CN111411153B - Method for detecting activity of various DNA glycosylase - Google Patents

Method for detecting activity of various DNA glycosylase Download PDF

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CN111411153B
CN111411153B CN201910008741.7A CN201910008741A CN111411153B CN 111411153 B CN111411153 B CN 111411153B CN 201910008741 A CN201910008741 A CN 201910008741A CN 111411153 B CN111411153 B CN 111411153B
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detection system
dna glycosylase
test
double
detection
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CN111411153A (en
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傅新元
闫娟
毛卓
郭剑南
杨盛莲
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Epitas Biosciences Shanghai Co Ltd
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Epitas Biosciences Shanghai Co Ltd
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Priority to CN201980087780.9A priority patent/CN113366114B/en
Priority to PCT/CN2019/128834 priority patent/WO2020140829A1/en
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Abstract

The present invention relates to a detection system for detecting a 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 comprises: at least one base, a fluorescent group and a separation tag that are recognized by the DNA glycosylase to be tested; and the fluorophore and the separation tag are located at both ends of the at least one base that is recognized by a DNA glycosylase; (b) A component that cleaves a glycoside-phosphate bond of a nucleic acid abasic site; and (c) a solid support bearing an isolated binding tag. The detection system has the advantages of high flux, low required sample amount, large signal window, simple and convenient operation, low cost and capability of detecting various DNA glycosylases.

Description

Method for detecting activity of various DNA glycosylase
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a method capable of detecting activities of various DNA glycosylase and application thereof.
Background
BER (base excision repair) pathway is the main pathway to repair endogenous DNA base damage caused by oxidation, alkylation and deamination. Among them, DNA glycosylase is an important link in BER pathway, including: UDG (uracil DNA glycosylase), TDG (thymine DNA glycosylase), OOG1 (8-hydroxyguanine DNA glycosylase), and the like. The DNA glycosylase can specifically cleave N-beta-glycosidic bonds on damaged or mismatched nucleotides, forming abasic sites (AP sites) on the DNA strand. The AP endonuclease then cleaves the glycoside-phosphate bond of the damaged nucleotide and removes small fragments of DNA including the AP site nucleotide, and a new fragment is synthesized by DNA polymerase I, and finally the two fragments are connected into a new repaired DNA chain by DNA ligase to complete DNA damage repair.
The abnormal expression of 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 is of great significance to clinical diagnosis and research and development of targeted inhibitors. The traditional methods such as gel electrophoresis method using fluorescence labeling substrate have low sensitivity, large sample amount and low flux, and are not suitable for quantitative analysis; the use of a radioisotope-labeled substrate instead of a fluorescent-labeled substrate can improve sensitivity and reduce sample size, but the isotope labeling can cause radioactive contamination; the high performance liquid chromatography has strong qualitative and quantitative capability, and needs a small amount of samples, but the pretreatment of the samples is complex, and the flux is also low; the enzyme-linked immunosorbent assay and the immunoblotting method require specific antibodies combined with proteins, have complex operation steps and are not suitable for detection of a large number of samples; fluorescence methods rely on external labelling with fluorophores and quenchers for homogeneous assays, which are suitable for high throughput, but whose signal window is relatively small and the substrate probe design is complex and expensive.
Therefore, there is an urgent need in the art to develop a method capable of detecting various 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 for detecting various DNA glycosylases, which has the advantages of high flux, low required sample size, large signal window, simple and convenient operation and low cost.
In a first aspect of the present invention, there is provided a detection system for detecting a DNA glycosylase, the detection 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 comprises: at least one base, a fluorescent group and a separation tag that are recognized by the DNA glycosylase to be tested;
and the fluorophore and the separation tag are located at both ends of the at least one base that is recognized by a DNA glycosylase;
(b) A component that cleaves a glycoside-phosphate bond of a nucleic acid abasic site; and
(C) A solid support with a separation binding tag.
In another preferred embodiment, the concentration of the double-stranded DNA probe in the detection system in the step (a) is 10-80nM, preferably 20-50nM, more preferably 25-35nM, still more preferably 30nM.
In another preferred embodiment, the double stranded DNA probe has a length of 5 to 200bp, preferably 10 to 100bp, more preferably 15 to 70bp, still more preferably 18 to 40bp.
In another preferred embodiment, the base recognized by the DNA glycosylase to be tested is selected from the group consisting of: uracil bases, cytosine bases, thymine bases, guanine bases, methylation-modified cytosine bases, 5-carboxycytosine, alkyladenine, and the like.
In another preferred embodiment, 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 an 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 T2 strand is a guanine base.
In another preferred embodiment, the fluorophore and the separation tag are each 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 to crosslink with a DNA probe.
In another preferred embodiment, the fluorophore is selected from the group consisting of: FAM, FITC, BODIPY-FL, G-Dye100, fluorX, cy3, cy5, texas Red, etc.
In another preferred embodiment, the separation tag is a tag that enables the nucleic acid sequence attached 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, peptide fragment or 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, an avidin, biotin, or a combination thereof.
In another preferred embodiment, the separation tag is biotin.
In another preferred embodiment, 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 in SEQ ID NO. 1, and the sequence of S2 is shown in SEQ ID NO. 2.
In another preferred embodiment, the detection system wherein (b) is selected from the group consisting of: alkaline mediator or abasic site endonucleases (AP endonucleases).
In another preferred embodiment, the alkaline medium is NaOH.
In another preferred embodiment, the final concentration of the alkaline medium in the detection system is 100-300mM, preferably 150-250mM, more preferably 200mM.
In another preferred embodiment, the solid support material in the detection system is selected from the group consisting of: metal, glass, gel, plastic, or combinations thereof.
In another preferred embodiment, the solid support material comprises: homopolymers, copolymers, or combinations thereof.
In another preferred embodiment, the solid support material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.
In another preferred embodiment, the solid support material is selected from the group consisting of: magnetic beads, microspheres, microwell plates, strips, test tubes, or combinations thereof.
In another preferred embodiment, the solid support 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 20mM.
In another preferred embodiment, the final concentration of EDTA is 0.5-2mM, preferably 1mM.
In another preferred embodiment, the final concentration of DTT is 0.5-2mM, preferably 1mM.
In another preferred embodiment, the detection system further comprises a DNA glycosylase to be tested.
In another preferred embodiment, the test DNA glycosylase is selected from the group consisting of: UDG, TDG, SMUG1, MBD4, OGG1, AAG, or a combination thereof.
In another preferred embodiment, the test DNA glycosylase is selected from the group consisting of: UDG, TDG or SMUG1.
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 20nM.
In another preferred embodiment, the DNA glycosylase is selected from the group consisting of: purified DNA glycosylase, a lysate thereof, a cell lysate, blood or an extract thereof, a body fluid or an extract thereof, or a combination thereof.
In another preferred embodiment, the cell lysate comprises a cancer cell lysate.
In another preferred embodiment, the cancer comprises lung cancer.
In another preferred embodiment, the detecting includes: qualitative 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 tested, performing a sufficient reaction;
(II) separating the nucleic acid fragment bearing the separation tag from the detection system using the separation tag described in the first aspect of the invention; and
(III) measuring a fluorescent signal by using a detection system after separating the nucleic acid fragments with the separation tags.
In another preferred embodiment, the detection of the method comprises: qualitative and quantitative detection.
In another preferred embodiment, the qualitative detection comprises: and detecting whether the sample to be detected contains DNA glycosylase.
In another preferred embodiment, the quantitative detection comprises: and detecting the concentration of the active DNA glycosylase in the sample to be detected.
In another preferred embodiment, the method further comprises performing a parallel control experiment with a blank sample that does not contain a DNA glycosylase and has a measured fluorescent signal of A0.
In another preferred embodiment, if the signal A1/A0>1.1 measured in step (III) is considered to be DNA glycosylase in the sample to be tested; if the signal A1/A0 measured in the step (III) is less than or equal to 1.1, the sample to be detected is considered to have no DNA glycosylase.
In another preferred example, when the method is used for quantitative detection of a DNA glycosylase, a DNA glycosylase solution of a known concentration is substituted for the sample to be detected, the operations of steps (I) to (III) are performed, and the method further comprises the steps of:
(IV) constructing a linear curve of fluorescence intensity versus known DNA glycosylase;
(V) repeating the steps (I) to (III) with the DNA glycosylase to be tested, bringing the obtained fluorescence intensity value into the linear curve obtained in the step (IV), and calculating the concentration of the active DNA glycosylase.
In another preferred embodiment, in step (I), 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 20nM.
In another preferred embodiment, in the step (I), the time for the sufficient reaction is 10 to 60min, preferably 20 to 40min, more preferably 30min.
In another preferred embodiment, in step (I), the temperature of the sufficient reaction is 20-40 ℃, preferably 22-30 ℃, more preferably 25 ℃.
In another preferred embodiment, in the step (I), the separation tag is biotin, and in the step (II), the separation binding tag is streptavidin.
In another preferred embodiment, in the step (II), the separation includes the sub-steps of:
(i) Adding component (c) of the detection system according to the first aspect of the present invention to the fully reacted detection system of step (I) for fully binding, wherein the separation binding tag is a tag capable of specifically binding to the separation tag;
(ii) Adding component (b) of the detection system according to the first aspect of the present invention to the fully bound detection system of sub-step (i) for performing 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 combinations thereof.
In another preferred embodiment, in the substep (i), the solid support material comprises: homopolymers, copolymers, or combinations thereof.
In another preferred embodiment, in the substep (i), the solid support material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.
In another preferred embodiment, in the substep (i), the solid support material is selected from the group consisting of: magnetic beads, microspheres, microwell plates, strips, test tubes, or combinations thereof.
In another preferred embodiment, in the substep (i), the solid support is a magnetic bead.
In another preferred embodiment, in said sub-step (i), said sufficient bonding time is from 0.5 to 2 hours, preferably from 0.8 to 1.5 hours, more preferably 1 hour.
In another preferred embodiment, in the substep (ii), the alkaline medium is NaOH.
In another preferred embodiment, the final concentration of the alkaline medium in the detection system is 100-300mM, preferably 150-250mM, more preferably 200mM.
In another preferred embodiment, in the substep (ii), the time of the nucleic acid cleavage reaction is 15-60min, preferably 20-40min, more preferably 30min.
In another preferred embodiment, in the step (III), the method further comprises transferring the fluorescent signal supernatant to be measured into a 96-well plate.
In another preferred embodiment, in the determination of the fluorescent signal of step (III), the determination is performed by a microplate reader.
In another preferred embodiment, the fluorescent group included in the T1 chain in the detection system is FAM, and in the determination of the fluorescent signal in the step (III), the excitation wavelength is 485nm, and the emission wavelength is 520nm.
In a third aspect of the invention, there is provided a kit for detecting a DNA glycosylase, the kit comprising:
(a) A first container and (a) in a detection system according to the first aspect of the invention in the first container;
(b) A second container and (b) in a detection system according to the first aspect of the invention in the second container; and
(C) A third container and (c) in a detection system according to the first aspect of the invention 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 understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a schematic diagram showing the mechanism of the double-stranded DNA probe for detecting various glycosylases according to the present invention.
FIG. 2 shows fluorescence values and analysis results after treatment with different concentrations of UDG using a G/U mismatched double stranded DNA probe as a substrate.
FIG. 3 shows fluorescence values and analysis results after treatment with different concentrations of TDG using a G/U mismatched double stranded DNA probe as a substrate.
FIG. 4 shows fluorescence values and analysis results after treatment with different concentrations of SMUG1 using a G/U mismatched double stranded DNA probe as substrate.
FIG. 5 shows the enzymatic 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 the fluorescence values measured in the present method for Calu-1 cell extract.
FIG. 6 shows the enzyme activity inhibition efficiency of TDG by Doxorubicin at different dilution concentrations.
FIG. 7 shows the enzyme activity inhibition ratios of different compounds to UDG in 384 well plate systems, the screening positive ratios were calculated with 30% and 50% inhibition ratios as thresholds, respectively, and values with inhibition ratios below 30% are not shown here.
Detailed Description
Through extensive and intensive research, the inventor develops a method capable of detecting various DNA glycosylases, which has the advantages of high flux, low required sample quantity, large signal window, simple and convenient operation and low cost for the first time through a large amount of screening. Specifically, the present inventors introduced one uracil into one strand of a double-stranded DNA probe, and labeled biotin and a fluorescent group on both sides of the uracil base, respectively, and further separated the fluorescent and biotin-labeled cleavage products 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, simple, high in flux and easy to operate, can be suitable for enzyme activity detection of various DNA glycosylases such as UDG, TDG, SMUG, OGG1 and the like by simply replacing a substrate probe, and has high accuracy. The present invention has been completed on the basis of this finding.
Terminology
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 double-stranded DNA probes of the invention for detecting DNA glycosylase.
In the present invention, 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; wherein, T1 chain includes: at least one base, a fluorescent group and a separation tag that are recognized by the DNA glycosylase to be tested; and the fluorescent group 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, still more preferably 30nM; the length of the double-stranded DNA probe is 5-200bp, preferably 10-100bp, more preferably 15-70bp, and still more preferably 18-40bp.
In the present invention, the base recognized by the DNA glycosylase to be tested is selected from, but not limited to, the group consisting of: uracil bases, cytosine bases, thymine bases, guanine bases, methylation-modified cytosine bases, 5-carboxycytosine, alkyladenine, and the like. In another preferred embodiment, 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 an 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 fluorescent group and the separation tag are each independently located at the 5 'end, the 3' end and the middle of the double-stranded DNA probe.
In the present invention, the fluorescent groups may be all fluorescent groups that can be used for crosslinking with the DNA probe. In another preferred embodiment, the fluorophore is selected from the group consisting of: FAM, FITC, BODIPY-FL, G-Dye100, fluorX, cy3, cy5, texas Red, etc.
In the present invention, the separation tag means a tag capable of separating a nucleic acid sequence attached to or containing the separation tag from a detection system, and may be selected from the group consisting of: a protein, peptide fragment or nucleic acid fragment.
In a preferred embodiment, the separation tag is selected from, but not limited to, the group consisting of: an antigen, an antibody, a ligand, a receptor, an avidin, biotin, or a combination thereof.
In a preferred embodiment, the separation tag is biotin.
In a preferred embodiment, in the double-stranded DNA probe, the sequence of the T1 strand is 5'-FAM-S1-biotin-3', and the sequence of the T2 strand is 5'-S2-3', wherein the sequence of S1 is shown in SEQ ID NO:1 and the sequence of S2 is shown in SEQ ID NO: 2.
DNA glycosylase
BER (base excision repair) pathway is the main pathway to repair endogenous DNA base damage caused by oxidation, alkylation and deamination, and DNA glycosylase is an important link in BER pathway. The DNA glycosylase can specifically cleave N-beta-glycosidic bonds on damaged or mismatched nucleotides, forming abasic sites (AP sites) on the DNA strand. The AP endonuclease then cleaves the glycoside-phosphate bond of the damaged nucleotide and removes small fragments of DNA including the AP site nucleotide, and a new fragment is synthesized by DNA polymerase I, and finally the two fragments are connected into a new repaired DNA chain by DNA ligase to complete DNA damage repair.
In one embodiment of the invention, the glycoside-phosphate bond of the abasic site is broken using NaOH solution as 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 single function uracil DNA glycosylase), MBD4 (methylated CpG binding domain protein 4), AAG (N-methylpurine DNA glycosylase, also known as MPG), and the like.
An important function of UDG (uracil-DNA glycosylase) is to prevent mutagenesis by cleaving the N-glycosyl bond and initiating the Base Excision Repair (BER) pathway to eliminate uracil from the DNA molecule. Uracil bases generally occur in 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. The enzyme also removes thymine from the C/T and T/T mismatches due to its lower activity. TDG can also remove uracil and 5-bromouracil from mispaired with guanine. The enzyme plays an important role in cellular defense against genetic mutations caused by spontaneous deamination of 5-methylcytosine and cytosine.
SMUG1 (Single-stranded, selectively monofunctional uracil DNA glycosylase) removes uracil and 5-hydroxymethyluracil from single-stranded and double-stranded DNA in nuclear chromatin, thereby facilitating base excision repair. In general, the repair activity for single-stranded DNA is stronger than that 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 to the major product of 5-methylcytosine CpG-TPGmismatch-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 a DNA glycosylase which is only active against 3-methyladenine, hypoxanthine and 1, N6-vinyladenine in mammals. Although AAG also has the ability to remove 8-oxyguanine DNA damage, it is not the primary glycosylase for 8-oxyguanine repair.
OGG1 (8-hydroxyguanine DNA glycosylase) releases free 8-hydroxyguanine from oxidatively mutagenized DNA and causes single strand breaks in double-stranded DNA on 8-hydroxyguanine residues paired with cytosine.
In one embodiment of the present invention, when the base recognized by the DNA glycosylase to be detected in the double-stranded DNA probe is uracil base, it can be used for detection of UDG, TDG, SMUG 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 detection of different DNA glycosylases.
The detection system of the invention
In the present invention, there is provided a detection system for detecting a 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 comprises: at least one base, a fluorescent group and a separation tag that are recognized by the DNA glycosylase to be tested; and the fluorophore and the separation tag are located at both ends of the at least one base that is recognized by a DNA glycosylase; (b) A component that cleaves the glycoside-phosphate bond of the abasic site of the nucleic acid.
In the detection system of the present invention, the (b) may be an alkaline mediator or an abasic site endonuclease (AP endonuclease).
In a preferred embodiment, the alkaline medium is NaOH and the final concentration in the detection system is 100-300mM, preferably 150-250mM, more preferably 200mM.
In the invention, the alkaline medium NaOH solution can be used as an alkaline medium to cut the abasic site.
Compared with the traditional abasic site endonuclease (AP endonuclease), the alkaline medium can be separated more efficiently in the separation step by utilizing the characteristics of reduced viscosity, increased buoyancy density, increased sedimentation speed and the like after nucleic acid denaturation.
In another preferred embodiment, the detection system further comprises: (c) a solid support bearing an isolated binding tag. The (c) is used for separating the nucleic acid fragment with the separation label from the detection system.
Wherein the solid support material is selected from, but not limited to, the group consisting of: metal, glass, gel, plastic, or combinations thereof. In another preferred embodiment, the solid support material comprises: homopolymers, copolymers, or combinations thereof. In another preferred embodiment, the solid support material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof. In another preferred embodiment, the solid support material is selected from the group consisting of: magnetic beads, microspheres, microwell plates, strips, 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, the reaction buffer comprising: 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 20mM. In a preferred embodiment, the final concentration of EDTA is 0.5-2mM, preferably 1mM. In a preferred embodiment, the final concentration of DTT is 0.5-2mM, preferably 1mM.
The detection method of the invention
In the present invention, there is provided a method for detecting a DNA glycosylase, comprising the steps of: (I) Providing components (a) and (b) in the detection system according to the first aspect of the present invention, and further comprising a DNA glycosylase to be tested, performing a sufficient reaction; (II) separating the nucleic acid fragment bearing the separation tag from the detection system using the separation tag described in the first aspect of the invention; and (III) carrying out fluorescent signal measurement on the detection system after the separation of the nucleic acid fragments with the separation labels.
The detection method of the invention comprises the following steps: qualitative and quantitative detection.
In the present invention, the qualitative detection includes: detecting whether a sample to be detected contains DNA glycosylase; the quantitative detection comprises the following steps: and 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 with a blank sample that does not contain DNA glycosylase and has a measured fluorescence signal of A0. The fluorescence signal obtained after the measurement by using the DNA glycosylase to be detected is A1, and if A1/A0 is more than 1.1, the DNA glycosylase is considered to exist in the sample to be detected; if A1/A0 is less than or equal to 1.1, the sample to be detected is considered to have no DNA glycosylase.
In another preferred example, when the method is used for quantitative detection of a DNA glycosylase, a DNA glycosylase solution of a known concentration is substituted for the sample to be detected, the operations of steps (I) to (III) are performed, and the method further comprises the steps of: (IV) constructing a linear curve of fluorescence intensity versus known DNA glycosylase; (V) repeating the steps (I) to (III) with the DNA glycosylase to be tested, bringing the obtained fluorescence intensity 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, more preferably 1 to 20nM.
In another preferred embodiment, in the step (I), the time for the sufficient reaction is 10 to 60min, preferably 20 to 40min, more preferably 30min. In another preferred embodiment, in step (I), the temperature of the sufficient reaction is 20-40 ℃, preferably 22-30 ℃, more preferably 25 ℃.
In a preferred embodiment of the present invention, in the step (I), the separation tag is biotin, and in the step (II), the separation binding tag is streptavidin.
In another preferred embodiment, in the step (II), the separation includes the sub-steps of:
(i) Adding the solid phase carrier with the separation binding label into the fully reacted detection system in the step (I) to fully bind, wherein the separation binding label is a label capable of specifically binding with the separation label; (ii) Adding an alkaline medium to the fully combined detection system in the sub-step (i) to perform a nucleic acid cleavage reaction.
In the substep (i), the solid support material is selected from, but not limited to, the group consisting of: metal, glass, gel, plastic, or combinations thereof. In another preferred embodiment, the solid support material comprises: homopolymers, copolymers, or combinations thereof. In another preferred embodiment, the solid support material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof. In another preferred embodiment, the solid support material is selected from the group consisting of: magnetic beads, microspheres, microwell plates, strips, test tubes, or combinations thereof.
In a preferred embodiment, in said substep (i), said solid support is a magnetic bead.
In another preferred embodiment, in said sub-step (i), said sufficient bonding time is from 0.5 to 2 hours, preferably from 0.8 to 1.5 hours, more preferably 1 hour.
In a preferred embodiment, in said substep (ii), said alkaline medium is NaOH, at a final concentration in the detection system of 100-300mM, preferably 150-250mM, more preferably 200mM; and the reaction time is 15 to 60 minutes, preferably 20 to 40 minutes, more preferably 30 minutes.
In another preferred embodiment, in the step (III), further comprising transferring the fluorescent signal supernatant to be measured into a 96-well plate; in the determination of the fluorescent signal of step (III), the determination is performed by an enzyme-labeled instrument.
In a preferred embodiment, the fluorescent group comprised by the T1 chain in the detection system is FAM and the excitation wavelength is 485nm and the emission wavelength is 520nm in the determination of the fluorescent signal of step (III).
In a preferred embodiment, after the double-stranded DNA probe and the sample to be detected are sufficiently reacted in a reaction buffer, streptavidin magnetic beads and a NaOH solution capable of denaturing nucleic acid are sequentially added, so that the double-stranded DNA of the abasic site is combined with the streptavidin-labeled magnetic beads; cutting an abasic site by an alkaline medium, leaving a nucleotide gap at the site, thereby disconnecting the DNA fragment with FAM at the 5 'end from the DNA fragment with biotin at the 3' end, and breaking the double helix structure of the DNA by the alkaline medium to cause separation of the FAM-labeled DNA fragment and the biotin-labeled DNA fragment from their complementary strands; the streptavidin marked magnetic beads can be adsorbed by a magnetic rack, and the DNA fragment with the fluorescent group FAM is released into the supernatant. The nucleic acid fragments with biotin labels are bound to a solid support, which is a magnetic bead, and thereby adsorbed to the tube wall, and then separated from the solution.
Thus, the cleaved nucleic acid probes remain in solution in the original detection system and each bear a fluorescent group. Therefore, the concentration of the DNA glycosylase in the liquid to be detected can be obtained by detecting the fluorescence signal.
The detection method provided by the invention has important significance for early diagnosis of abnormal expression of DNA glycosylase and research and development of inhibitors targeting the DNA glycosylase.
Kit for detecting a substance in a sample
The invention provides a kit for detecting DNA glycosylase, which is characterized by comprising the following components: (a) A first container and (a) in a detection system according to the first aspect of the invention in the first container; (b) A second container and (b) in a detection system according to the first aspect of the invention in the second container; and (c) a third container and (c) in a detection system according to the first aspect of the invention 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 for detecting the activities of various DNA glycosylases, and adopts a double-chain DNA fluorescent probe containing corresponding DNA glycosylase recognition sites in the technical scheme of the invention, and the principle is applicable to various DNA glycosylases;
(2) The operation is simple, and the positive rate is high: in the scheme, no additional amplification step is needed, and the traditional method for detecting the glycosylase is often combined with a complex amplification step, so that false positive signals are easy to appear in the amplification process; the method used in the invention does not need participation of other enzymes;
(3) High flux, low cost: the method in the scheme can be applied to a high-throughput screening system, can be used for screening 384-well plates horizontally, and has the cost lower than 1 yuan/well.
(4) The alkaline medium can be used as a nucleic acid denaturing agent, and compared with the traditional abasic site endonuclease (AP endonuclease), the alkaline medium can utilize the characteristics of viscosity reduction, buoyancy density increase, sedimentation speed increase and the like after nucleic acid denaturation, so that the cut nucleic acid fragments are separated more efficiently in the separation step.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise indicated, the materials and reagents used in the examples were all commercially available products.
Experimental reagent and instrument
Experimental reagent:
The DNA oligonucleotides were synthesized and purified by the Jieli bioengineering Co.Ltd (Shanghai). Uracil DNA Glycosylase (UDG), single strand selective single function uracil DNA glycosylase (SMUG 1) and alkyl adenine DNA glycosylase (AAG) were purchased from NEB (U.S., massa), thymine glycosylase (TDG) over-expressed from E.coli and purified; streptavidin magnetic beads are purchased from Thermo Fisher Co., ltd (USA, hemp, japan), magnetic frames are purchased from Boehringer, shenzhen, and sodium hydroxide is purchased from the national drug group; the ultrapure water used in the preparation of the solution was obtained from a Millipore Milli-Q water purification system.
Experimental instrument:
Fluorescence detection was performed using a Kendi Infinite-200 fluorescence spectrometer (Kendi, switzerland); the excitation wavelength is 485nm, and the emission wavelength is 520nm; the excited slit width was 20nm and the emitted slit width was 10nm.
Example 1: pure UDG Activity assay
Firstly, designing a double-stranded DNA probe T1-T2 containing uracil base, wherein the nucleotide sequence of the 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 strand is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 strand and the T2 strand complement each other to form a double-stranded DNA probe T1-T2.
The double-stranded DNA probes T1-T2 and UDG at different concentrations (0 nM,15nM,30nM,60nM,120 nM) were added to reaction buffers each having the following composition: 20mM Tris-Cl pH8.0,1mM EDTA,1mM DTT; the final volume was 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 of streptavidin magnetic beads were added to the reaction solution to fully bind 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 a magnetic rack, and the supernatant was transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorometry.
A linear curve between fluorescence intensity and UDG concentration was constructed, and the result is shown in FIG. 2, R 2 = 0.9902, and the UDG protein concentration is in a linear relation with the fluorescence value between 0 and 120 nM. It was demonstrated that the present method can detect UGD protein as low as 15 nM.
Example 2: pure TDG Activity detection
Firstly, designing a double-stranded DNA probe T1-T2 containing uracil base, wherein the nucleotide sequence of the 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 strand is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 strand and the T2 strand complement each other to form a double-stranded DNA probe T1-T2.
The double-stranded DNA probes T1-T2 and TDG at different concentrations (0 nM,1.85nM,5.56nM,16.67nM,50 nM) were added to reaction buffers each having the following composition: 20mM HEPES pH7.5, 100mM NaCl,0.2mM EDTA,2.5mM MgCl 2; the final volume was 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 of streptavidin magnetic beads were added to the reaction solution to fully bind 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 a magnetic rack, and the supernatant was transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorometry.
A linear curve between fluorescence intensity and TDG concentration was constructed, and the result is shown in FIG. 3, R 2 = 0.9968, and the TDG protein concentration is in linear relation with fluorescence value between 0 and 50 nM. The method was demonstrated to detect TGD protein 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 the 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 strand is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 strand and the T2 strand are complementary to form a double-stranded DNA probe T1-T2;
The double-stranded DNA probes T1-T2 and SMUG1 at different concentrations (0 nM,12.5nM,25nM,50nM,100 nM) were added to reaction buffers each having the following composition: 10mM Tris-Cl pH7.0, 10mM MgCl 2, 1mM DTT; the final volume was 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 of streptavidin magnetic beads were added to the reaction solution to fully bind 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 60 minutes, the streptavidin magnetic beads were adsorbed to the tube wall under the action of a magnetic rack, and the supernatant was transferred to a new 96-well plate and placed in a Tecan microplate reader for fluorometry.
A linear curve between fluorescence intensity and TDG concentration was constructed, and the result is shown in FIG. 4, R 2 = 0.9945, SMUG protein concentration between 0-100 nM is linear with fluorescence value. The present method was demonstrated to detect SMUG proteins as low as 12.5 nM.
Example 4: detection of TDG Activity in Lung cancer (Calu-1) cell lysate
The detection method of the invention is adopted to analyze and detect the TDG activity in Calu-1 cell lysate, and comprises the following specific steps:
Calu-1 cell samples were isolated by centrifugation (5 min,1000rpm,4 ℃) and resuspended by lysis buffer (20 mM Tris-Cl pH 8.0,1.5mM MgCl 2, 10mM KCl,1mM DTT,1mM EDTA); standing the mixed solution on ice for 10min and centrifuging at 3500rpm for 10min, removing supernatant, re-suspending and precipitating with cell nucleus lysate (20 mM Tris-Cl pH 8.0, 420mM NaCl,10mM KCl,1mM DTT,1mM EDTA), standing on ice for 20min, centrifuging at 12000rpm for 20min, and collecting supernatant as Calu-1 nuclear cell lysate; the Calu-1 nuclear cell lysate can be directly subjected to TDG activity detection without further treatment; the assay was identical to the assay for pure TDG activity in example 2.
As shown in FIG. 5, the measured fluorescence values were taken into a linear curve of the concentration of TDG protein and the fluorescence values, and it was calculated that 10 7 Calu-1 cells contained approximately 9.12nM of TDG protein.
Example 5: inhibition of TDG activity assay
Doxorubicin is a small molecule compound that binds to double-stranded DNA and, when bound to DNA, can affect the ability of TDG to recognize double-stranded DNA. 50nM TDG was mixed with different concentrations of Doxokumicin and incubated at 37℃for 30min; double-stranded DNA probes T1-T2 were added to the mixture to give a system with a final volume of 100. Mu.L, and incubated at 25℃for 30min; then adding 1 mu L of streptavidin magnetic beads into the mixed system, and fully reacting for 1 hour; naOH is added into the reaction solution to a final concentration of 200mM, the reaction solution is incubated for 1 hour at room temperature, streptavidin magnetic beads are adsorbed to the tube wall under the action of a magnetic rack, the supernatant is transferred into a new 96-well plate and placed into a Tecan microplate reader for fluorescence measurement; all experiments were repeated twice.
After background subtraction of the measured fluorescence values, the fluorescence values of the different concentrations of Doxorubicin were calculated as the ratio of activity relative to the solvent control, taking the solvent control fluorescence value as 100%. As shown in fig. 6, the relative fluorescence of the system decreased with increasing concentration of Doxorubicin, IC 50 = 2.12nM. The results indicate that the method can be used for detecting the activity inhibitor of TDG.
Example 6: high throughput screening of DNA glycosylase inhibitors
The method can be applied to 384-well plate systems for high throughput screening of DNA glycosylase inhibitors, taking UDG as an example, wherein UDG is mixed with a reaction buffer (20mM HEPES pH 7.5, 100mM NaCl,0.2mM EDTA,2.5mM MgCl 2) at a final concentration of 50nm/L, the mixture is added into 384-well plates through a pipetting workstation, the volume of the mixture in each well is 19.6 mu L, a compound (1 mM) distributed in the 384-well plates is added into 384-well plates containing TDG mixed solution through the pipetting workstation (0.4 mu L of compound/well), and the final concentration of the compound is 20 mu M; pre-incubating the enzyme with the compound for 30min at room temperature, and adding 10 μl of double-stranded DNA probe to a final concentration of 30nm/L, incubating at 25 ℃ for 30min, creating abasic sites; streptavidin beads were added to the mixture (0.6. Mu.L/well) and incubated for 1 hour, naOH solution was added to a final concentration of 200mM and incubated for 30min; streptavidin magnetic beads are adsorbed by a magnetic frame, the supernatant solution is transferred to a new 384-well plate, and the 384-well plate is placed in a Tecan microplate reader for fluorescence measurement.
After background subtraction of the measured fluorescence values, the inhibition ratios of the fluorescence values of the different compounds relative to the solvent control group were calculated with the solvent control group fluorescence value being 100% and the inhibition ratio being 0%. As shown in FIG. 7, a screening positive rate of 1.5% was obtained with a 30% enzyme activity inhibition rate as a threshold value, and a screening positive rate of 0.9% was obtained with a 50% enzyme activity inhibition rate as a threshold value. The experiment proves that the method can be used for high-throughput screening of DNA glycosylase inhibitors.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
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Claims (52)

1. A detection system for detecting a DNA glycosylase, the detection 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 comprises: at least one base, a fluorescent group and a separation tag that are recognized by the DNA glycosylase to be tested;
and the fluorophore and the separation tag are located at both ends of the at least one base that is recognized by a DNA glycosylase;
(b) A component capable of cleaving a glycoside-phosphate bond of an abasic site of a nucleic acid, the component being an alkaline medium NaOH; and
(C) A solid support with a separation binding tag.
2. The detection system of claim 1, wherein the concentration of double-stranded DNA probe in (a) in the detection system is 10nM to 80nM.
3. The detection system of claim 2, wherein the concentration of double-stranded DNA probe in (a) in the detection system is 20-50nM.
4. The detection system of claim 2, wherein the concentration of double-stranded DNA probe in (a) in the detection system is 25-35nM.
5. The detection system of claim 2, wherein the concentration of double-stranded DNA probe in (a) in the detection system is 30nM.
6. The detection system of claim 1, wherein the double-stranded DNA probe is 5-200bp in length.
7. The detection system of claim 6, wherein the double-stranded DNA probe has a length of 10-100bp.
8. The detection system of claim 6, wherein the double-stranded DNA probe has a length of 15-70bp.
9. The detection system of claim 6, wherein the double-stranded DNA probe has a length of 18-40bp.
10. The test system according to claim 1, wherein the base recognized by the test DNA glycosylase is selected from the group consisting of: uracil bases, cytosine bases, thymine bases, guanine bases, methylation modified cytosine bases, 5-carboxycytosine, alkyladenine.
11. The test system according to claim 1, wherein the at least one base recognized by the DNA glycosylase to be tested is a uracil base.
12. The test system according to claim 1, wherein 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.
13. The detection system of claim 1, wherein the fluorophore and the separation tag are each independently located at the 5 'end, the 3' end, and the middle of the double-stranded DNA probe.
14. The detection system of claim 1, wherein the fluorophore comprises a fluorophore that can be used to crosslink with a DNA probe.
15. The detection system of claim 1, wherein the fluorophore is selected from the group consisting of: FAM, FITC, BODIPY-FL, G-Dye100, fluorX, cy3, cy5, texas Red.
16. The detection system of claim 1, wherein the separation tag is a tag that enables a nucleic acid sequence attached to or comprising the separation tag to be separated from the detection system.
17. The detection system of claim 1, wherein the separation tag is selected from the group consisting of: a protein, peptide fragment or nucleic acid fragment.
18. The detection system of claim 1, wherein the separation tag is selected from the group consisting of: an antigen, an antibody, a ligand, a receptor, an avidin, biotin, or a combination thereof.
19. The detection system of claim 1, wherein the separation tag is biotin.
20. The assay system of claim 1, wherein the alkaline medium is present in the assay system at a final concentration of 100 to 300mM.
21. The assay system of claim 20, wherein the alkaline medium is present in the assay system at a final concentration of 150 to 250mM.
22. The assay system of claim 20, wherein the alkaline medium is present in the assay system at a final concentration of 200mM.
23. The assay system of claim 1 wherein in the assay system, the solid support material is selected from the group consisting of: metal, glass, colloid, plastic, or combinations thereof.
24. The detection system of claim 1, wherein the solid support material comprises: homopolymers, copolymers, or combinations thereof.
25. The detection system of claim 1, wherein the solid support material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.
26. The detection system of claim 1, wherein the solid support material is selected from the group consisting of: magnetic beads, microspheres, microwell plates, strips, test tubes, or combinations thereof.
27. The detection system of claim 1, wherein the solid support is a magnetic bead.
28. The test system of claim 1, wherein the final volume of the test system is 50 to 200 μl.
29. The test system of claim 28, wherein the final volume of the test system is 60 to 150 μl.
30. The test system of claim 28, wherein the final volume of the test system is 80 to 120 μl.
31. The test system of claim 28, wherein the final volume of the test system is 100 μl.
32. The detection system of claim 1, wherein the detection system further comprises a reaction buffer.
33. The assay system of claim 32, wherein the reaction buffer comprises: tris-ClpH8.0, EDTA, DTT.
34. The assay system of claim 33, wherein the final concentration of Tris-clph8.0 is 10-50mM.
35. The assay system of claim 34, wherein the final concentration of Tris-clph8.0 is 15-30mM.
36. The assay system of claim 34, wherein the final concentration of Tris-clph8.0 is 20mM.
37. The assay system of claim 33, wherein the final concentration of EDTA is 0.5-2mM.
38. The test system of claim 37, wherein the final concentration of EDTA is 1mM.
39. The assay system of claim 33, wherein said DTT is present at a final concentration of 0.5-2mM.
40. The assay system of claim 39, wherein the final concentration of DTT is 1mM.
41. The test system of claim 1, further comprising a DNA glycosylase to be tested.
42. The test system of claim 1, wherein the test DNA glycosylase is selected from the group consisting of: UDG, TDG, SMUG1, MBD4, OGG1, AAG, or a combination thereof.
43. The test system of claim 1 or 41, wherein the test DNA glycosylase is selected from the group consisting of: UDG, TDG or SMUG1.
44. The test system of claim 1 or 41, wherein the concentration of the test DNA glycosylase is in the range of 1 nM to 500nM.
45. The test system of claim 44, wherein the concentration of the test DNA glycosylase is in the range of 1 nM to 50nM.
46. The test system of claim 44, wherein the concentration of the test DNA glycosylase is in the range of 1 nM to 20nM.
47. The detection system according to claim 1, wherein 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 in SEQ ID NO. 1 and the sequence of S2 is shown in SEQ ID NO. 2.
48. The detection system of claim 1, wherein the DNA glycosylase is selected from the group consisting of: purified DNA glycosylase, a lysate thereof, a cell lysate, blood or an extract thereof, a body fluid or an extract thereof, or a combination thereof.
49. The test system of claim 48, wherein the cell lysate comprises a cancer cell lysate.
50. The test system of claim 49, wherein the cancer comprises lung cancer.
51. The assay system of claim 1, wherein said detecting comprises: qualitative and quantitative detection.
52. Use of the detection system according to any one of claims 1 to 51 for the detection of DNA glycosylase for non-disease diagnostic purposes, or for the preparation of a DNA glycosylase detection reagent or detection kit.
CN201910008741.7A 2019-01-04 2019-01-04 Method for detecting activity of various DNA glycosylase Active CN111411153B (en)

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CN201980087780.9A CN113366114B (en) 2019-01-04 2019-12-26 Method for detecting activity of various DNA glycosylase
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