CN111411153A - Method for detecting activity of various DNA glycosylases - Google Patents

Method for detecting activity of various DNA glycosylases Download PDF

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CN111411153A
CN111411153A CN201910008741.7A CN201910008741A CN111411153A CN 111411153 A CN111411153 A CN 111411153A CN 201910008741 A CN201910008741 A CN 201910008741A CN 111411153 A CN111411153 A CN 111411153A
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dna glycosylase
dna
another preferred
detection system
double
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傅新元
闫娟
毛卓
郭剑南
杨盛莲
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Shanghai Epitas Biotechnology Co ltd
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Shanghai Epitas Biotechnology Co ltd
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Priority to CN201980087780.9A priority patent/CN113366114A/en
Priority to PCT/CN2019/128834 priority patent/WO2020140829A1/en
Publication of CN111411153A publication Critical patent/CN111411153A/en
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Abstract

The invention relates to a detection system for detecting DNA glycosylase, which comprises: (a) a double-stranded DNA probe comprising two strands of T1 and T2, and the two strands of T1 and T2 may form a double-stranded DNA structure; wherein the T1 chain includes: at least one basic group, a fluorescent group and a separation label which can be recognized by the DNA glycosylase to be detected; and the fluorophore and the separation tag are located at both ends of the at least one base recognized by the DNA glycosylase, respectively; (b) a component that can cleave a glycoside-phosphate bond at an abasic site of a nucleic acid; and (c) a solid support bearing the separation-binding tag. The detection system has high flux, low sample quantity, large signal window, simple and convenient operation and low cost, and can detect various DNA glycosylases.

Description

Method for detecting activity of various DNA glycosylases
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a method for detecting activities of various DNA glycosylases and application thereof.
Background
The DNA glycosylase can specifically excise N- β -glycosidic bonds on damaged or mismatched nucleotides to form abasic sites (AP sites) on the DNA strand, then the AP endonuclease cleaves the glycosidic-phosphate bonds of the damaged nucleotides and removes small pieces of DNA including the nucleotides at the AP sites, and a new fragment is synthesized by DNA polymerase I, and finally the two are connected into a new repaired DNA strand by DNA ligase to complete DNA damage repair.
The abnormal expression of the DNA glycosylase is closely related to human diseases, so that the rapid, accurate, convenient and high-throughput detection of the activity of the DNA glycosylase has important significance for clinical diagnosis and the research and development of targeted inhibitors. The conventional method such as a gel electrophoresis method using a fluorescence labeled substrate has low sensitivity, large sample amount and low flux, and is not suitable for quantitative analysis; the sensitivity can be improved and the sample amount can be reduced by using the radioactive isotope labeled substrate to replace the fluorescent labeled substrate, but the radioactive contamination can be caused by the isotope labeling; the high performance liquid chromatography has stronger qualitative and quantitative capability, needs a small amount of samples, but has complex pretreatment of the samples and low flux; the enzyme-linked immunosorbent assay and the immunoblotting method need specific antibodies combined with proteins, have complicated operation steps and are not suitable for detecting a large number of samples; fluorescence methods rely on external labeling with fluorophores and quenchers for homogeneous assays, are suitable for high throughput, but have relatively small signal windows and complex and expensive substrate probe designs.
Therefore, there is an urgent need in the art to develop a method capable of detecting a variety of DNA glycosylases with high throughput, low sample size, large signal window, simple operation, and low cost.
Disclosure of Invention
The invention aims to provide a method which has high flux, low sample quantity, large signal window, simple and convenient operation and low cost and can detect various DNA glycosylases.
In a first aspect of the present invention, there is provided an assay system for the detection of DNA glycosylase, the assay system comprising:
(a) a double-stranded DNA probe comprising two strands of T1 and T2, and the two strands of T1 and T2 may form a double-stranded DNA structure;
wherein the T1 chain includes: at least one basic group, a fluorescent group and a separation label which can be recognized by the DNA glycosylase to be detected;
and the fluorophore and the separation tag are located at both ends of the at least one base recognized by the DNA glycosylase, respectively;
(b) a component that can cleave a glycoside-phosphate bond at an abasic site of a nucleic acid; and
(c) a solid support bearing a separation-binding tag.
In another preferred embodiment, in the detection system, the concentration of the double-stranded DNA probe in (a) is 10-80nM, preferably 20-50nM, more preferably 25-35nM, and most preferably 30 nM.
In another preferred embodiment, the double-stranded DNA probe has a length of 5-200bp, preferably 10-100bp, more preferably 15-70bp, still more preferably 18-40 bp.
In another preferred embodiment, the base recognized by the DNA glycosylase to be tested is selected from the group consisting of: uracil base, cytosine base, thymine base, guanine base, methylation modified cytosine base, 5-carboxyl cytosine, alkyl adenine etc..
In another preferred example, the base recognized by the DNA glycosylase to be tested is a uracil base.
In another preferred embodiment, the at least one base recognized by the DNA glycosylase to be tested is a uracil base.
In another preferred embodiment, the at least one base recognized by the DNA glycosylase to be tested is a uracil base, and the corresponding position on the strand T2 is a guanine base.
In another preferred embodiment, the fluorophore and the separation tag are independently located at the 5 'end, the 3' end, and the middle of the double-stranded DNA probe.
In another preferred embodiment, the fluorophore comprises a fluorophore that can be used for cross-linking with a DNA probe.
In another preferred embodiment, the fluorophore is selected from the group consisting of FAM, FITC, BODIPY-F L, G-Dye100, FluorX, Cy3, Cy5, Texas Red, and the like.
In another preferred embodiment, the separation tag is a tag that enables a nucleic acid sequence linked to or comprising the separation tag to be separated from the detection system.
In another preferred embodiment, the separation tag is selected from the group consisting of: a protein, a peptide fragment, or a nucleic acid fragment.
In another preferred embodiment, the separation tag is selected from the group consisting of: an antigen, an antibody, a ligand, a receptor, avidin, biotin, or a combination thereof.
In another preferred embodiment, the separation tag is biotin.
In another preferred example, the sequence of the T1 chain is 5 '-FAM-S1-biotin-3' and the sequence of the T2 chain is 5 '-S2-3', wherein the sequence of S1 is shown as SEQ ID NO. 1 and the sequence of S2 is shown as SEQ ID NO. 2.
In another preferred embodiment, in the detection system, the (b) is selected from the group consisting of: alkaline medium or abasic site endonuclease (AP endonuclease).
In another preferred embodiment, the alkaline medium is NaOH.
In another preferred embodiment, the final concentration of the basic medium in the detection system is 100-300mM, preferably 150-250mM, and more preferably 200 mM.
In another preferred embodiment, in the detection system, the solid phase carrier material is selected from the group consisting of: metal, glass, gel, plastic, or a combination thereof.
In another preferred embodiment, the solid phase carrier material comprises: a homopolymer, a copolymer, or a combination thereof.
In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.
In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: magnetic beads, microspheres, microplates, slats, test tubes, or combinations thereof.
In another preferred embodiment, the solid phase carrier is a magnetic bead.
In another preferred embodiment, the final volume of the detection system is 50-200. mu. L, preferably 60-150. mu. L, more preferably 80-120. mu. L, more preferably 100. mu. L.
In another preferred embodiment, the detection system further comprises a reaction buffer.
In another preferred embodiment, the reaction buffer comprises: Tris-Cl pH8.0, EDTA, DTT, etc.
In another preferred embodiment, the final concentration of Tris-Cl pH8.0 is 10-50mM, preferably 15-30mM, more preferably 20 mM.
In another preferred embodiment, the final concentration of EDTA is between 0.5 and 2mM, preferably 1 mM.
In another preferred embodiment, the final concentration of DTT is 0.5-2mM, preferably 1 mM.
In another preferred embodiment, the detection system further comprises a DNA glycosylase to be detected.
In another preferred embodiment, the DNA glycosylase to be tested is selected from the group consisting of: UDG, TDG, SMUG1, MBD4, OGG1, AAG, or a combination thereof.
In another preferred embodiment, the DNA glycosylase to be tested is selected from the group consisting of: UDG, TDG or SMUG 1.
In another preferred embodiment, the concentration of the DNA glycosylase to be tested is in the range of 1 to 500nM, preferably 1 to 50nM, more preferably 1 to 20 nM.
In another preferred embodiment, 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 a combination thereof.
In another preferred embodiment, the cell lysate comprises cancer cell lysate.
In another preferred embodiment, the cancer comprises lung cancer.
In another preferred example, the detecting includes: qualitative detection and quantitative detection.
In a second aspect of the invention, there is provided a method for detecting a DNA glycosylase, comprising the steps of:
(I) providing (a) and (b) in the detection system according to the first aspect of the present invention, and further comprising a DNA glycosylase to be detected, and performing a sufficient reaction;
(II) isolating the nucleic acid fragments carrying the isolation tag from the test system using the isolation tag of the first aspect of the invention; and
(III) measuring the fluorescence signal of the detection system after the nucleic acid fragment with the separation label is separated.
In another preferred example, the detecting of the method includes: qualitative detection and quantitative detection.
In another preferred example, the qualitative detection includes: and (3) detecting whether the sample to be detected contains DNA glycosylase.
In another preferred embodiment, the quantitative detection comprises: and (3) detecting the concentration of the active DNA glycosylase in the sample to be detected.
In another preferred example, the method further comprises performing a parallel control experiment on a blank sample, wherein the blank sample does not contain the DNA glycosylase and the measured fluorescence signal is a 0.
In another preferred embodiment, if the signal A1/A0>1.1 is determined in step (III), then the sample to be tested is considered to contain DNA glycosylase; and (5) if the signal A1/A0 measured in the step (III) is less than or equal to 1.1, determining that the DNA glycosylase does not exist in the sample to be detected.
In another preferred example, when the method is used for quantitative detection of DNA glycosylase, the operations of steps (I) to (III) are performed by replacing the sample to be detected with a DNA glycosylase solution of different concentration with known concentration, and further comprising the steps of:
(IV) constructing a linear curve of the fluorescence intensity and the known DNA glycosylase;
and (V) repeating the steps (I) to (III) on the DNA glycosylase to be detected, substituting the obtained fluorescence intensity numerical value into the linear curve obtained in the step (IV), and calculating the concentration of the active DNA glycosylase.
In another preferred embodiment, in the step (I), the concentration of the DNA glycosylase to be tested is in the range of 1 to 500nM, preferably 1 to 50nM, and more preferably 1 to 20 nM.
In another preferred embodiment, in the step (I), the time for sufficient reaction is 10-60min, preferably 20-40min, and more preferably 30 min.
In another preferred embodiment, in the step (I), the temperature for sufficient reaction is 20-40 ℃, preferably 22-30 ℃, and more preferably 25 ℃.
In another preferred example, in the step (I), the separation tag is biotin, and in the step (II), the separation binding tag is streptavidin.
In another preferred example, in the step (II), the separating includes the sub-steps of:
(i) adding the component (c) in the detection system according to the first aspect of the present invention into the detection system after the sufficient reaction of step (I) for sufficient binding, wherein the separation binding tag is a tag capable of specifically binding to the separation tag;
(ii) (ii) adding the component (b) of the detection system according to the first aspect of the present invention to the detection system after sufficient binding in substep (i) to perform a nucleic acid cleavage reaction.
In another preferred embodiment, in the substep (i), the solid support material is selected from the group consisting of: metal, glass, gel, plastic, or a combination thereof.
In another preferred embodiment, in the substep (i), the solid carrier material comprises: a homopolymer, a copolymer, or a combination thereof.
In another preferred embodiment, in the substep (i), the solid carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.
In another preferred embodiment, in the substep (i), the solid carrier material is selected from the group consisting of: magnetic beads, microspheres, microplates, slats, test tubes, or combinations thereof.
In another preferred embodiment, in the substep (i), the solid phase carriers are magnetic beads.
In another preferred embodiment, in the sub-step (i), the time for sufficient bonding is 0.5-2h, preferably 0.8-1.5h, and more preferably 1 h.
In another preferred example, in the substep (ii), the alkaline medium is NaOH.
In another preferred embodiment, the final concentration of the basic medium in the detection system is 100-300mM, preferably 150-250mM, and more preferably 200 mM.
In another preferred embodiment, in the sub-step (ii), the time of the nucleic acid cleavage reaction is 15-60min, preferably 20-40min, and more preferably 30 min.
In another preferred example, in the step (III), the supernatant to be measured for the fluorescence signal is transferred to a 96-well plate.
In another preferred example, in the determination of the fluorescence signal in the step (III), the determination is performed by a microplate reader.
In another preferred example, the fluorophore included in the T1 chain in the detection system is FAM, and the excitation wavelength is 485nm and the emission wavelength is 520nm in the measurement of the fluorescence signal in the step (III).
In a third aspect of the present invention, there is provided a kit for detecting a DNA glycosylase, the kit comprising:
(a) a first container and (a) in a test system according to the first aspect of the invention located in the first container;
(b) a second container and (b) in a detection system according to the first aspect of the present invention in the second container; and
(c) a third container and (c) in a detection system according to the first aspect of the invention located in the third container.
In another preferred embodiment, the kit further comprises instructions for use.
In a fourth aspect of the invention, there is provided the use of a detection system according to the first aspect of the invention for detecting a DNA glycosylase.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a schematic diagram of the mechanism of the double-stranded DNA probe of the invention for detecting various glycosylases.
FIG. 2 shows the fluorescence values and the analysis results of different concentrations of UDG treated with a G/U mismatched double-stranded DNA probe as a substrate.
FIG. 3 shows the fluorescence values and analysis results of TDG treatment at different concentrations using a G/U mismatched double-stranded DNA probe as a substrate.
FIG. 4 shows the fluorescence values and the analysis results of different concentrations of SMUG1 treated with a G/U mismatched double-stranded DNA probe as a substrate.
FIG. 5 shows the enzyme activity of DNA glycosylase contained in Calu-1 cells. The black dots and lines are standard curves drawn using purified TDG protein, and the red dots are fluorescence values determined in the method for Calu-1 cell extract.
FIG. 6 shows the inhibitory effect of Doxorubicin on TDG enzyme activity at various dilution concentrations.
FIG. 7 shows the enzyme activity inhibition rates of different compounds on UDG in a 384-well plate system, and screening positive rates are calculated by taking 30% inhibition rate and 50% inhibition rate as threshold values respectively, and values of the inhibition rates lower than 30% are not shown here.
Detailed Description
The inventor of the invention develops a method which has high flux, low sample quantity, large signal window, simple operation and low cost and can detect various DNA glycosylases for the first time through extensive and intensive research and a large amount of screening. Specifically, the inventor introduces uracil into one strand of the double-stranded DNA probe, labels biotin and fluorescent groups on two sides of the uracil base respectively, and further separates enzyme digestion products labeled by fluorescence and biotin by using streptavidin magnetic beads and sodium hydroxide solution. The result shows that the detection method can effectively improve the detection sensitivity and the signal window, is safe and simple, has high flux and easy operation, can be suitable for enzyme activity detection of various DNA glycosylases such as UDG, TDG, SMUG, OGG1 and the like by simply replacing the substrate probe, and has high accuracy. The present invention has been completed based on this finding.
Term(s) for
Double-stranded DNA probe
As used herein, the terms "nucleic acid probe", "nucleic acid probe of the invention", "double-stranded DNA probe", and the like, are used interchangeably to refer to a double-stranded DNA probe of the invention for use in detecting DNA glycosylase.
In the invention, the double-stranded DNA probe comprises two strands of T1 and T2, and the two strands of T1 and T2 can form a double-stranded DNA structure; wherein, the T1 chain includes: at least one basic group, a fluorescent group and a separation label which can be recognized by the DNA glycosylase to be detected; and the fluorophore and the separation tag are located at both ends of the at least one base recognized by the DNA glycosylase, respectively.
In the present invention, the concentration of the double-stranded DNA probe in the detection system is 10-80nM, preferably 20-50nM, more preferably 25-35nM, and most preferably 30 nM; the length of the double-stranded DNA probe is 5-200bp, preferably 10-100bp, more preferably 15-70bp, and more preferably 18-40 bp.
In the present invention, the base recognized by the DNA glycosylase to be tested is selected from the following group: uracil base, cytosine base, thymine base, guanine base, methylation modified cytosine base, 5-carboxyl cytosine, alkyl adenine etc.. In another preferred example, the base recognized by the DNA glycosylase to be tested is a uracil base.
In a preferred embodiment, the at least one base recognized by the DNA glycosylase to be tested is a uracil base.
In a preferred embodiment, the at least one base recognized by the DNA glycosylase to be tested is a uracil base and the corresponding position on the T2 strand is a guanine base.
In the present invention, the fluorophore and the separation tag are independently located at the 5 'end, the 3' end and the middle of the double-stranded DNA probe, respectively.
In another preferred embodiment, the fluorophore is selected from the group consisting of FAM, FITC, BODIPY-F L, G-Dye100, FluorX, Cy3, Cy5, Texas Red, and the like.
In the present invention, the separation tag is a tag capable of separating the nucleic acid sequence connected to or comprising the separation tag from the detection system, and may be selected from the group consisting of: a protein, a peptide fragment, or a nucleic acid fragment.
In a preferred embodiment, the separation tag is selected from the group consisting of, but not limited to: an antigen, an antibody, a ligand, a receptor, avidin, biotin, or a combination thereof.
In a preferred embodiment, the isolation tag is biotin.
In a preferred embodiment, in the double-stranded DNA probe, the sequence of the T1 chain is 5 '-FAM-S1-biotin-3', and the sequence of the T2 chain is 5 '-S2-3', wherein the sequence of S1 is shown as SEQ ID NO. 1, and the sequence of S2 is shown as SEQ ID NO. 2.
DNA glycosylase
DNA glycosylases specifically cleave the N- β -glycosidic bond on damaged or mismatched nucleotides to form abasic sites (AP sites) on the DNA strand.
In one embodiment of the present invention, the glycoside-phosphate bond at the abasic site is cleaved using NaOH solution as an alkaline medium or nucleic acid denaturing agent.
Known DNA glycosylases include: UDG (uracil DNA glycosylase), TDG (thymine DNA glycosylase), OGG1 (8-hydroxyguanine DNA glycosylase), SMUG1 (single-strand-selective monofunctional uracil DNA glycosylase), MBD4 (methylated CpG binding domain protein 4), AAG (N-methylpurine DNA glycosylase, also called MPG), and the like.
An important function of UDG (uracil-DNA glycosylase) is to prevent mutagenesis by cleaving N-glycosyl bonds and initiating the Base Excision Repair (BER) pathway to eliminate uracil from a DNA molecule. Uracil bases typically occur during cytosine deamination or misincorporation of dUMP residues.
TDG (thymine-DNA glycosylase) removes thymine moieties from G/T mismatches by hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of DNA and the mismatched thymine. Due to the lower activity, the enzyme also removes thymine from C/T and T/T mismatches. TDG can also use guanine to remove uracil and 5-bromouracil from mismatch. This enzyme plays an important role in cellular defense against genetic mutations caused by spontaneous deamination of 5-methylcytosine and cytosine.
SMUG1 (single strand selective monofunctional uracil DNA glycosylase) removes uracil and 5-hydroxymethyluracil from single-and double-stranded DNA in chromatin, facilitating base excision repair. Generally, the repair activity is stronger for single-stranded DNA than for double-stranded DNA.
MBD4 (methylated CpG binding domain protein 4) contains a methyl-CpG binding domain that can effectively remove thymine or uracil from mismatched CpG sites in vitro. In addition, the methyl-CpG binding domain of MBD4 preferentially binds the major product of 5-methylcytosine CpG-TpG mismatch-methyl-CpG deamination. The combined specificity of binding and catalysis suggests that the enzyme may act to minimize methyl-CpG mutations.
AAG (N-methylpurine DNA glycosylase, also called MPG) is the only DNA glycosylase active on 3-methyladenine, hypoxanthine and 1, N6-vinyladenine in mammals. Although AAG also has the ability to remove 8-oxoguanine DNA damage, it is not the primary glycosylase for 8-oxoguanine repair.
OGG1 (8-hydroxyguanine DNA glycosylase) releases free 8-hydroxyguanine from the oxidatively mutagenized DNA and causes single strand breaks in double stranded DNA at the 8-hydroxyguanine residue that pairs with cytosine.
In one embodiment of the invention, when the base recognized by the DNA glycosylase to be detected in the double-stranded DNA probe is a uracil base, the double-stranded DNA probe can be used for detection of UDG, TDG, SMUG1 or MBD 4.
In the invention, the base which can be recognized by the DNA glycosylase to be detected in the double-stranded DNA probe can be designed according to different recognition sites of different DNA glycosylases so as to realize the detection of different DNA glycosylases.
Detection System of the invention
In the present invention, there is provided an assay system for detecting DNA glycosylase, comprising: (a) a double-stranded DNA probe comprising two strands of T1 and T2, and the two strands of T1 and T2 may form a double-stranded DNA structure; wherein the T1 chain includes: at least one basic group, a fluorescent group and a separation label which can be recognized by the DNA glycosylase to be detected; and the fluorophore and the separation tag are located at both ends of the at least one base recognized by the DNA glycosylase, respectively; (b) a component which can cleave a glycoside-phosphate bond at an abasic site of a nucleic acid.
In the detection system of the present invention, the (b) may be an alkaline medium or an abasic site endonuclease (AP endonuclease).
In a preferred embodiment, the alkaline medium is NaOH and the final concentration in the assay system is 100-300mM, preferably 150-250mM, more preferably 200 mM.
In the invention, the alkaline medium NaOH solution can be used as an alkaline medium at the same time to cut abasic sites.
Compared with the traditional abasic site endonuclease (AP endonuclease), the alkaline medium in the invention can be more efficiently separated in the separation step by utilizing the characteristics of reduced viscosity, increased buoyancy density, accelerated sedimentation speed and the like after nucleic acid denaturation.
In another preferred embodiment, the detection system further comprises: (c) a solid support bearing a separation-binding tag. The method of (c) is used for isolating the nucleic acid fragment with the isolation tag from the test system.
Wherein, the solid phase carrier material is selected from but not limited to the following group: metal, glass, gel, plastic, or a combination thereof. In another preferred embodiment, the solid phase carrier material comprises: a homopolymer, a copolymer, or a combination thereof. In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof. In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: magnetic beads, microspheres, microplates, slats, test tubes, or combinations thereof.
In a preferred embodiment, the solid support is a magnetic bead.
In the present invention, the final volume of the detection system is 50 to 200. mu. L, preferably 60 to 150. mu. L, more preferably 80 to 120. mu. L, and still more preferably 100. mu. L.
In another preferred embodiment, the detection system further comprises a reaction buffer, and the reaction buffer comprises: Tris-Cl pH8.0, EDTA, DTT, etc. In a preferred embodiment, the final concentration of Tris-Cl pH8.0 is 10-50mM, preferably 15-30mM, more preferably 20 mM. In a preferred embodiment, the final concentration of EDTA is between 0.5 and 2mM, preferably 1 mM. In a preferred embodiment, the final concentration of DTT is 0.5-2mM, preferably 1 mM.
Detection method of the invention
In the present invention, there is provided a method for detecting a DNA glycosylase, characterized by comprising the steps of: (I) providing components (a) and (b) in the detection system according to the first aspect of the invention, and further comprising a DNA glycosylase to be detected, and carrying out a sufficient reaction; (II) isolating the nucleic acid fragments carrying the isolation tag from the test system using the isolation tag of the first aspect of the invention; and (III) measuring the fluorescence signal of the detection system after the nucleic acid fragment with the separation label is separated.
The detection method comprises the following steps: qualitative detection and quantitative detection.
In the present invention, the qualitative detection comprises: detecting whether a sample to be detected contains DNA glycosylase or not; the quantitative detection comprises the following steps: and (3) detecting the concentration of the active DNA glycosylase in the sample to be detected.
In a preferred embodiment, the method further comprises performing a parallel control experiment on a blank sample that does not contain a DNA glycosylase and the measured fluorescence signal is a 0. The fluorescence signal obtained after the DNA glycosylase to be detected is used for determination is A1, if A1/A0 is more than 1.1, the DNA glycosylase is considered to be in the sample to be detected; and if A1/A0 is less than or equal to 1.1, determining that the DNA glycosylase does not exist in the sample to be detected.
In another preferred example, when the method is used for quantitative detection of DNA glycosylase, the operations of steps (I) to (III) are performed by replacing the sample to be detected with a DNA glycosylase solution of different concentration with known concentration, and further comprising the steps of: (IV) constructing a linear curve of the fluorescence intensity and the known DNA glycosylase; and (V) repeating the steps (I) to (III) on the DNA glycosylase to be detected, substituting the obtained fluorescence intensity numerical value into the linear curve obtained in the step (IV), and calculating the concentration of the active DNA glycosylase.
In the present invention, the concentration of the DNA glycosylase to be tested is in the range of 1 to 500nM, preferably 1 to 50nM, and more preferably 1 to 20 nM.
In another preferred embodiment, in the step (I), the time for sufficient reaction is 10-60min, preferably 20-40min, and more preferably 30 min. In another preferred embodiment, in the step (I), the temperature for sufficient reaction is 20-40 ℃, preferably 22-30 ℃, and more preferably 25 ℃.
In a preferred embodiment of the present invention, in step (I), the separation tag is biotin, and in step (II), the separation binding tag is streptavidin.
In another preferred example, in the step (II), the separating includes the sub-steps of:
(i) adding a solid phase carrier with a separation and combination label into the detection system after the full reaction in the step (I) for full combination, wherein the separation and combination label is a label capable of being specifically combined with the separation label; (ii) (ii) adding an alkaline medium to the fully bound detection system of substep (i) to perform a nucleic acid cleavage reaction.
In the substep (i), the solid support material is selected from, but not limited to, the following group: metal, glass, gel, plastic, or a combination thereof. In another preferred embodiment, the solid phase carrier material comprises: a homopolymer, a copolymer, or a combination thereof. In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof. In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: magnetic beads, microspheres, microplates, slats, test tubes, or combinations thereof.
In a preferred embodiment, in the substep (i), the solid phase carriers are magnetic beads.
In another preferred embodiment, in the sub-step (i), the time for sufficient bonding is 0.5-2h, preferably 0.8-1.5h, and more preferably 1 h.
In a preferred embodiment, in sub-step (ii), the alkaline medium is NaOH, and the final concentration in the detection system is 100-; and the reaction time is 15-60min, preferably 20-40min, more preferably 30 min.
In another preferred embodiment, the step (III) further comprises transferring the supernatant to be assayed for the fluorescent signal to a 96-well plate; in the measurement of the fluorescent signal in the step (III), the measurement is performed by a microplate reader.
In a preferred embodiment, the fluorophore included in the T1 chain in the detection system is FAM, and the excitation wavelength is 485nm and the emission wavelength is 520nm in the measurement of the fluorescence signal in the step (III).
In a preferred embodiment, after the double-stranded DNA probe and a sample to be detected are fully reacted in a reaction buffer solution, streptavidin magnetic beads and a NaOH solution capable of denaturing nucleic acid are sequentially added, so that double-stranded DNA at abasic sites is combined with the streptavidin-labeled magnetic beads; the alkaline medium cuts the abasic site, a gap of nucleotide is left at the site, so that the DNA fragment with FAM at the 5 'end is disconnected with the DNA fragment with biotin at the 3' end, and the double helix structure of the DNA is damaged by the alkaline medium, so that the FAM-labeled DNA fragment and the biotin-labeled DNA fragment are separated from the complementary strand of the FAM-labeled DNA fragment and the biotin-labeled DNA fragment; the streptavidin-labeled magnetic beads can be adsorbed by a magnetic rack, and the DNA fragments with the fluorescent group FAM are released into the supernatant. The nucleic acid fragments with biotin labels are bound to a solid support, magnetic beads, and thus adsorbed to the tube wall, and then separated from the solution.
Therefore, the cleaved nucleic acid probes remain in the solution of the original detection system and are all provided with fluorescent groups. Therefore, the concentration of the DNA glycosylase in the solution to be detected can be obtained by detecting the fluorescence signal.
The detection method provided by the invention is of great significance for early diagnosis of abnormal expression of the DNA glycosylase and development of an inhibitor of the targeted DNA glycosylase.
Reagent kit
The invention provides a kit for detecting DNA glycosylase, which is characterized by comprising: (a) a first container and (a) in a test system according to the first aspect of the invention located in the first container; (b) a second container and (b) in a detection system according to the first aspect of the present invention in the second container; and (c) a third container and (c) in a detection system according to the first aspect of the invention located in the third container.
In another preferred embodiment, the kit further comprises instructions for use.
The main advantages of the invention include:
(1) the invention designs a method capable of detecting the activity of various DNA glycosylases, adopts a double-stranded DNA fluorescent probe containing a corresponding DNA glycosylase recognition site in the technical scheme of the invention, and the principle is suitable for various DNA glycosylases;
(2) the operation is simple, and the positive rate is high: in the scheme, an additional amplification step is not needed, the traditional method for detecting the glycosylase is often combined with a complex amplification step, and false positive signals are easy to appear in the amplification process; the method used in the invention does not need the participation of other enzymes;
(3) high throughput, low cost: the method in the scheme can be applied to a high-throughput screening system, can be used for horizontal screening of 384-hole plates, and has the cost lower than 1 yuan/hole.
(4) Compared with the traditional abasic site-removed endonuclease (AP endonuclease), the alkaline medium can be used as a nucleic acid denaturant, and the characteristics of reduced viscosity, increased buoyancy density, accelerated sedimentation speed and the like after nucleic acid denaturation can be utilized, so that the cut nucleic acid fragments can be separated more efficiently in the separation step.
The present invention is further illustrated by the following examples, which are intended to illustrate and not to limit the scope of the invention, the experimental procedures, without specific conditions noted in the following examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to manufacturer's recommendations.
The materials and reagents used in the examples were all commercially available products unless otherwise specified.
Experimental reagents and instruments
Experimental reagent:
DNA oligonucleotides were synthesized and purified by Czeri bioengineering, Inc. (Shanghai). Uracil DNA Glycosylase (UDG), single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) and alkyl adenine DNA glycosylase (AAG) were purchased from NEB (USA, Massachusetts), and thymine glycosylase (TDG) was overexpressed from E.coli and purified; streptavidin magnetic beads were purchased from Thermo Fisher, inc (ma, usa), magnetic scaffolds were purchased from bosch (shenzhen), sodium hydroxide were purchased from the national drug group; the ultrapure water used in the solution preparation was obtained from a Millipore Milli-Q water purification system.
An experimental instrument:
fluorescence detection was measured using an Infinite-200 fluorescence spectrometer (Kendi, Switzerland); the excitation wavelength is 485nm, and the emission wavelength is 520 nm; the excitation slit width was 20nm and the emission slit width was 10 nm.
Example 1: pure UDG Activity assay
Firstly, designing a double-stranded DNA probe T1-T2 containing uracil base, wherein the nucleotide sequence of a T1 chain is as follows: 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), and the T1 chain and the T2 chain are complementary to form a double-stranded DNA probe T1-T2.
Double-stranded DNA probe T1-T2 and UDG with different concentrations (0nM, 15nM, 30nM, 60nM, 120nM) were added to reaction buffer solution respectively, the reaction buffer solution consisted of 20mM Tris-Cl pH8.0, 1mM EDTA, 1mM DTT, and a final volume of 100. mu. L, the concentration of the double-stranded DNA probe T1-T2 in the system was 30nM, the system was incubated at 25 ℃ for 30 minutes to allow base excision reaction to occur, then 1. mu. L streptavidin magnetic beads were added to the reaction solution to bind well with biotin-labeled DNA for 1 hour, NaOH was added to the reaction solution to a final concentration of 200mM, incubated at room temperature for 30 minutes, the streptavidin magnetic beads were adsorbed to the tube wall under the action of magnetic frame, the supernatant was transferred to a new 96-well plate, and placed in a Tecan microplate reader to perform fluorescence measurement.
A linear curve between fluorescence intensity and UDG concentration was constructed, and the results are shown in FIG. 2, R20.9902, the concentration of UDG protein is in a linear relationship with the fluorescence value between 0 and 120 nM. The method is proved to detect UGD protein as low as 15 nM.
Example 2: pure TDG Activity assay
Firstly, designing a double-stranded DNA probe T1-T2 containing uracil base, wherein the nucleotide sequence of a T1 chain is as follows: 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), and the T1 chain and the T2 chain are complementary to form a double-stranded DNA probe T1-T2.
The double-stranded DNA probe T1-T2 and TDG at different concentrations (0nM, 1.85nM, 5.56nM, 16.67nM, 50nM) were added to the reaction buffer, respectively, which consisted of: 20mM HEPES pH7.5, 100mM NaCl, 0.2mM EDTA, 2.5mM MgCl2The final volume is 100 mu L, the concentration of the double-stranded DNA probe T1-T2 in the system is 30nM, the system is incubated for 30 minutes at 25 ℃ to enable the base excision reaction to occur, then 1 mu L streptavidin magnetic beads are added into the reaction liquid to be fully combined with the biotin-labeled DNA for 1 hour, NaOH is added into the reaction liquid to achieve the final concentration of 200mM, the incubation is carried out for 30 minutes at room temperature, the streptavidin magnetic beads are adsorbed to the tube wall under the action of a magnetic frame, the supernatant is transferred to a new 96-well plate, and the 96-well plate is placed into a Tecan microplate reader to carry out fluorescence measurement.
A linear curve between fluorescence intensity and TDG concentration was constructed, and the results are shown in FIG. 3, R20.9968, the concentration of TDG protein is in a linear relationship with the fluorescence value between 0 and 50 nM. The method was demonstrated to detect TGD proteins as low as 1.85 nM.
Example 3: pure SMUG1 activity assay
Firstly, designing a double-stranded DNA probe T1-T2 containing uracil base, wherein the nucleotide sequence of a T1 chain is as follows: 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), and the T1 chain and the T2 chain are complementary to form a double-stranded DNA probe T1-T2;
the double-stranded DNA probe T1-T2 and different concentrations (0nM, 12.5nM, 25nM, 50nM, 100nM) of SMUG1 were added to the reaction buffer, respectively, which consisted of: 10mM Tris-Cl pH7.0, 10mM MgCl21mM DTT, the final volume is 100 mu L, the concentration of the double-stranded DNA probe T1-T2 in the system is 30nM, the system is incubated for 30 minutes at 25 ℃ to enable the base excision reaction to occur, then 1 mu L streptavidin magnetic beads are added into the reaction solution to be fully combined with the biotin-labeled DNA for 1 hour,adding NaOH into the reaction solution to a final concentration of 200mM, incubating at room temperature for 60 minutes, adsorbing the streptavidin magnetic beads to the tube wall under the action of a magnetic frame, transferring the supernatant into a new 96-well plate, and placing the plate into a Tecan enzyme-linked immunosorbent assay for fluorescence determination.
A linear curve between fluorescence intensity and TDG concentration was constructed, and the results are shown in FIG. 4, R2The concentration of the SMUG protein is 0-100 nM and is in linear relation with the fluorescence value. The method proves that the SMUG protein can be detected as low as 12.5 nM.
Example 4: detection of TDG Activity in Lung cancer (Calu-1) cell lysate
The detection method provided by the invention is used for analyzing and detecting the TDG activity in the Calu-1 cell lysate, and comprises the following specific steps:
calu-1 cell samples were separated by centrifugation (5min, 1000rpm, 4 ℃) and passed through lysis buffer (20mM Tris-Cl pH8.0, 1.5mM MgCl210mM KCl, 1mM DTT, 1mM EDTA); the mixture was left on ice for 10min and centrifuged at 3500rpm for 10min, the supernatant was removed, the pellet was resuspended in a cell nucleus lysate (20mM Tris-Cl pH8.0, 420mM NaCl, 10mM KCl, 1mM DTT, 1mM EDTA) and left on ice for 20min, and centrifuged at 12000rpm for 20min, the supernatant was Calu-1 nuclear cell lysate; the Calu-1 nuclear cell lysate can be directly used for detecting the activity of the TDG without further treatment; the detection method was the same as that for the pure TDG activity in example 2.
As shown in FIG. 5, the measured fluorescence value was substituted into a well-plotted TDG protein concentration-fluorescence value linear curve, and 10 was obtained by calculation7Each Calu-1 cell probably contained the TDG protein equivalent to 9.12 nM.
Example 5: inhibitory assay for TDG Activity
The method comprises the steps of mixing 50nM TDG with different concentrations of Doxorubicin and incubating at 37 ℃ for 30min, adding double-stranded DNA probes T1-T2 into the mixture to obtain a system with a final volume of 100 mu L, incubating at 25 ℃ for 30min, adding 1 mu L streptavidin magnetic beads into the mixed system, fully reacting for 1 hour, adding NaOH into a reaction solution to a final concentration of 200mM, incubating at room temperature for 1 hour, adsorbing the streptavidin magnetic beads to the tube wall under the action of a magnetic frame, transferring the supernatant into a new 96-well plate, placing into a Tecan microplate reader for fluorescence measurement, and repeating all experiments twice.
After background subtraction of the measured fluorescence values, the activity ratio was calculated as the fluorescence value of the solvent control group being 100% and the fluorescence value of the different concentrations of Doxorubicin relative to the solvent control group. As shown in FIG. 6, the relative fluorescence of the system decreased with increasing concentration of Doxorubicin, IC502.12 nM. The results show that the method can be used for detecting the activity inhibitor of the TDG.
Example 6: high throughput screening of DNA glycosylase inhibitors
The method can be applied to a 384-pore plate system for high-throughput screening of DNA glycosylase inhibitors, and takes UDG as an example, the UDG is mixed with reaction buffer solution (20mM HEPES pH7.5, 100mM NaCl, 0.2mM EDTA, 2.5mM MgCl) at the final concentration of 50 nm/L2) Mixing, adding to a 384 well plate by a pipetting station, the volume of the mixture in each well being 19.6. mu. L, adding the compound (1mM) arranged in the 384 well plate to the 384 well plate containing the TDG mixture (0.4. mu. L compound/well) by the pipetting station, the final concentration of the compound being 20. mu.M, pre-incubating the enzyme with the compound at room temperature for 30min and adding 10. mu. L double-stranded DNA probe to the final concentration of 30 nm/L, incubating at 25 ℃ for 30min to generate abasic sites, adding streptavidin magnetic beads to the mixture (0.6. mu. L/well) and incubating for 1h, adding NaOH solution to the final concentration of 200mM and incubating for 30min, adsorbing the supernatant solution by a magnetic rack, transferring the supernatant solution to a new 384 well plate, placing the 384 well plate in a Tecan reader for fluorometry.
After the background is subtracted from the measured fluorescence value, the fluorescence value of the solvent control group is taken as 100%, the inhibition rate is taken as 0%, and the inhibition rate of the fluorescence value of different compounds relative to the solvent control group is calculated. As shown in FIG. 7, the screening positive rate was 1.5% when 30% of the enzyme activity inhibition rate was used as a threshold, and 0.9% when 50% of the enzyme activity inhibition rate was used as a threshold. The experiment proves that the method can be used for high-throughput screening of the DNA glycosylase inhibitor.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<120> a method for detecting the activity of various DNA glycosylases
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Claims (10)

1. A test system for detecting DNA glycosylase, the test system comprising:
(a) a double-stranded DNA probe comprising two strands of T1 and T2, and the two strands of T1 and T2 may form a double-stranded DNA structure;
wherein the T1 chain includes: at least one basic group, a fluorescent group and a separation label which can be recognized by the DNA glycosylase to be detected;
and the fluorophore and the separation tag are located at both ends of the at least one base recognized by the DNA glycosylase, respectively;
(b) a component that can cleave a glycoside-phosphate bond at an abasic site of a nucleic acid; and
(c) a solid support bearing a separation-binding tag.
2. The test system of claim 1, wherein the at least one base recognized by the DNA glycosylase to be tested is a uracil base.
3. The detection system of claim 1, wherein (b) is a NaOH solution.
4. The test system of claim 1, further comprising a test DNA glycosylase.
5. The test system of claim 1 or 4, wherein the test DNA glycosylase is selected from the group consisting of: UDG, TDG or SMUG 1.
6. The test system according to claim 1, 4 or 5, wherein the concentration of the DNA glycosylase to be tested is in the range of 1 to 500nM, preferably 1 to 50nM, more preferably 1 to 20 nM.
7. The detection system as claimed in claim 1, wherein the sequence of T1 chain is 5 '-FAM-S1-biotin-3' and the sequence of T2 chain is 5 '-S2-3', wherein the sequence of S1 is shown as SEQ ID NO. 1 and the sequence of S2 is shown as SEQ ID NO. 2.
8. A method for detecting DNA glycosylase comprising the steps of:
(I) providing (a) and (b) in the test system according to claim 1, and further comprising a test DNA glycosylase, and performing a sufficient reaction;
(II) isolating the nucleic acid fragments bearing the isolation tag from the test system using the isolation tag of claim 1; and
(III) measuring the fluorescence signal of the detection system after the nucleic acid fragment with the separation label is separated.
9. A kit for detecting DNA glycosylase, the kit comprising:
(a) a first container and a detection system according to claim 1 in the first container;
(b) a second container and (b) in the detection system of claim 1 in the second container; and
(c) a third container and (c) in the detection system of claim 1 in the third container.
10. Use of a test system according to claim 1 for the detection of DNA glycosylases.
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