UDG (uracil DNA glycosylase), TDG (thymine DNA glycosylase) and OOG1 (8-hydroxyguanine DNA glycosylase) )Wait.
  • UDG uracil DNA glycosylase
  • TDG thymine DNA glycosylase
  • OOG1 8-hydroxyguanine DNA glycosylase
  • DNA ligase connects the two into a new repaired DNA strand to complete DNA damage repair.
  • DNA glycosylase is closely related to human diseases, so the rapid, accurate, convenient and high-throughput detection of DNA glycosylase activity is of great significance for clinical diagnosis and the development of targeted inhibitors.
  • Existing traditional methods such as gel electrophoresis using fluorescent-labeled substrates have low sensitivity, require large sample volume, and low throughput, which is not suitable for quantitative analysis; replacing fluorescent-labeled substrates with radioisotope-labeled substrates can increase sensitivity and reduce Sample volume, but isotope labeling can cause radioactive contamination; qualitative and quantitative capabilities of high-performance liquid chromatography are strong, and the sample volume is not large, but the sample preparation is complicated and the throughput is also not high; enzyme-linked immunosorbent assay and Immunoblotting requires a specific antibody that binds to the protein, and the operation steps are cumbersome and not suitable for the detection of a large number of samples; the fluorescent method relies on external labeling with fluorophores and quenchers for homogeneous determination, suitable for high-throughput, but
  • the object of the present invention is to provide a method capable of detecting multiple DNA glycosylation enzymes with high throughput, low sample volume, large signal window, simple operation and low cost.
  • a detection system for detecting DNA glycosylase comprising:
  • a double-stranded DNA probe the double-stranded DNA probe includes two strands of T1 and T2, and the two strands of T1 and T2 may form a double-stranded DNA structure;
  • the T1 chain includes: at least one base that can be recognized by the DNA glycosylation enzyme to be tested, a fluorescent group, and a separation tag;
  • fluorescent group and the separation label are respectively located at both ends of the at least one base that can be recognized by DNA glycosylase;
  • the concentration of the double-stranded DNA probe is 10-80 nM, preferably 20-50 nM, more preferably 25-35 nM, more preferably The ground is 30nM.
  • the length of the double-stranded DNA probe is 5-200 bp, preferably 10-100 bp, more preferably 15-70 bp, more preferably 18-40 bp.
  • the base recognized by the test DNA glycosylase is selected from the group consisting of uracil base, cytosine base, thymine base, guanine base, methylation Modified cytosine base, 5-carboxycytosine, alkyl adenine, etc.
  • the base recognized by the DNA glycosylation enzyme to be tested is a uracil base.
  • the at least one base recognized by the DNA glycosylation enzyme to be tested is a uracil base.
  • the at least one base recognized by the DNA glycosylation enzyme to be tested is a uracil base, and the corresponding position on the T2 chain is a guanine base.
  • the fluorophore and the separation tag are independently located at the 5'end, 3'end, and middle of the double-stranded DNA probe.
  • the fluorescent group includes a fluorescent group that can be used for cross-linking with a DNA probe.
  • the fluorescent group is selected from the group consisting of FAM, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Texas Red, etc.
  • the separation tag refers to a tag capable of separating the nucleic acid sequence connected to or containing the separation tag from the detection system.
  • the separation tag is selected from the group consisting of proteins, peptides or nucleic acid fragments.
  • the separation tag is selected from the group consisting of antigen, antibody, ligand, receptor, avidin, biotin, or a combination thereof.
  • the separation tag is biotin.
  • sequence of the T1 chain is 5'-FAM-S1-biotin-3'
  • sequence of the T2 chain is 5'-S2-3'
  • sequence of S1 is as SEQ ID NO:1
  • sequence of S2 is shown in SEQ ID NO:2.
  • the (b) is selected from the group consisting of alkaline media or abasic site endonuclease (AP endonuclease).
  • the alkaline medium is NaOH.
  • the final concentration of the alkaline medium in the detection system is 100-300 mM, preferably 150-250 mM, and more preferably 200 mM.
  • the solid phase carrier material is selected from the group consisting of metal, glass, colloid, plastic, or a combination thereof.
  • the material of the solid phase carrier includes: homopolymer, copolymer, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of polystyrene, polyethylene, polypropylene, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of magnetic beads, microspheres, microplates, slats, test tubes, or a combination thereof.
  • the solid carrier is magnetic beads.
  • the final volume of the detection system is 50-200 ⁇ L, preferably 60-150 ⁇ L, more preferably 80-120 ⁇ L, more preferably 100 ⁇ L.
  • the detection system further includes a reaction buffer.
  • reaction buffer includes: Tris-Cl pH 8.0, EDTA, DTT, etc.
  • the final concentration of Tris-Cl pH 8.0 is 10-50 mM, preferably 15-30 mM, and more preferably 20 mM.
  • the final concentration of the EDTA is 0.5-2mM, preferably 1mM.
  • the final concentration of DTT is 0.5-2mM, preferably 1mM.
  • the detection system further includes a DNA glycosylation enzyme to be tested.
  • the DNA glycosylation enzyme to be tested is selected from the group consisting of UDG, TDG, SMUG1, MBD4, OGG1, AAG, or a combination thereof.
  • the DNA glycosylation enzyme to be tested is selected from the group consisting of UDG, TDG or SMUG1.
  • the concentration range of the DNA glycosylase to be tested is 1 to 500 nM, preferably 1 to 50 nM, and more preferably 1 to 20 nM.
  • the DNA glycosylase is selected from the group consisting of purified DNA glycosylase and its lysate, cell lysate, blood or its extract, body fluid or its extract, or Its combination.
  • the cell lysate includes cancer cell lysate.
  • the cancer includes lung cancer.
  • the detection includes: qualitative detection and quantitative detection.
  • the fluorescent signal is measured by the detection system after separating the nucleic acid fragments with separation tags.
  • the detection of the method includes: qualitative detection and quantitative detection.
  • the qualitative detection includes: detecting whether the sample to be tested contains DNA glycosylase.
  • the quantitative detection includes: detecting the concentration of the active DNA glycosylase in the sample to be detected.
  • the method further includes performing a parallel control experiment on a blank sample, the blank sample does not include DNA glycosylase, and the measured fluorescence signal is A0.
  • step (III) if the signal A1/A0>1.1 measured in step (III), it is considered that there is DNA glycosylase in the sample to be detected; if the signal A1/A0 ⁇ 1.1 measured in step (III), It is considered that there is no DNA glycosylase in the sample to be detected.
  • the DNA glycosylation enzyme solutions of different concentrations with known concentrations are substituted for the samples to be detected, and steps (I) to ( III) operation, and also includes steps:
  • step (V) Repeat steps (I) to (III) of the DNA glycosylase to be tested, and bring the obtained fluorescence intensity value into the linear curve obtained in step (IV) to calculate the concentration of active DNA glycosylase.
  • the concentration of the DNA glycosylation enzyme to be tested ranges from 1 to 500 nM, preferably from 1 to 50 nM, and more preferably from 1 to 20 nM.
  • the sufficient reaction time is 10-60 min, preferably 20-40 min, and more preferably 30 min.
  • the temperature of the sufficient reaction is 20-40°C, preferably 22-30°C, more preferably 25°C.
  • the separation label in the step (I), is biotin, and in the step (II), the separation binding label is streptavidin.
  • the separation includes sub-steps:
  • step (i) The component (c) in the detection system according to the first aspect of the present invention is added to the fully reacted detection system of step (I) to perform sufficient binding, wherein the separation-binding label refers to A label specifically bound by the separation label;
  • the solid phase carrier material is selected from the group consisting of metal, glass, colloid, plastic, or a combination thereof.
  • the material of the solid phase carrier includes: a homopolymer, a copolymer, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of polystyrene, polyethylene, polypropylene, or a combination thereof.
  • the solid-phase carrier material is selected from the group consisting of magnetic beads, microspheres, microplates, slats, test tubes, or a combination thereof.
  • the solid phase carrier is a magnetic bead.
  • the sufficient binding time is 0.5-2h, preferably 0.8-1.5h, more preferably 1h.
  • the alkaline medium is NaOH.
  • the final concentration of the alkaline medium in the detection system is 100-300 mM, preferably 150-250 mM, and more preferably 200 mM.
  • the time of the nucleic acid cleavage reaction is 15-60 min, preferably 20-40 min, more preferably 30 min.
  • the step (III) further includes transferring the supernatant of the fluorescent signal to be measured to a 96-well plate.
  • the measurement is performed by a microplate reader.
  • the fluorescent group included in the T1 chain in the detection system is FAM, and in the measurement of the fluorescent signal in step (III), the excitation wavelength is 485 nm and the emission wavelength is 520 nm.
  • kits for detecting DNA glycosylase comprising:
  • kit further includes instructions for use.
  • the detection system according to the first aspect of the present invention for detecting DNA glycosylase.
  • FIG. 1 is a schematic diagram of the mechanism of the double-stranded DNA probe of the present invention for detecting various glycosylation enzymes.
  • Figure 2 shows the fluorescence values and analysis results of U/G mismatched double-stranded DNA probes as substrates after UDG treatment at different concentrations.
  • Figure 3 shows the fluorescence values and analysis results of G/U mismatched double-stranded DNA probe as a substrate after TDG treatment at different concentrations.
  • Figure 4 shows the fluorescence values and analysis results of G/U mismatched double-stranded DNA probes as substrates after treatment with different concentrations of SMUG1.
  • Figure 5 shows the DNA glycosylation enzyme activity of Calu-1 cells.
  • the black dots and lines are the standard curve drawn with purified TDG protein, and the red dots are the fluorescence value of Calu-1 cell extract measured in this method.
  • Figure 6 shows the inhibitory efficiency of Doxorubicin at different dilutions on TDG.
  • Fig. 7 shows the inhibition rate of UDG enzyme activity by different compounds in the 384-well plate system.
  • the screening positive rate was calculated based on the threshold of 30% and 50% inhibition rate, respectively, and the value of inhibition rate less than 30% was not shown here.
  • the present inventors developed for the first time a method capable of detecting multiple DNA glycosylation enzymes with high throughput, low sample volume, large signal window, simple operation, and low cost. Specifically, the inventor introduced a uracil into one of the strands of the double-stranded DNA probe, and labeled biotin and fluorescent groups on both sides of the uracil base, respectively, and then used streptavidin magnetic beads Separate the fluorescent and biotin-labeled cleavage products with sodium hydroxide solution. The results show that the detection method of the present invention can effectively improve the detection sensitivity and increase the signal window, and the method is safe, simple, high-throughput, and easy to operate. By simply replacing the substrate probe, it can be applied to a variety of DNA glycosylation Enzymes such as UDG, TDG, SMUG and OGG1 are detected with high accuracy. On this basis, the present invention has been completed.
  • nucleic acid probe As used herein, the terms “nucleic acid probe”, “nucleic acid probe of the present invention”, “double-stranded DNA probe” and the like are used interchangeably, and refer to the double-stranded DNA probe used in the present invention for detecting DNA glycosylase .
  • the double-stranded DNA probe includes two strands of T1 and T2, and the two strands of T1 and T2 can form a double-stranded DNA structure; wherein, the T1 strand includes: at least one can be A base, a fluorescent group and a separation label recognized by the DNA glycosylase to be tested; and the fluorescent group and the separation label are respectively located at both ends of the at least one base recognized by the DNA glycosylase.
  • the concentration of the double-stranded DNA probe in the detection system is 10-80nM, preferably 20-50nM, more preferably 25-35nM, more preferably 30nM; the double-stranded
  • the length of the DNA probe is 5-200 bp, preferably 10-100 bp, more preferably 15-70 bp, more preferably 18-40 bp.
  • the base recognized by the DNA glycosylation enzyme to be tested is selected from but not limited to the following group: uracil base, cytosine base, thymine base, guanine base, methyl group Modified cytosine base, 5-carboxycytosine, alkyl adenine, etc.
  • the base recognized by the DNA glycosylation enzyme to be tested is a uracil base.
  • the at least one base recognized by the DNA glycosylation enzyme to be tested is a uracil base.
  • the at least one base recognized by the DNA glycosylation enzyme to be tested is a uracil base, and the corresponding position on the T2 chain is a guanine base.
  • the fluorescent group and the separation tag are independently located at the 5'end, 3'end, and middle of the double-stranded DNA probe.
  • the fluorescent group may be all fluorescent groups available for cross-linking with DNA probes.
  • the fluorescent group is selected from the group consisting of FAM, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Texas Red, etc.
  • the separation tag refers to a tag capable of separating the nucleic acid sequence connected to or containing the separation tag from the detection system, and may be selected from: protein, peptide or nucleic acid fragment.
  • the separation tag is selected from but not limited to the following group: antigen, antibody, ligand, receptor, avidin, biotin, or a combination thereof.
  • the separation tag is biotin.
  • the sequence of the T1 strand is 5'-FAM-S1-biotin-3', and the sequence of the T2 strand is 5'-S2-3 ', where the sequence of S1 is shown in SEQ ID NO: 1, and the sequence of S2 is shown in SEQ ID NO: 2.
  • the BER (base excision repair) pathway is the main pathway for repairing endogenous DNA base damage caused by oxidation, alkylation and deamination, and DNA glycosylase is an important link in the BER pathway.
  • DNA glycosylase can specifically excise N- ⁇ -glycosidic bonds on damaged or mismatched nucleotides, forming abasic sites (AP sites) on the DNA strand. Then AP endonuclease will cut the glycoside-phosphate bond of the damaged nucleotide, and remove the small DNA including AP site nucleotide, and the new fragment is synthesized by DNA polymerase I.
  • DNA ligase connects the two into a new repaired DNA strand to complete DNA damage repair.
  • a NaOH solution is used as an alkaline medium or nucleic acid denaturant to break the glycosidic-phosphate bond at the abasic site.
  • DNA glycosylases include: UDG (uracil DNA glycosylase), TDG (thymine DNA glycosylase), OGG1 (8-hydroxyguanine DNA glycosylase), SMUG1 (single-stranded Selective monofunctional uracil DNA glycosylase), MBD4 (methylated CpG binding domain protein 4), AAG (N-methylpurine DNA glycosylase, also known as MPG), etc.
  • UDG uracil-DNA glycosylase
  • BER base excision repair
  • TDG thymine-DNA glycosylase
  • SMUG1 single-strand selective single-functional uracil DNA glycosylase
  • SMUG1 single-strand selective single-functional uracil DNA glycosylase
  • the repair activity of single-stranded DNA is stronger than that of double-stranded DNA.
  • MBD4 (methylated CpG binding domain protein 4) contains a methyl-CpG binding domain, which can effectively remove thymine or uracil from mismatched CpG sites in vitro.
  • the methyl-CpG binding domain of MBD4 preferentially binds the main product of 5-methylcytosine CpG-TpG mismatch-methyl-CpG deamination. The combined specificity of binding and catalysis indicates that this enzyme can play a role in minimizing methyl-CpG mutations.
  • AAG N-methylpurine DNA glycosylase, also known as MPG
  • MPG N-methylpurine DNA glycosylase
  • AAG also has the ability to remove 8-oxoguanine DNA damage, it is not the main glycosylation enzyme for 8-oxoguanine repair.
  • OGG1 (8-hydroxyguanine DNA glycosylase) releases free 8-hydroxyguanine from oxidized mutagenized DNA, and double-stranded DNA on the 8-hydroxyguanine residue paired with cytosine Causes a single chain break.
  • the base recognized by the test DNA glycosylase is a uracil base
  • it can be used for UDG, TDG, SMUG1 or MBD4 Of detection.
  • the bases recognized by the DNA glycosylation enzyme to be tested in the double-stranded DNA probe can be designed according to different recognition sites of different DNA glycosylases to achieve different DNA glycosylases Detection of enzymes.
  • a detection system for detecting DNA glycosylases which includes: (a) a double-stranded DNA probe including two strands of T1 and T2, and the The two strands of T1 and T2 can form a double-stranded DNA structure; wherein, the T1 strand includes: at least one base that can be recognized by the DNA glycosylase to be tested, a fluorescent group, and a separation tag; and the fluorescent group And the separation tag are respectively located at both ends of the at least one base recognized by the DNA glycosylase; (b) a component that can break the glycosidic-phosphate bond of the abasic site of the nucleic acid.
  • the (b) may be an alkaline medium or an abasic site endonuclease (AP endonuclease).
  • AP endonuclease abasic site endonuclease
  • the alkaline medium is NaOH
  • the final concentration in the detection system is 100-300 mM, preferably 150-250 mM, more preferably 200 mM.
  • the alkaline medium NaOH solution can also be used as an alkaline medium to cut the abasic site.
  • the alkaline medium in the present invention can take advantage of the characteristics such as reduced viscosity, increased buoyancy density and accelerated sedimentation speed after denaturation of nucleic acids. It is separated more efficiently in the separation step.
  • the detection system further includes: (c) a solid phase carrier with a separation and binding tag.
  • the above (c) is used to separate the nucleic acid fragments with separation tags from the detection system.
  • the material of the solid phase carrier is selected from but not limited to the following group: metal, glass, colloid, plastic or a combination thereof.
  • the material of the solid phase carrier includes: homopolymer, copolymer, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of polystyrene, polyethylene, polypropylene, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of magnetic beads, microspheres, microplates, slats, test tubes, or a combination thereof.
  • the solid carrier is magnetic beads.
  • the final volume of the detection system is 50-200 ⁇ L, preferably 60-150 ⁇ L, more preferably 80-120 ⁇ L, more preferably 100 ⁇ L.
  • the detection system further includes a reaction buffer
  • the reaction buffer includes: Tris-Cl pH 8.0, EDTA, DTT and the like.
  • the final concentration of Tris-Cl pH 8.0 is 10-50 mM, preferably 15-30 mM, more preferably 20 mM.
  • the final concentration of EDTA is 0.5-2 mM, preferably 1 mM.
  • the final concentration of DTT is 0.5-2 mM, preferably 1 mM.
  • a method for detecting DNA glycosylase characterized in that it includes the steps of: (I) providing the component (a) in the detection system according to the first aspect of the present invention and (b), and also includes the DNA glycosylase to be tested, to perform a sufficient reaction; (II) using the separation tag described in the first aspect of the present invention, to separate the nucleic acid fragments bearing the separation tag from the detection system; and (III) The fluorescent signal is measured by the detection system after separating the nucleic acid fragments with separation tags.
  • the detection method of the present invention includes: qualitative detection and quantitative detection.
  • the qualitative detection includes: detecting whether the sample to be tested contains DNA glycosylase; the quantitative detection includes: detecting the concentration of the active DNA glycosylase in the sample to be detected.
  • the method further includes performing a parallel control experiment on a blank sample, the blank sample does not contain DNA glycosylase, and the measured fluorescence signal is A0.
  • the fluorescence signal obtained after measurement using the DNA glycosylation enzyme to be tested is A1. If A1/A0>1.1, it is considered that there is DNA glycosylation enzyme in the sample to be tested; if A1/A0 ⁇ 1.1, the sample to be tested is considered There is no DNA glycosylase.
  • the DNA glycosylation enzyme solutions of different concentrations with known concentrations are substituted for the samples to be detected, and steps (I) to ( III) operation, and also includes steps: (IV) construct a linear curve of fluorescence intensity and known DNA glycosylase; (V) repeat steps (I) to (III) of the DNA glycosylation enzyme to be tested, The obtained fluorescence intensity value is taken into the linear curve obtained in step (IV), and the concentration of active DNA glycosylase is calculated.
  • the concentration range of the DNA glycosylase to be tested is 1 to 500 nM, preferably 1 to 50 nM, and more preferably 1 to 20 nM.
  • the sufficient reaction time is 10-60 min, preferably 20-40 min, and more preferably 30 min.
  • the temperature of the sufficient reaction is 20-40°C, preferably 22-30°C, more preferably 25°C.
  • the separation label in the step (I), is biotin, and in the step (II), the separation binding label is streptavidin .
  • the separation includes sub-steps:
  • the solid phase carrier with the separation and binding tag is added to the fully reacted detection system of step (I) to perform sufficient binding, wherein the separation and binding tag refers to a label that can specifically bind to the separation tag (Ii)
  • an alkaline medium is added to perform a nucleic acid cleavage reaction.
  • the solid-phase carrier material is selected from but not limited to the following group: metal, glass, colloid, plastic, or a combination thereof.
  • the material of the solid phase carrier includes: homopolymer, copolymer, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of polystyrene, polyethylene, polypropylene, or a combination thereof.
  • the material of the solid phase carrier is selected from the group consisting of magnetic beads, microspheres, microplates, slats, test tubes, or a combination thereof.
  • the solid support is magnetic beads.
  • the sufficient binding time is 0.5-2h, preferably 0.8-1.5h, more preferably 1h.
  • the alkaline medium is NaOH
  • the final concentration in the detection system is 100-300 mM, preferably 150-250 mM, more preferably 200 mM
  • the reaction time is 15-60min, preferably 20-40min, more preferably 30min.
  • the step (III) further includes transferring the supernatant of the fluorescent signal to be measured to a 96-well plate; in the measurement of the fluorescent signal of the step (III), the determination Performed by a microplate reader.
  • the fluorescent group included in the T1 chain in the detection system is FAM, and in the measurement of the fluorescent signal in step (III), the excitation wavelength is 485 nm and the emission wavelength is 520 nm.
  • streptavidin magnetic beads and a NaOH solution capable of denaturing nucleic acid are sequentially added, and then removed
  • the double-stranded DNA at the base site is combined with streptavidin-labeled magnetic beads;
  • the alkaline medium cleaves the abasic site, leaving a nucleotide gap at the site, thereby making the 5'
  • the DNA fragment with FAM at the end is disconnected from the DNA fragment with biotin at the 3'end, and the DNA double helix structure is destroyed by the alkaline medium, causing the FAM-labeled DNA fragment and the biotin-labeled DNA fragment to separate from their complementary strands;
  • the streptavidin-labeled magnetic beads can be adsorbed by the magnetic stand, and the DNA fragment with the fluorescent group FAM is released into the supernatant.
  • the biotin-labeled nucleic acid fragment is bound to a solid carrier such as magnetic
  • the cleaved nucleic acid probes remain in the original detection system solution, and they all carry fluorescent groups. Therefore, by detecting the fluorescence signal, the concentration of DNA glycosylase in the test solution can be obtained.
  • the detection method provided by the present invention is of great significance for early diagnosis of abnormal expression of DNA glycosylase and development of inhibitors targeting DNA glycosylase.
  • the present invention provides a kit for detecting DNA glycosylase, characterized in that the kit includes: (a) a first container and the first container in the first container according to the first aspect of the present invention (A) in the detection system; (b) the second container and (b) in the detection system according to the first aspect of the invention located in the second container; and (c) the third container and the third container (C) in the detection system according to the first aspect of the present invention.
  • kit further includes instructions for use.
  • the present invention has designed a method that can detect the activity of multiple DNA glycosylases.
  • a double-stranded DNA fluorescent probe containing a corresponding DNA glycosylase recognition site is used. This principle is applicable to Various DNA glycosylases;
  • High-throughput and low cost The method in this solution can be applied to a high-throughput screening system, which can do 384-well plate-level screening, and the cost is less than 1 yuan/well.
  • the alkaline medium in the present invention can also be used as a nucleic acid denaturant.
  • AP endonuclease abasic site endonuclease
  • the viscosity and buoyancy can be reduced after denaturation of nucleic acid
  • the characteristics of increased density and accelerated sedimentation speed make the cut nucleic acid fragments more efficiently separated in the separation step.
  • DNA oligonucleotides are synthesized and purified by Jerry Bioengineering Co., Ltd. (Shanghai).
  • Uracil DNA glycosylase (UDG), single-stranded selective monofunctional uracil DNA glycosylase (SMUG1) and alkyl adenine DNA glycosylase (AAG) were purchased from NEB (Massachusetts, USA), Thymidine Glycosylase (TDG) was obtained by overexpression and purification in E. coli; streptavidin magnetic beads were purchased from Thermo Fisher Co., Ltd. (USA, Massachusetts), and the magnetic stand was purchased from Borsch (Shenzhen), Sodium hydroxide was purchased from Sinopharm Group; the ultrapure water used in the preparation of the solution came from Millipore Milli-Q water purification system.
  • Fluorescence detection was performed using a Kendi Infinite-200 fluorescence spectrometer (Switzerland, Kendi); excitation wavelength was 485nm, emission wavelength was 520nm; excitation slit width was 20nm, and emission slit width was 10nm.
  • T1-T2 containing uracil base
  • the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1);
  • the nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), the T1 chain and the T2 chain complement each other to form a double-stranded DNA probe T1-T2.
  • the composition of the reaction buffer is: 20mM Tris-Cl pH8.0, 1mM EDTA , 1mM DTT; the final volume is 100 ⁇ L, the concentration of double-stranded DNA probes T1-T2 in the system is 30nM, the system is incubated at 25 °C for 30 minutes, so that the base excision reaction occurs, and then 1 ⁇ L streptavidin magnetic beads
  • the reaction solution was added and the biotin-labeled DNA was fully combined for 1 hour. NaOH was added to the reaction solution to a final concentration of 200 mM.
  • the streptavidin magnetic beads were adsorbed to the tube wall under the action of a magnetic stand.
  • the supernatant was transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorescence measurement.
  • T1-T2 containing uracil base
  • the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1);
  • the nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), the T1 chain and the T2 chain complement each other to form a double-stranded DNA probe T1-T2.
  • the composition of the reaction buffer is: 20mM HEPES pH7.5, 100mM NaCl, 0.2 mM EDTA, 2.5 mM MgCl 2 ; the final volume is 100 ⁇ L, the concentration of the double-stranded DNA probe T1-T2 in the system is 30 nM, and the system is incubated at 25° C. for 30 minutes to allow the base excision reaction to occur, and then 1 ⁇ L Streptavidin magnetic beads were added to the reaction solution to fully bind the biotin-labeled DNA for 1 hour.
  • NaOH was added to the reaction solution to a final concentration of 200 mM. Incubate at room temperature for 30 minutes. Under the action of a magnetic stand, streptavidin magnetic The beads are adsorbed to the wall of the tube, and the supernatant is transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorescence measurement.
  • T1-T2 containing uracil base
  • the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1);
  • the nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), the T1 chain and the T2 chain complement each other to form a double-stranded DNA probe T1-T2;
  • the composition of the reaction buffer is: 10mM Tris-Cl pH 7.0, 10mM MgCl 2 , 1 mM DTT; the final volume is 100 ⁇ L, the concentration of double-stranded DNA probes T1-T2 in the system is 30 nM, and the system is incubated at 25° C. for 30 minutes to cause base excision reaction, and then 1 ⁇ L of streptavidin Add the magnetic beads to the reaction solution and fully bind the biotin-labeled DNA for 1 hour.
  • Example 4 Detection of TDG activity in lysates of lung cancer (Calu-1) cells
  • the detection method of the present invention was used to analyze and detect the TDG activity in Calu-1 cell lysate.
  • the specific steps are as follows:
  • Calu-1 cell samples were separated by centrifugation (5 min, 1000 rpm, 4° C.) and resuspended in lysis buffer (20 mM Tris-Cl pH 8.0, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM DTT, 1 mM EDTA); the mixture was on ice Leave on top for 10 min and centrifuge at 3500 rpm for 10 minutes, remove the supernatant, resuspend the pellet with nuclear lysate (20 mM Tris-Cl pH 8.0, 420 mM NaCl, 10 mM KCl, 1 mM DTT, 1 mM EDTA) and resuspend the pellet in Allow to stand on ice for 20min and centrifuge at 12000rpm for 20 minutes.
  • the supernatant is Calu-1 nuclear cell lysate; Calu-1 nuclear cell lysate can be directly tested for TDG activity without further processing; detection The method is the same as the pure T
  • the measured fluorescence value is brought into the drawn linear curve of TDG protein concentration-fluorescence value, and it is calculated that 10 7 Calu-1 cells probably contain TDG protein equivalent to 9.12 nM.
  • Doxorubicin is a small molecule compound that binds to double-stranded DNA. It can affect the ability of TDG to recognize double-stranded DNA after it is combined with DNA. 50nM TDG was mixed with different concentrations of Doxorubicin and incubated at 37°C for 30min; double-stranded DNA probes T1-T2 were added to the mixture to obtain a system with a final volume of 100 ⁇ L, and incubated at 25°C for 30min; then 1 ⁇ L of streptavidin The magnetic beads were added to the mixed system and reacted fully for 1 hour; NaOH was added to the reaction solution to a final concentration of 200mM and incubated at room temperature for 1 hour. Under the action of the magnetic stand, the streptavidin magnetic beads were adsorbed to the tube wall, The clear solution was transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorescence measurement; all experiments were repeated twice.
  • the fluorescence value of the solvent control group is 100%, and the fluorescence value of Doxorubicin at different concentrations is calculated as the activity ratio relative to the solvent control group. 6
  • UDG has a final concentration of 50 nm/L and reaction buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.2 mM EDTA, 2.5mM MgCl 2 ) mixed, added to a 384-well plate through a pipetting station, the volume of the mixture in each well was 19.6 ⁇ L, and the compound (1 mM) arranged in the 384-well plate was added to the TDG containing pipette through the pipetting station In the 384-well plate of the mixed solution (0.4 ⁇ L compound/well), the final concentration of the compound is 20 ⁇ M; the enzyme and the compound are pre-incubated at room temperature for 30 min, and 10 ⁇ L of double-stranded DNA probe is added to the final concentration of 30 nm/L, and incubated at 25°C for 30 Minutes, abasic sites are generated
  • the fluorescence value of the solvent control group was 100% and the inhibition rate was 0%, and the inhibition rate of the fluorescence value of different compounds relative to the solvent control group was calculated.
  • the 30% enzyme activity inhibition rate is a threshold, a 1.5% screening positive rate can be obtained, and if the 50% enzyme activity inhibition rate is a threshold, a 0.9% screening positive rate can be obtained.

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Abstract

一种用于检测DNA糖基化酶的检测体系，包括：(a)双链DNA探针，所述双链DNA探针包括T1和T2两条链，并且所述T1和T2两条链可形成双链DNA结构；其中，所述T1链包括：至少一个可被待测DNA糖基化酶识别的碱基、荧光基团和分离标签；并且所述荧光基团和分离标签分别位于所述至少一个可被DNA糖基化酶识别的碱基的两端；(b)可使核酸脱碱基位点的糖苷-磷酸键断裂的组分；和(c)带有分离结合标签的固相载体。所述检测体系通量高、需要样品量低、信号窗口大、操作简便、成本低、且能够检测多种DNA糖基化酶。

Description

一种可检测多种DNA糖基化酶活性的方法 技术领域
本发明属于生物分析技术领域，具体涉及一种可检测多种DNA糖基化酶活性的方法及其应用。
背景技术
BER(碱基切除修复)通路是修复由氧化、烷基化和脱氨基所引起的内源性DNA碱基损伤的主要途径。其中DNA糖基化酶是BER通路中的重要环节，包括：UDG(尿嘧啶DNA糖基化酶)、TDG(胸腺嘧啶DNA糖基化酶)和OOG1(8-羟基鸟嘌呤DNA糖基化酶)等。DNA糖基化酶可以特异性切除受损或错配核苷酸上的N-β-糖苷键，在DNA链上形成脱碱基位点(AP位点)。接着AP核酸内切酶会把受损核苷酸的糖苷-磷酸键切开，并移去包括AP位点核苷酸在内的小片段DNA，并由DNA聚合酶I合成新的片段，最终由DNA连接酶把两者连成新的被修复DNA链，完成DNA损伤修复。
DNA糖基化酶的异常表达与人类疾病密切相关，因此对DNA糖基化酶活性的快速、准确、方便、高通量检测对于临床诊断和靶向抑制剂的研发具有重要意义。现有的传统方法如使用荧光标记底物的凝胶电泳法灵敏度偏低，需要样品量大，通量低，不适合定量分析；用放射同位素标记底物代替荧光标记底物可以提高灵敏度，降低样品量，但同位素标记会引起放射性污染；高效液相色谱法的定性和定量能力较强，需要的样品量不多，但样品的前处理复杂，通量同样不高；酶联免疫吸附法和免疫印迹法需要与蛋白结合的特异性抗体，操作步骤繁琐，不适合大量样品的检测；荧光方法依赖于用荧光团和猝灭剂进行外部标记，用于均相测定，适合高通量，但其信号窗口相对较小，且底物探针设计复杂费用高昂。
因此，本领域迫切需要开发一种通量高、需要样品量低、信号窗口大、操作简便、成本低的能够检测多种DNA糖基化酶的方法。
发明内容
本发明的目的就是提供一种通量高、需要样品量低、信号窗口大、操作简便、成本低的能够检测多种DNA糖基化酶的方法。
在本发明的第一方面，提供了一种用于检测DNA糖基化酶的检测体系，所述检测体系包括：
(a)双链DNA探针，所述双链DNA探针包括T1和T2两条链，并且所述T1和T2两条链可形成双链DNA结构；
其中，所述T1链包括：至少一个可被待测DNA糖基化酶识别的碱基、荧光基团和分离标签；
并且所述荧光基团和分离标签分别位于所述至少一个可被DNA糖基化酶识别的碱基的两端；
(b)可使核酸脱碱基位点的糖苷-磷酸键断裂的组分；和
(c)带有分离结合标签的固相载体。
在另一优选例中，所述检测体系中，所述(a)中，双链DNA探针的浓度为10-80nM，较佳地为20-50nM，更佳地为25-35nM，更佳地为30nM。
在另一优选例中，所述双链DNA探针的长度为5-200bp，较佳地为10-100bp，更佳地为15-70bp，更佳地为18-40bp。
在另一优选例中，所述可被待测DNA糖基化酶识别的碱基选自下组：尿嘧啶碱基、胞嘧啶碱基、胸腺嘧啶碱基、鸟嘌呤碱基、甲基化修饰的胞嘧啶碱基、5-羧基胞嘧啶、烷基腺嘌呤等。
在另一优选例中，所述可被待测DNA糖基化酶识别的碱基为尿嘧啶碱基。
在另一优选例中，所述至少一个可被待测DNA糖基化酶识别的碱基为一个尿嘧啶碱基。
在另一优选例中，所述至少一个可被待测DNA糖基化酶识别的碱基为一个尿嘧啶碱基，并且T2链上的相应位置为鸟嘌呤碱基。
在另一优选例中，所述荧光基团和分离标签各自独立地位于所述双链DNA探针的5’端、3’端和中部。
在另一优选例中，所述荧光基团包括可以用于与DNA探针交联的荧光基团。
在另一优选例中，所述荧光基团选自下组：FAM、FITC、BODIPY-FL、G-Dye100、FluorX、Cy3、Cy5、Texas Red等。
在另一优选例中，所述分离标签是指能够使连接于或包含所述分离标签的 核酸序列从检测体系中被分离出来的标签。
在另一优选例中，所述分离标签选自下组：蛋白质、肽段或核酸片段。
在另一优选例中，所述分离标签选自下组：抗原、抗体、配体、受体、亲和素、生物素，或其组合。
在另一优选例中，所述分离标签为生物素。
在另一优选例中，所述T1链的序列为5’-FAM-S1-biotin-3’，并且所述T2链的序列为5’-S2-3’，其中，S1的序列如SEQ ID NO:1所示，S2的序列如SEQ ID NO:2所示。
在另一优选例中，所述检测体系中，所述(b)选自下组：碱性介质或脱碱基位点核酸内切酶(AP核酸内切酶)。
在另一优选例中，所述碱性介质为NaOH。
在另一优选例中，所述碱性介质在检测体系中的终浓度为100-300mM，较佳地为150-250mM，更佳地为200mM。
在另一优选例中，所述检测体系中，所述固相载体材质选自下组：金属、玻璃、胶体、塑料或其组合。
在另一优选例中，所述的固相载体材质包括：均聚物、共聚物、或其组合。
在另一优选例中，所述的固相载体材质选自下组：聚苯乙烯、聚乙烯、聚丙烯、或其组合。
在另一优选例中，所述的固相载体材质选自下组：磁珠、微球、微孔板、板条、试管、或其组合。
在另一优选例中，所述的固相载体为磁珠。
在另一优选例中，所述检测体系的终体积为50-200μL，较佳地为60-150μL，更佳地为80-120μL，更佳地为100μL。
在另一优选例中，所述检测体系还包括反应缓冲液。
在另一优选例中，所述反应缓冲液包括：Tris-Cl pH8.0、EDTA、DTT等。
在另一优选例中，所述Tris-Cl pH8.0的终浓度为10-50mM，较佳地为15-30mM，更佳地为20mM。
在另一优选例中，所述EDTA的终浓度为0.5-2mM，较佳地为1mM。
在另一优选例中，所述DTT的终浓度为0.5-2mM，较佳地为1mM。
在另一优选例中，所述检测体系还包括待测DNA糖基化酶。
在另一优选例中，所述待测DNA糖基化酶选自下组：UDG、TDG、SMUG1、MBD4、OGG1、AAG，或其组合。
在另一优选例中，所述待测DNA糖基化酶选自下组：UDG、TDG或SMUG1。
在另一优选例中，所述待测DNA糖基化酶的浓度范围为1至500nM，较佳地为1至50nM，更佳地为1至20nM。
在另一优选例中，所述DNA糖基化酶选自下组：纯化的DNA糖基化酶及其溶解液、细胞裂解液、血液或其抽提物、体液或其抽提物，或其组合。
在另一优选例中，所述细胞裂解液包括癌症细胞裂解液。
在另一优选例中，所述癌症包括肺癌。
在另一优选例中，所述的检测包括：定性检测和定量检测。
在本发明的第二方面，提供了一种用于检测DNA糖基化酶的方法，包括步骤：
(I)提供如本发明第一方面所述的检测体系中的(a)和(b)，并且还包括待测DNA糖基化酶，进行充分反应；
(II)利用本发明第一方面中所述的分离标签，将带有分离标签的核酸片段从检测体系中分离；和
(III)将分离出带有分离标签的核酸片段后的检测体系进行荧光信号的测定。
在另一优选例中，所述方法的检测包括：定性检测和定量检测。
在另一优选例中，所述定性检测包括：对待检测样品中是否含有DNA糖基化酶的检测。
在另一优选例中，所述定量检测包括：对待检测样品中有活性的DNA糖基化酶的浓度的检测。
在另一优选例中，所述方法还包括进行空白样品的平行对照实验，所述空白样品中不包含DNA糖基化酶，且测得的荧光信号为A0。
在另一优选例中，若步骤(III)测得的信号A1/A0>1.1，则认为待检测样品中有DNA糖基化酶；若步骤(III)测得的信号A1/A0≤1.1，则认为待检测样品中没 有DNA糖基化酶。
在另一优选例中，当所述方法用于定量检测DNA糖基化酶时，将浓度已知的不同浓度的DNA糖基化酶溶液替换所述待检测样品，进行步骤(I)至(III)的操作，并且还包括步骤：
(IV)构建荧光强度与已知的DNA糖基化酶的线性曲线；
(V)将待测DNA糖基化酶重复步骤(I)至(III)，将所得其荧光强度数值带入步骤(IV)所得的线性曲线，计算有活性的DNA糖基化酶的浓度。
在另一优选例中，所述步骤(I)中，所述待测DNA糖基化酶的浓度范围为1至500nM，较佳地为1至50nM，更佳地为1至20nM。
在另一优选例中，所述步骤(I)中，所述充分反应的时间为10-60min，较佳地为20-40min，更佳地为30min。
在另一优选例中，所述步骤(I)中，所述充分反应的温度为20-40℃，较佳地为22-30℃，更佳地为25℃。
在另一优选例中，所述步骤(I)中，所述的分离标签为生物素，并且所述步骤步骤(II)中，所述的分离结合标签为链霉亲和素。
在另一优选例中，所述步骤(II)中，所述分离包括子步骤：
(i)将如本发明第一方面所述的检测体系中的组分(c)加入步骤(I)的充分反应后的检测体系，进行充分结合，其中，所述分离结合标签是指可与所述分离标签特异性结合的标签；
(ii)向子步骤(i)的充分结合后的检测体系中，加入如本发明第一方面所述的检测体系中的组分(b)，进行核酸切割反应。
在另一优选例中，所述子步骤(i)中，所述固相载体材质选自下组：金属、玻璃、胶体、塑料或其组合。
在另一优选例中，所述子步骤(i)中，所述的固相载体材质包括：均聚物、共聚物、或其组合。
在另一优选例中，所述子步骤(i)中，所述的固相载体材质选自下组：聚苯乙烯、聚乙烯、聚丙烯、或其组合。
在另一优选例中，所述子步骤(i)中，所述的固相载体材质选自下组：磁珠、微球、微孔板、板条、试管、或其组合。
在另一优选例中，所述子步骤(i)中，所述的固相载体为磁珠。
在另一优选例中，所述子步骤(i)中，所述充分结合的时间为0.5-2h，较佳地0.8-1.5h，更佳地1h。
在另一优选例中，所述子步骤(ii)中，所述碱性介质为NaOH。
在另一优选例中，所述碱性介质在检测体系中的终浓度为100-300mM，较佳地为150-250mM，更佳地为200mM。
在另一优选例中，所述子步骤(ii)中，所述核酸切割反应的时间为15-60min，较佳地为20-40min，更佳地为30min。
在另一优选例中，所述步骤(III)中，还包括将待测定荧光信号上清液转移至96孔板中。
在另一优选例中，所述步骤(III)的荧光信号的测定中，所述测定通过酶标仪进行。
在另一优选例中，所述检测体系中T1链所包括的荧光基团为FAM，并且所述步骤(III)的荧光信号的测定中，激发波长为485nm，发射波长为520nm。
在本发明第三方面，提供了一种用于检测DNA糖基化酶的试剂盒，所述试剂盒包括：
(a)第一容器以及位于第一容器中的如本发明第一方面所述的检测体系中的(a)；
(b)第二容器以及位于第二容器中的如本发明第一方面所述的检测体系中的(b)；和
(c)第三容器以及位于第三容器中的如本发明第一方面所述的检测体系中的(c)。
在另一优选例中，所述试剂盒还包括使用说明书。
在本发明第四方面，提供了一种如本发明第一方面所述的检测体系的用途，用于检测DNA糖基化酶。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方 案。限于篇幅，在此不再一一累述。
附图说明
图1为本发明所述双链DNA探针用于检测多种糖基化酶的机理示意图。
图2为以G/U错配双链DNA探针为底物，不同浓度UDG处理后的荧光值及分析结果。
图3为以G/U错配双链DNA探针为底物，不同浓度TDG处理后的荧光值及分析结果。
图4为以G/U错配双链DNA探针为底物，不同浓度SMUG1处理后的荧光值及分析结果。
图5为Calu-1细胞中所含DNA糖基化酶酶活。黑色点及线为利用纯化TDG蛋白绘制的标准曲线，红色点为Calu-1细胞提取物在本方法中测定的荧光值。
图6为不同稀释浓度的Doxorubicin对TDG的酶活抑制效率。
图7为384孔板体系中，不同化合物对UDG的酶活抑制率，分别以30％及50％抑制率为阈值计算筛选阳性率，抑制率低于30％的值未在此显示。
具体实施方式
本发明人经过广泛而深入的研究，经过大量筛选，首次开发了一种通量高、需要样品量低、信号窗口大、操作简便、成本低的能够检测多种DNA糖基化酶的方法。具体地，本发明人将双链DNA探针的其中一条链中引入一个尿嘧啶，并且在该尿嘧啶碱基的两侧分别标记生物素和荧光基团，进而利用链霉亲和素磁珠和氢氧化钠溶液对荧光和生物素标记的酶切产物进行分离。结果表明，使用本发明的检测方法，可以有效的提高检测灵敏度，提高信号窗口，且方法安全、简单、通量高、易操作，通过简单更换底物探针可以适用于多种DNA糖基化酶如UDG、TDG、SMUG和OGG1等的酶活检测，且精确度高。在此基础上完成了本发明。
术语
双链DNA探针
如本文所用，术语“核酸探针”、“本发明核酸探针”、“双链DNA探针”等可互换使用，指本发明中用于检测DNA糖基化酶的双链DNA探针。
在本发明中，所述的双链DNA探针包括T1和T2两条链，并且所述T1和T2两条链可形成双链DNA结构；其中，所述的T1链包括：至少一个可被待测DNA糖基化酶识别的碱基、荧光基团和分离标签；并且所述荧光基团和分离标签分别位于所述至少一个可被DNA糖基化酶识别的碱基的两端。
在本发明中，所述的双链DNA探针在检测体系中的浓度为10-80nM，较佳地为20-50nM，更佳地为25-35nM，更佳地为30nM；所述双链DNA探针的长度为5-200bp，较佳地为10-100bp，更佳地为15-70bp，更佳地为18-40bp。
在本发明中，所述可被待测DNA糖基化酶识别的碱基选自但不限于下组：尿嘧啶碱基、胞嘧啶碱基、胸腺嘧啶碱基、鸟嘌呤碱基、甲基化修饰的胞嘧啶碱基、5-羧基胞嘧啶、烷基腺嘌呤等。在另一优选例中，所述可被待测DNA糖基化酶识别的碱基为尿嘧啶碱基。
在一个优选的实施方式中，所述至少一个可被待测DNA糖基化酶识别的碱基为一个尿嘧啶碱基。
在一个优选的实施方式中，所述至少一个可被待测DNA糖基化酶识别的碱基为一个尿嘧啶碱基，并且T2链上的相应位置为鸟嘌呤碱基。
在本发明中，所述荧光基团和分离标签各自独立地位于所述双链DNA探针的5’端、3’端和中部。
在本发明中，所述荧光基团可以是所有可用于与DNA探针交联的荧光基团。在另一优选例中，所述荧光基团选自下组：FAM、FITC、BODIPY-FL、G-Dye100、FluorX、Cy3、Cy5、Texas Red等。
在本发明中，所述分离标签是指能够使连接于或包含所述分离标签的核酸序列从检测体系中被分离出来的标签，可以选自：蛋白质、肽段或核酸片段。
在一个优选的实施方式中，所述分离标签选自但不限于下组：抗原、抗体、配体、受体、亲和素、生物素，或其组合。
在一个优选的实施方式中，所述分离标签为生物素。
在一个优选的实施方式中，所述双链DNA探针中，所述T1链的序列为5’-FAM-S1-biotin-3’，并且所述T2链的序列为5’-S2-3’，其中，S1的序列如SEQ ID NO:1所示，S2的序列如SEQ ID NO:2所示。
DNA糖基化酶
BER(碱基切除修复)通路是修复由氧化、烷基化和脱氨基所引起的内源性DNA碱基损伤的主要途径，而DNA糖基化酶是BER通路中的重要环节。DNA糖基化酶可以特异性切除受损或错配核苷酸上的N-β-糖苷键，在DNA链上形成脱碱基位点(AP位点)。接着AP核酸内切酶会把受损核苷酸的糖苷-磷酸键切开，并移去包括AP位点核苷酸在内的小片段DNA，并由DNA聚合酶I合成新的片段，最终由DNA连接酶把两者连成新的被修复DNA链，完成DNA损伤修复。
在本发明的一个实施方式中，使用NaOH溶液作为碱性介质或核酸变性剂，使脱碱基位点的的糖苷-磷酸键断裂。
已知的DNA糖基化酶包括：UDG(尿嘧啶DNA糖基化酶)、TDG(胸腺嘧啶DNA糖基化酶)、OGG1(8-羟基鸟嘌呤DNA糖基化酶)、SMUG1(单链选择性单功能尿嘧啶DNA糖基化酶)、MBD4(甲基化CpG结合结构域蛋白4)、AAG(N-甲基嘌呤DNA糖基化酶，也称MPG)等。
UDG(尿嘧啶-DNA糖基化酶)的一个重要功能是通过切割N-糖基键并启动碱基切除修复(BER)途径从DNA分子中消除尿嘧啶来防止诱变。尿嘧啶碱基一般发生在胞嘧啶脱氨基或dUMP残基的错误掺入中。
TDG(胸腺嘧啶-DNA糖基化酶)通过水解DNA的糖-磷酸骨架和错配的胸腺嘧啶之间的碳-氮键从G/T错配中去除胸腺嘧啶部分。由于活性较低，该酶还可从C/T和T/T错配中去除胸腺嘧啶。TDG还可以用鸟嘌呤从错误配对中去除尿嘧啶和5-溴尿嘧啶。该酶在细胞防御由5-甲基胞嘧啶和胞嘧啶的自发脱氨作用引起的遗传突变中起重要作用。
SMUG1(单链选择性单功能尿嘧啶DNA糖基化酶)，可从核染色质中的单链和双链DNA中去除尿嘧啶及5-羟甲基尿嘧啶，从而有助于碱基切除修复。一般来说对单链DNA的修复活性比双链DNA强。
MBD4(甲基化CpG结合结构域蛋白4)含有甲基-CpG结合结构域，其可以在体外从错配的CpG位点有效地去除胸腺嘧啶或尿嘧啶。此外，MBD4的甲基-CpG结合结构域优先结合5-甲基胞嘧啶CpG-TpG错配-甲基-CpG脱氨的主要产物。结合和催化的组合特异性表明该酶可以起到最小化甲基-CpG突变的作用。
AAG(N-甲基嘌呤DNA糖基化酶，也称MPG)是哺乳动物体内是唯一对3-甲 基腺嘌呤、次黄嘌呤和1，N6-乙烯基腺嘌呤具有活性的DNA糖基化酶。虽然AAG也具有去除8-氧鸟嘌呤DNA损伤的能力，但它不是8-氧鸟嘌呤修复的主要糖基化酶。
OGG1(8-羟基鸟嘌呤DNA糖基化酶)从被氧化诱变的DNA中释放出游离的8-羟基鸟嘌呤，并在与胞嘧啶配对的8-羟基鸟嘌呤残基上的双链DNA中造成单链断裂。
在本发明的一个实施方式中，当所述双链DNA探针中，所述可被待测DNA糖基化酶识别的碱基为尿嘧啶碱基时，可用于UDG、TDG、SMUG1或MBD4的检测。
在本发明中，可根据不同DNA糖基化酶的不同识别位点，设计双链DNA探针中所述的可被待测DNA糖基化酶识别的碱基，以实现不同的DNA糖基化酶的检测。
本发明的检测体系
在本发明中，提供了一种用于检测DNA糖基化酶的检测体系，包括：(a)双链DNA探针，所述双链DNA探针包括T1和T2两条链，并且所述T1和T2两条链可形成双链DNA结构；其中，所述T1链包括：至少一个可被待测DNA糖基化酶识别的碱基、荧光基团和分离标签；并且所述荧光基团和分离标签分别位于所述至少一个可被DNA糖基化酶识别的碱基的两端；(b)可使核酸脱碱基位点的糖苷-磷酸键断裂的组分。
在本发明的检测体系中，所述的(b)可以是碱性介质或脱碱基位点核酸内切酶(AP核酸内切酶)。
在一个优选的实施方式中，所述碱性介质为NaOH，并且在检测体系中的终浓度为100-300mM，较佳地为150-250mM，更佳地为200mM。
在本发明中，所述的碱性介质NaOH溶液，同时可作为碱性介质，对脱碱基位点进行切割。
本发明中的碱性介质，相较于传统的脱碱基位点核酸内切酶(AP核酸内切酶)，可利用核酸变性以后粘度下降、浮力密度升高、沉降速度加快等特点，在分离步骤中更加高效的被分离。
在另一优选例中，所述的检测体系中还包括：(c)带有分离结合标签的固相载体。所述的(c)用于将带有分离标签的核酸片段从检测体系中分离。
其中，所述固相载体材质选自但不限于下组：金属、玻璃、胶体、塑料或其组合。在另一优选例中，所述的固相载体材质包括：均聚物、共聚物、或其组合。在另一优选例中，所述的固相载体材质选自下组：聚苯乙烯、聚乙烯、聚丙烯、或其组合。在另一优选例中，所述的固相载体材质选自下组：磁珠、微球、微孔板、板条、试管、或其组合。
在一个优选的实施方式中，所述的固相载体为磁珠。
在本发明中，所述检测体系的终体积为50-200μL，较佳地为60-150μL，更佳地为80-120μL，更佳地为100μL。
在另一优选例中，所述检测体系还包括反应缓冲液，所述的反应缓冲液包括：Tris-Cl pH8.0、EDTA、DTT等。在一个优选的实施方式中，所述Tris-Cl pH8.0的终浓度为10-50mM，较佳地为15-30mM，更佳地为20mM。在一个优选的实施方式中，所述EDTA的终浓度为0.5-2mM，较佳地为1mM。在一个优选的实施方式中，所述DTT的终浓度为0.5-2mM，较佳地为1mM。
本发明的检测方法
在本发明中，提供了一种用于检测DNA糖基化酶的方法，其特征在于，包括步骤：(I)提供如本发明第一方面所述的检测体系中的组分(a)和(b)，并且还包括待测DNA糖基化酶，进行充分反应；(II)利用本发明第一方面中所述的分离标签，将带有分离标签的核酸片段从检测体系中分离；和(III)将分离出带有分离标签的核酸片段后的检测体系进行荧光信号的测定。
本发明的检测方法包括：定性检测和定量检测。
在本发明中，所述定性检测包括：对待检测样品中是否含有DNA糖基化酶的检测；所述定量检测包括：对待检测样品中有活性的DNA糖基化酶的浓度的检测。
在一个优选的实施方式中，所述方法还包括进行空白样品的平行对照实验，所述空白样品中不包含DNA糖基化酶，且测得的荧光信号为A0。使用待测DNA糖基化酶进行测定后所得的荧光信号为A1，若A1/A0>1.1，则认为待检测 样品中有DNA糖基化酶；若A1/A0≤1.1，则认为待检测样品中没有DNA糖基化酶。
在另一优选例中，当所述方法用于定量检测DNA糖基化酶时，将浓度已知的不同浓度的DNA糖基化酶溶液替换所述待检测样品，进行步骤(I)至(III)的操作，并且还包括步骤：(IV)构建荧光强度与已知的DNA糖基化酶的线性曲线；(V)将待测DNA糖基化酶重复步骤(I)至(III)，将所得其荧光强度数值带入步骤(IV)所得的线性曲线，计算有活性的DNA糖基化酶的浓度。
在本发明中，所述待测DNA糖基化酶的浓度范围为1至500nM，较佳地为1至50nM，更佳地为1至20nM。
在另一优选例中，所述步骤(I)中，所述充分反应的时间为10-60min，较佳地为20-40min，更佳地为30min。在另一优选例中，所述步骤(I)中，所述充分反应的温度为20-40℃，较佳地为22-30℃，更佳地为25℃。
在本发明的一个优选的实施方式中，所述步骤(I)中，所述的分离标签为生物素，并且所述步骤步骤(II)中，所述的分离结合标签为链霉亲和素。
在另一优选例中，所述步骤(II)中，所述分离包括子步骤：
(i)将带有分离结合标签的固相载体加入步骤(I)的充分反应后的检测体系，进行充分结合，其中，所述分离结合标签是指可与所述分离标签特异性结合的标签；(ii)向子步骤(i)的充分结合后的检测体系中，加入碱性介质，进行核酸切割反应。
在所述的子步骤(i)中，所述固相载体材质选自但不限于下组：金属、玻璃、胶体、塑料或其组合。在另一优选例中，所述的固相载体材质包括：均聚物、共聚物、或其组合。在另一优选例中，所述的固相载体材质选自下组：聚苯乙烯、聚乙烯、聚丙烯、或其组合。在另一优选例中，所述的固相载体材质选自下组：磁珠、微球、微孔板、板条、试管、或其组合。
在一个优选的实施方式中，所述子步骤(i)中，所述的固相载体为磁珠。
在另一优选例中，所述子步骤(i)中，所述充分结合的时间为0.5-2h，较佳地0.8-1.5h，更佳地1h。
在一个优选的实施方式中，所述子步骤(ii)中，所述碱性介质为NaOH，在检测体系中的终浓度为100-300mM，较佳地为150-250mM，更佳地为200mM； 并且反应的时间为15-60min，较佳地为20-40min，更佳地为30min。
在另一优选的实施方式中，所述步骤(III)中，还包括将待测定荧光信号上清液转移至96孔板中；所述步骤(III)的荧光信号的测定中，所述测定通过酶标仪进行。
在一个优选的实施方式中，所述检测体系中T1链所包括的荧光基团为FAM，并且所述步骤(III)的荧光信号的测定中，激发波长为485nm，发射波长为520nm。
在一个优选的实施方式中，将所述双链DNA探针与待检测样品在反应缓冲液中进行充分反应后，依次加入链霉亲和素磁珠和能够使核酸变性的NaOH溶液，于是脱碱基位点的双链DNA与链霉亲和素标记的磁珠结合；碱性介质对脱碱基位点进行切割，在该位点处留下一个核苷酸的缺口，从而使5’端带有FAM的DNA片段与3’端带有生物素的DNA片段断开，DNA双螺旋结构被碱性介质破坏从而导致FAM标记的DNA片段和生物素标记的DNA片段与其互补链分离；所述链霉亲和素标记的磁珠可以被磁力架吸附，带有荧光基团FAM的DNA片段释放至上清液中。带有生物素标记的核酸片段被结合到磁珠这一固相载体上，从而被吸附至管壁，随后从溶液中分离。
因此，被切开的核酸探针则留在原检测体系的溶液中，并且均带有荧光基团。因此，检测其荧光信号则可得到待测液中DNA糖基化酶的浓度。
本发明提供的检测方法对于DNA糖基化酶的异常表达早期诊断和靶向DNA糖基化酶的抑制剂研发对重要意义。
试剂盒
本发明提供了一种用于检测DNA糖基化酶的试剂盒，其特征在于，所述试剂盒包括：(a)第一容器以及位于第一容器中的如本发明第一方面所述的检测体系中的(a)；(b)第二容器以及位于第二容器中的如本发明第一方面所述的检测体系中的(b)；和(c)第三容器以及位于第三容器中的如本发明第一方面所述的检测体系中的(c)。
在另一优选例中，所述试剂盒还包括使用说明书。
本发明的主要优点包括：
(1)本发明设计了一种可检测多种DNA糖基化酶活性的方法，本发明技术方案中采用含有相应DNA糖基化酶识别位点的双链DNA荧光探针，该原理适用于各种DNA糖基化酶；
(2)操作简单，阳性率高：本方案中不需要额外的扩增步骤，传统的检测糖基化酶的方法往往结合复杂的扩增步骤，扩增过程中容易出现假阳性信号；本发明所用的方法不需要其它酶的参与；
(3)高通量，成本低：本方案中方法可以被应用于高通量筛选体系，可以做384孔板水平的筛选，成本低于1元/孔。
(4)本发明中的碱性介质，同时也可作为核酸变性剂，相较于传统的脱碱基位点核酸内切酶(AP核酸内切酶)，可利用核酸变性以后粘度下降、浮力密度升高、沉降速度加快等特点，使得被切开的核酸片段在分离步骤中更加高效的被分离。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例所用的材料和试剂均为市售产品。
实验试剂和仪器
实验试剂：
DNA寡核苷酸是由捷瑞生物工程有限公司(上海)合成和纯化的。尿嘧啶DNA糖基化酶(UDG),单链选择性单功能尿嘧啶DNA糖基化酶(SMUG1)和烷基腺嘌呤DNA糖基化酶(AAG)购自NEB(美国，麻省)，胸腺嘧啶糖基化酶(TDG)由大肠杆菌中过表达并纯化所得；链霉亲和素磁珠购于Thermo Fisher有限公司(美国，麻省)，磁力架购自博尔希(深圳)，氢氧化钠购自国药集团；溶液制备时所用超纯水均来自Millipore Milli-Q水净化系统。
实验仪器：
荧光检测使用肯帝Infinite-200荧光光谱仪进行测定(瑞士，肯帝公司)；激发波长为485nm，发射波长为520nm；激发的狭缝宽度为20nm，发射的狭隙宽度为10nm。
实施例1：纯的UDG活性检测
首先设计一个含有尿嘧啶碱基的双链DNA探针T1-T2，其中T1链的核苷酸序列为：5’-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3’(SEQ ID NO:1)；T2链的核苷酸序列为5’-ATT TCA GCT CCA TTC ACG TTA-3’(SEQ ID NO:2)，T1链和T2链互补形成双链DNA探针T1-T2。
将双链DNA探针T1-T2及不同浓度(0nM，15nM，30nM，60nM，120nM)的UDG分别加入到反应缓冲液中，反应缓冲液的组成为：20mM Tris-Cl pH8.0，1mM EDTA，1mM DTT；终体积为100μL，体系中双链DNA探针T1-T2的浓度为30nM,体系在25℃下孵育30分钟，使碱基切除反应发生，然后将1μL链霉亲和素磁珠加入反应液与生物素标记的DNA充分结合1小时，将NaOH加入到反应液中至终浓度200mM，室温孵育30分钟，在磁力架的作用下链霉亲和素磁珠被吸附至管壁，将上清液转移至新的96孔板中，置入Tecan酶标仪中进行荧光测定。
构建荧光强度与UDG浓度之间的线性曲线，结果如图2所示，R 2＝0.9902，UDG蛋白浓度在0～120nM之间与荧光值呈线性关系。证明本方法可检测低至15nM的UGD蛋白。
实施例2：纯的TDG活性检测
首先设计一个含有尿嘧啶碱基的双链DNA探针T1-T2，其中T1链的核苷酸序列为：5’-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3’(SEQ ID NO:1)；T2链的核苷酸序列为5’-ATT TCA GCT CCA TTC ACG TTA-3’(SEQ ID NO:2)，T1链和T2链互补形成双链DNA探针T1-T2。
将双链DNA探针T1-T2及不同浓度(0nM，1.85nM，5.56nM，16.67nM，50nM) 的TDG分别加入到反应缓冲液中，反应缓冲液的组成为：20mM HEPES pH7.5，100mM NaCl，0.2mM EDTA，2.5mM MgCl 2；终体积为100μL，体系中双链DNA探针T1-T2的浓度为30nM,体系在25℃下孵育30分钟，使碱基切除反应发生，然后将1μL链霉亲和素磁珠加入反应液与生物素标记的DNA充分结合1小时，将NaOH加入到反应液中至终浓度200mM，室温孵育30分钟，在磁力架的作用下链霉亲和素磁珠被吸附至管壁，将上清液转移至新的96孔板中，置入Tecan酶标仪中进行荧光测定。
构建荧光强度与TDG浓度之间的线性曲线，结果如图3所示，R 2＝0.9968，TDG蛋白浓度在0～50nM之间与荧光值呈线性关系。证明本方法可检测低至1.85nM的TGD蛋白。
实施例3：纯的SMUG1活性检测
首先设计一个含有尿嘧啶碱基的双链DNA探针T1-T2，其中T1链的核苷酸序列为：5’-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3’(SEQ ID NO:1)；T2链的核苷酸序列为5’-ATT TCA GCT CCA TTC ACG TTA-3’(SEQ ID NO:2)，T1链和T2链互补形成双链DNA探针T1-T2；
将双链DNA探针T1-T2及不同浓度(0nM，12.5nM，25nM，50nM，100nM)的SMUG1分别加入到反应缓冲液中，反应缓冲液的组成为：10mM Tris-Cl pH7.0，10mM MgCl 2，1mM DTT；终体积为100μL，体系中双链DNA探针T1-T2的浓度为30nM,体系在25℃下孵育30分钟，使碱基切除反应发生，然后将1μL链霉亲和素磁珠加入反应液与生物素标记的DNA充分结合1小时，将NaOH加入到反应液中至终浓度200mM，室温孵育60分钟，在磁力架的作用下链霉亲和素磁珠被吸附至管壁，将上清液转移至新的96孔板中，置入Tecan酶标仪中进行荧光测定。
构建荧光强度与TDG浓度之间的线性曲线，结果如图4所示，R 2＝0.9945，SMUG蛋白浓度在0～100nM之间与荧光值呈线性关系。证明本方法可检测低至12.5nM的SMUG蛋白。
实施例4：肺癌(Calu-1)细胞裂解液中的TDG活性检测
采用本发明的检测方法对Calu-1细胞裂解液中的TDG活性进行了分析检测，具体步骤如下：
Calu-1细胞样品通过离心分离(5min，1000rpm，4℃)并通过裂解缓冲液(20mM Tris-Cl pH 8.0，1.5mM MgCl 2，10mM KCl，1mM DTT，1mM EDTA)重悬；混合液在冰上静置10min并以3500rpm的速度进行离心分离10分钟，将上清液去除，用细胞核裂解液(20mM Tris-Cl pH 8.0，420mM NaCl，10mM KCl，1mM DTT，1mM EDTA)重悬沉淀并在冰上静置20min，以12000rpm的速度进行离心分离20分钟，所述上清液即为Calu-1核细胞裂解液；Calu-1核细胞裂解液可以直接进行TDG活性检测，无需进一步处理；检测方法和实施例2中纯的TDG活性检测方法相同。
如图5所示，将测定的荧光值带入绘制好的TDG蛋白浓度-荧光值线性曲线中，通过计算得到10 7个Calu-1细胞中大概含有相当于9.12nM的TDG蛋白。
实施例5：TDG的活性抑制检测
Doxorubicin是一种和双链DNA结合的小分子化合物，其与DNA结合以后可以影响TDG对双链DNA的识别能力。50nM TDG与不同浓度Doxorubicin混合并于37℃下孵育30min；双链DNA探针T1-T2被加入到混合物中得到终体积为100μL的体系，于25℃孵育30min；然后将1μL链霉亲和素磁珠加入混合体系中，充分反应1小时；将NaOH加入到反应液中至终浓度200mM，室温孵育1小时，在磁力架的作用下链霉亲和素磁珠被吸附至管壁，将上清液转移至新的96孔板中并置入Tecan酶标仪中进行荧光测定；所有实验均重复两次。
测定的荧光值扣除本底以后，以溶剂对照组荧光值为100％，不同浓度Doxorubicin的荧光值相对于溶剂对照组计算为活性比例。如图6所示，系统的相对荧光随Doxorubicin的浓度的增加而减小，IC 50＝2.12nM。结果表明本方法可用于检测TDG的活性抑制剂。
实施例6：DNA糖基化酶抑制剂的高通量筛选
本方法可应用于384孔板体系进行DNA糖基化酶抑制剂的高通量筛选，以UDG为例，UDG以50nm/L的终浓度与反应缓冲液(20mM HEPES pH 7.5，100mM  NaCl，0.2mM EDTA，2.5mM MgCl 2)混合，通过移液工作站加入到384孔板中，每孔中混合物的体积为19.6μL，排布于384孔板的化合物(1mM)通过移液工作站加入到含有TDG混合液的384孔板中(0.4μL化合物/孔)，化合物的终浓度为20μM；酶与化合物在室温预孵育30min，并加入10μL双链DNA探针至终浓度30nm/L，25℃孵育30分钟，产生脱碱基位点；链霉亲和素磁珠被加入混合液(0.6μL/孔)并孵育1小时，加入NaOH溶液至终浓度200mM并孵育30min；链霉亲和素磁珠被磁力架吸附，上清溶液被转移至新的384孔板，将384孔板置入Tecan酶标仪中进行荧光测定。
测定的荧光值扣除本底以后，以溶剂对照组荧光值为100％，抑制率为0％，计算不同化合物荧光值相对溶剂对照组的抑制率。如图7所示，若以30％酶活抑制率为阈值可得到1.5％筛选阳性率，若以50％酶活抑制率为阈值则可得到0.9％筛选阳性率。本实验证明此方法可用于高通量筛选DNA糖基化酶抑制剂。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于检测DNA糖基化酶的检测体系，其特征在于，所述检测体系包括：
    (a)双链DNA探针，所述双链DNA探针包括T1和T2两条链，并且所述T1和T2两条链可形成双链DNA结构；
    其中，所述T1链包括：至少一个可被待测DNA糖基化酶识别的碱基、荧光基团和分离标签；
    并且所述荧光基团和分离标签分别位于所述至少一个可被DNA糖基化酶识别的碱基的两端；
    (b)可使核酸脱碱基位点的糖苷-磷酸键断裂的组分；和
    (c)带有分离结合标签的固相载体。
  2. 如权利要求1所述的检测体系，其特征在于，所述至少一个可被待测DNA糖基化酶识别的碱基为一个尿嘧啶碱基。
  3. 如权利要求1所述的检测体系，其特征在于，所述(b)为NaOH溶液。
  4. 如权利要求1所述的检测体系，其特征在于，所述检测体系还包括待测DNA糖基化酶。
  5. 如权利要求1或4所述的检测体系，其特征在于，所述待测DNA糖基化酶选自下组：UDG、TDG或SMUG1。
  6. 如权利要求1、4或5所述的检测体系，其特征在于，所述待测DNA糖基化酶的浓度范围为1至500nM，较佳地为1至50nM，更佳地为1至20nM。
  7. 如权利要求1所述的检测体系，其特征在于，所述T1链的序列为5’-FAM-S1-biotin-3’，并且所述T2链的序列为5’-S2-3’，其中，S1的序列如SEQ ID NO:1所示，S2的序列如SEQ ID NO:2所示。
  8. 一种用于检测DNA糖基化酶的方法，其特征在于，包括步骤：
    (I)提供如权利要求1所述的检测体系中的(a)和(b)，并且还包括待测DNA糖基化酶，进行充分反应；
    (II)利用权利要求1中所述的分离标签，将带有分离标签的核酸片段从检测体系中分离；和
    (III)将分离出带有分离标签的核酸片段后的检测体系进行荧光信号的测定。
  9. 一种用于检测DNA糖基化酶的试剂盒，其特征在于，所述试剂盒包括：
    (a)第一容器以及位于第一容器中的如权利要求1所述的检测体系中的(a)；
    (b)第二容器以及位于第二容器中的如权利要求1所述的检测体系中的(b)；和
    (c)第三容器以及位于第三容器中的如权利要求1所述的检测体系中的(c)。
  10. 一种如权利要求1所述的检测体系的用途，其特征在于，用于检测DNA糖基化酶。
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