CN113151420B

CN113151420B - One-step fluorescence detection system, and detection method and application of DNA glycosylase activity

Info

Publication number: CN113151420B
Application number: CN202110361586.4A
Authority: CN
Inventors: 张春阳; 胡娟; 刘雯
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-06-21
Anticipated expiration: 2041-04-02
Also published as: CN113151420A

Abstract

The invention discloses a one-step fluorescence detection system, a detection method of DNA glycosylase activity and application thereof, comprising a substrate, a template, APE1 enzyme, DNA polymerase, dNTP/dUTP mixture, UDG enzyme and fluorescent dye, wherein the substrate and the template are single-stranded DNA, the substrate contains 8-oxoguanine, and NH is used at the 3' end of the substrate₂Modifying; the template is formed by cross-linking at least one piece of DNA1 and at least one piece of DNA2, wherein the DNA1 and the DNA2 do not contain adenine, and the joint of the DNA1 and the DNA2 contains adenine deoxynucleotide; the substrate is complementary to DNA1 and a portion of DNA2, and cytosine complementary to 8-oxoguanine is located in DNA 1; dNTPs do not contain dTTP. The detection system can simply and quickly complete detection; the exponential amplification reaction can be carried out, and the detection sensitivity of hOGG1 is improved; avoids the use of nicking incision enzyme, thereby effectively preventing nonspecific amplification.

Description

One-step fluorescence detection system, and detection method and application of DNA glycosylase activity

Technical Field

The invention belongs to the technical field of analysis and inspection, and relates to a one-step fluorescence detection system, a detection method of DNA glycosylase activity and application.

Background

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Human 8-hydroxyguanine DNA glycosylase 1(hOGG1) is an important glycosylase in nuclei and mitochondria, acts on 8-oxoguanine (8-oxoG) paired with cytosine, and initiates the excision repair process of oxidized guanine bases in all organisms. Since abnormalities in the level of hOGG1 are associated with various diseases, accurate and simple detection of hOGG1 activity is of great significance in disease prediction and diagnosis.

According to the research of the inventors, isothermal exponential amplification reaction (EXPAR) is a promising molecular diagnostic technique due to its simple operation, fast reaction and high amplification efficiency in recent years. In classical EXPAR experiments, nicking endonucleases are commonly used to cyclically cleave phosphodiester bonds, allowing the 3' end of the nicking site to serve as a primer to continually initiate a new round of replication. However, the use of nicking endonucleases in EXPAR assays tends to cause unwanted non-specific amplification, which limits the reliability and specificity of detection in practical applications. Additionally increasing the number of nicking endonucleases also decreases the reaction rate of the EXPAR.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a one-step fluorescence detection system, a detection method and application of DNA glycosylase activity, wherein the detection system can complete detection more simply and rapidly; the exponential amplification reaction can be carried out, and the detection sensitivity of hOGG1 is improved; the use of nicking endonucleases is avoided, thereby effectively preventing non-specific amplification observed in conventional EXPAR systems.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, a one-step fluorescence detection system comprises,

substrate and template: the substrate and the template are single-stranded DNA, the substrate contains 8-oxoguanine, and NH is used at the 3' end of the substrate₂Modifying; the template is formed by cross-linking at least one piece of DNA1 and at least one piece of DNA2, wherein the DNA1 and the DNA2 do not contain adenine, and the joint of the DNA1 and the DNA2 contains adenine deoxynucleotide; the substrate is complementary to DNA1 and a portion of DNA2, and cytosine complementary to 8-oxoguanine is located in DNA 1;

APE1 enzyme: for cleavage of the AP site;

DNA polymerase and dNTP/dUTP mix: used for carrying out extension reaction at the 3' OH end after the AP locus is cut off, and dNTP does not contain dTTP;

UDG enzyme: used for removing uracil in the extension reaction to obtain an extension product;

fluorescent dye: for generating fluorescence with the amplification product.

In the present invention, NH is used for the 3' end of the substrate₂The modification is used to prevent non-specific amplification. The substrate contains 8-oxoguanine, cytosine complementary to 8-oxoguanine is located in DNA1, and is capable of allowing hOGG1 to recognize and excise 8-oxoguanine at a specific position to generate an AP site, generating a 3' OH end by enzymatic cleavage with APE1, then performing an extension reaction by DNA polymerase and dNTP/dUTP mixture, allowing uracil nucleotide to be incorporated into the extension product due to adenine deoxynucleotide at the junction, and then passing U throughAnd (3) carrying out amplification by recognizing and cutting uracil by using a DG enzyme and then cutting to generate a 3' OH end by using an APE1 enzyme, wherein an amplification product can be detected by using a fluorescent dye, so that the activity of hOGG1 can be detected.

The number of DNA1 and DNA2 in the template affects the amplification multiplicity, for example, when there is only one DNA1 and one DNA2, there is only one junction, and only single amplification can be performed by the action of adenine bases in the junction; when two DNAs 1 and one DNA2 are present, there are two junctions, i.e., 2 adenine bases, triple amplification can be achieved; when two

DNAs

1 and 2 are present, if there are three junctions, i.e., 3 adenine bases, quadruple amplification can be achieved; that is, the number of DNA1 and DNA2 affects the number of adenine bases and thus the amplification efficiency, and the detection effect is better as the amplification efficiency is higher.

On the other hand, a method for detecting the activity of DNA glycosylase is to add a sample to be detected containing hOGG1 into the one-step fluorescence detection system for incubation, and then perform fluorescence detection.

In a third aspect, a kit for detecting the activity of DNA glycosylase comprises the one-step fluorescence detection system and a buffer solution.

In a fourth aspect, the use of the one-step fluorescence detection system for screening for an inhibitor of hOGG1 is provided.

In a fifth aspect, the application of the one-step fluorescence detection system in preparing a reagent for detecting cancer cells is provided.

The invention has the beneficial effects that:

1. compared with the traditional method which consumes much time and operation steps, the one-step fluorescence detection system provided by the invention only needs one-step operation in the process of detecting the activity of hOGG1, the total detection time is about 40min, additional primer probe and modification separation steps are not needed, and the one-step fluorescence detection system has the characteristics of high selectivity and sensitivity and zero background.

2. The one-step fluorescent detection system provided by the invention utilizes the high amplification efficiency of the high-specificity excision of 8-oxoG base induced by hOGG1 and the multiple-cycle enzyme repair amplification in the process of detecting the activity of hOGG1 so as to realize rapidness and simplicityThe mix-read format sensitively detects hOGG1 with a detection limit of 2.97X 10^-8U/μL。

3. The one-step fluorescence detection system provided by the invention can also be used for screening enzyme inhibitors and detecting hOGG1 in human cancer cells. Due to the simplicity of detection of the one-step fluorescence detection system and the ability to improve detection efficiency, the mix-read assay is a promising platform for further use in the development of diagnostic reagents.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of the mechanism for detecting hOGG1 by the hybrid-read method in the embodiment of the present invention.

FIG. 2 is a graph showing the results of experiments for detecting the feasibility of hOGG1 by the mixed-read method in the examples of the present invention, (A) analysis of cleavage reaction of hOGG1 by polyacrylamide gel (PAGE), (B) real-time monitoring of the fluorescence profile of the reaction products in the presence of hOGG1+ APE1+ UDG (experiment), hOGG1+ APE1, hOGG1+ UDG and APE1+ UDG, respectively, (C) analysis of RCA reaction products by polyacrylamide gel (PAGE), and (D) measurement of the fluorescence spectra of the reaction products in the presence of hOGG1+ APE1+ UDG (experiment), hOG 1+ APE1, hOGG1+ UDG and APE1+ UDG, respectively.

FIG. 3 is a schematic diagram of the mechanism for detecting hOGG1 using other template designs in an embodiment of the present invention, (A) template-2, and (B) template-3.

FIG. 4 shows fluorescence spectra of hOGG1 detected using other template designs in an embodiment of the present invention, (A) template-1, (B) template-2, (C) template-3, and (D) three templates (F-F)₀)/F₀The result of the comparison of the values.

FIG. 5 is a graph showing the results of an optimization experiment for detecting hOGG1 according to an embodiment of the present invention, where (A) the fluorescence values corresponding to different amounts of template-1, (B) the fluorescence values corresponding to different combinations of buffers, (C) the fluorescence values corresponding to different amounts of APE1 enzyme, (D) the fluorescence values corresponding to different amounts of UDG enzyme, (E) the fluorescence values corresponding to different amounts of KF enzyme, (F) the fluorescence values corresponding to different concentrations of dNTP/dUTP, and (G) the fluorescence values corresponding to different reaction times.

FIG. 6 is a graph showing the results of the detection of the sensitivity and specificity of hOGG1 in the present example, (A) the fluorescence spectrum curve with the change of hOGG1 concentration, (B) the fluorescence intensity curve with the change of hOGG1 concentration, the inset is shown at 1 × 10^-7To 5X 10^-5Fluorescence intensity in the U/. mu.L range is linearly related to the logarithm of hOGG1 concentration, (C) fluorescence spectroscopy was performed under conditions of 0.02U/. mu.L hOGG1, 0.02 g/. mu.L bovine serum albumin, 0.02 g/. mu.L IgG, 0.02U/. mu.L AAG, 0.02U/. mu.L TDG, and reaction buffer (control).

FIG. 7 is a graph showing the relative activity of hOGG1 at various concentrations of O8 inhibitor in accordance with the present invention.

FIG. 8 is a graph showing the results of measurement of actual samples according to the example of the present invention, (A) the fluorescence intensities of A549 cells, HeLa cells, HL-7702 cells, HEK-293T cell extracts and heat-inactivated A549 cell extracts were measured, and (B) the fluorescence intensities were linearly related to the number of A549 cells.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the fact that nicking endonuclease easily causes unnecessary nonspecific amplification and influences the reaction rate of EXPAR, the invention provides a one-step fluorescence detection system, a detection method of DNA glycosylase activity and application.

In an exemplary embodiment of the present invention, there is provided a one-step fluorescence detection system comprising,

APE1 enzyme: for cleavage of the AP site;

fluorescent dye: for generating fluorescence with the amplification product.

NH for 3' end of substrate₂The modification is used to prevent non-specific amplification. The substrate contains 8-oxoguanine, cytosine complementary to the 8-oxoguanine is located in DNA1, hOGG1 can identify that 8-oxoguanine is cut off at a specific position to generate an AP site, APE1 enzyme cutting generates a 3 'OH end, then DNA polymerase and dNTP/dUTP mixture are used for extension reaction, uracil nucleotide is doped into an extension product due to adenine deoxynucleotide at the connection position, UDG enzyme identification requests cutting of uracil, APE1 enzyme cutting generates a 3' OH end, amplification is carried out, an amplification product can be detected through fluorescent dye, and therefore detection of hOGG1 activity is achieved.

DNAs

1 and 2 are present, quadruple amplification can be achieved if three junctions, i.e., 3 adenine bases, are present; that is, the number of DNA1 and DNA2 affects the number of adenine bases and thus the amplification efficiency, and the detection effect is better as the amplification efficiency is higher. Thus in some embodiments of this embodiment, the template is formed by the cross-linking of two DNAs 1 and two DNAs 2.

In some embodiments of this embodiment, the DNA polymerase is Klenow large fragment polymerase.

Make it

In some embodiments of this embodiment, the fluorescent dye is SYBR Green II.

In some embodiments of this embodiment, the substrate has the nucleotide sequence set forth in SEQ ID NO.1 and the template has the nucleotide sequence set forth in SEQ ID NO. 2.

Optimization experiments show that the amount of the template influences the fluorescence intensity and thus the detection result, and in some examples of the embodiment, the concentration of the template is 14-16 nM when the concentration of the substrate is 10 nM. When the template with the concentration is adopted, the fluorescence intensity of fluorescence detection is better.

The amount of APE1 enzyme used also affects the fluorescence intensity, and in some examples of this embodiment, the amount of substrate is 2X 10^-4When nmol, the dosage of APE1 enzyme is 0.75-0.85U. The fluorescence intensity of fluorescence detection was better with this amount of APE1 enzyme.

The amount of UDG enzyme used also affects the fluorescence intensity, and in some examples of this embodiment, the amount of substrate is 2X 10^- ⁴In nmol, the amount of UDG enzyme is not less than 2U. When the amount of the UDG enzyme is 2U, the amount of the UDG enzyme is not greatly increased, so that the amount of the UDG enzyme is preferably 2.0-2.2U, and the addition of the UDG enzyme is reduced on the premise of ensuring the fluorescence intensity of detection.

The amount of Klenow large fragment polymerase also affects the fluorescence intensity, and in some embodiments, the amount of substrate is 2X 10^-4In nmol, the amount of Klenow large fragment polymerase is 1.9-2.1U. Experiments show that the fluorescence intensity of fluorescence detection is better under the condition.

The concentration of the dNTP/dUTP mixture also affects the fluorescence intensity, and in some embodiments, the concentration of the dNTP/dUTP mixture is 58-62. mu.M at a substrate concentration of 10 nM. Experiments show that the fluorescence intensity of fluorescence detection is better under the condition.

In another embodiment of the invention, a method for detecting the activity of DNA glycosylase is provided, wherein a sample to be detected containing hOGG1 is added into the one-step fluorescence detection system for incubation, and then fluorescence detection is carried out.

In some embodiments of this embodiment, the incubation temperature is 35-39 ℃.

Experiments show that the detection effect is better when the incubation time is not less than 40 min. And when the incubation time is 40-40.5 min, the detection effect can be ensured, and the detection time can be reduced.

In some embodiments of this embodiment, the buffer is a combination of buffer A and buffer B, wherein buffer A comprises Tris-HCl with a concentration of 18-22 mM and a pH of 7.9-8.1, EDTA with a concentration of 0.9-1.1 mM, DTT with a concentration of 0.9-1.1 mM, BSA with a concentration of 0.09-0.11 mg/ml; the buffer solution B comprises 45-55 mM potassium acetate (potassium acetate), 18-22 mM Tris-acetate, 9-11 mM magnesium acetate (magnesium acetate) and 0.9-1.1 mM DTT.

In some examples of this embodiment, the excitation wavelength used in the fluorescence detection is 487-489 nm.

In a third embodiment of the invention, a kit for detecting the activity of DNA glycosylase is provided, which comprises the above one-step fluorescence detection system and a buffer solution.

In a fourth embodiment of the present invention, there is provided a use of the above one-step fluorescence detection system for screening for an inhibitor of hOGG 1.

In a fifth embodiment of the present invention, an application of the above one-step fluorescence detection system in preparing a reagent for detecting cancer cells is provided.

Specifically, the cancer cell is an A549 cell and/or a HeLa cell.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Examples

The nucleotide sequences employed in the examples are shown in Table 1.

TABLE 1 nucleotide sequences

In the substrate, the underlined letter O denotes 8-oxoG. In the template, the portion complementary to the substrate is underlined.

One-step assay of hOGG 1: the entire reaction was carried out in 20. mu.l of a solution containing 10nM substrate, 15nM template-1, hOGG1 at different concentrations, 0.8U of APE1, 2U of UDG, 2U of Klenow large fragment polymerase, 60. mu.M of dNTP/dUTP mix (dATP, dGTP, dCTP, dUTP, each at 60. mu.M concentration), buffer A (20mM Tris-HCl,1mM EDTA,1mM DTT,0.1mg/ml BSA, pH 8.0), buffer B (50mM potassium acetate,20mM Tris-Ac, 10mM magnesium acetate,1mM DTT, pH 7.9). After reacting for 40 minutes at 37 ℃, the amplification product is mixed with SYBR Green II, diluted to a final volume of 50 microliters, and when the excitation wavelength is 488 nanometers, the spectrum is recorded between 502 and 650 nanometers by using a fluorescence spectrometer. Data analysis was performed with maximum fluorescence emission intensity at 525 nm.

Gel electrophoresis experiment: after the amplification reaction, the product was mixed with a fluorescence indicator (SYBR Gold) and added to a 10% polyacrylamide gel, and the gel was electrophoresed in tris-boronic acid (TBE buffer: 89mM Tris-HCl, 89mM boric acid, 2mM EDTA, pH 8.3) at a constant pressure of 110V at room temperature for 40 min. The gels were analyzed by a ChemiDoc MP imaging system. To study the cleavage repair process of hOGG1 alone, 10% polyacrylamide gel electrophoresis analysis of the reaction product of Cy 3-labeled substrate was performed using an EPI-Green light source (520-545 nm excitation) and a 577-613 nm filter.

Inhibitor experiments: different concentrations of the O8 inhibitor were reacted with 0.02U/. mu.L of hOGG1 and other reagents at 37 ℃ followed by fluorescence measurements.

Cell culture and cell extraction: the lung adenocarcinoma cell line A549 (or HeLa cells, HL-7702 cells, HEK-293T cells) is cultured in DMEM medium, and 10% fetal bovine serum and 1% penicillin streptomycin are added into the DMEM medium. The medium was incubated at 37 ℃ in an incubator containing 5% carbon dioxide until the cells were mature. After the cells were matured, the cell extract was extracted using a nuclear extraction kit (Active Motif) according to the instructions. The extract obtained was used for subsequent testing.

The quadruple amplification detection mechanism diagram is shown in figure 1, firstly, a single-stranded DNA substrate modified by 8-oxoG base is designed, and the 3' end of the single-stranded DNA substrate is NH₂And modified so that non-specific amplification can be prevented. The substrate hybridizes to template-1 to form partially double-stranded DNA, wherein 8-oxoG/C base pairs are available for recognition of hOGG 1. In the presence of hOGG1, hOGG1 can bind efficiently to the recognition site, thereby excising the impaired 8-oxoG base from the 8-oxoG/C base pair, resulting in an AP site. The AP site can be cleaved by APE1, resulting in the generation of a cleaved substrate 3' OH terminus. When a DNA polymerase and a dNTP/dUTP mix (i.e., dATP, dGTP, dCTP, and dUTP) are present, the extension reaction is initiated at the 3' OH and uracil (U) nucleotides are incorporated into the extension product, forming two damage sites. Uracil lesions can be cleared by UDG, creating AP sites. APE1 then cleaves these AP sites, releasing both trigger strands (trigger strand-X, trigger strand-Y). Hybridization of the cleavage substrate to template-1 promotes another round of enzymatic repair amplification and produces a large number of trigger strands (first stage). The generated trigger strand-X can continue to hybridize with the free template-1, producing an extension product containing three damage sites, which under the synergistic effect of UDG and APE1 results in a new cyclic enzyme repair amplification process, releasing a large amount of trigger strand-X and trigger strand-Y (second stage); the resulting trigger strand-Y can continue to hybridize to free template-1, producing an extension product containing two damage sites, resulting in a new cyclic enzymatic repair amplification process, also releasing a large amount of trigger strand-X and trigger strand-Y (third stage). At the same time, the generated trigger strand-X can also bind to template-1 to generate new dsDNA that is different from the second stage (fourth stage). The presence of hOGG1 can induce multiple rounds of enzymatic repair amplification including cleavage of substrate, trigger strand-X and triggerStrand-Y initiated amplification, ultimately leads to exponential amplification and the generation of more and more trigger strands. The amplification product was easily detected using SYBR Green II as a fluorescent dye. In contrast, in the absence of hOGG1, no 8-oxoG base excision reaction occurred, nor was a cleavage substrate containing the 3' OH terminus formed, which resulted in no amplification of enzymatic repair, and thus no SYBR Green II signal could be observed.

Feasibility test

To verify the viability of the assay, gel electrophoresis analysis was used to verify the viability of the experiment, as shown in figure 2A. The cleavage process of hOGG1 was studied with a single-stranded DNA substrate modified with 8-oxoG bases and labeled with Cy3 (FIG. 2A, lane 5). The reaction products were analyzed with direct excitation of Cy 3. In the absence of hOGG1, only one 107nt band was observed (which contained Cy3 labeled substrate and template-1), indicating that no cleavage reaction occurred (FIG. 2A, lanes 1 and 2). In the presence of hOGG1, a new 5nt band was observed, indicating that hOGG1 can recognize and cleave 8-oxoG containing substrates (FIG. 2A, lanes 3 and 4).

To investigate the necessity of the UDG enzyme in the cyclic enzyme repair amplification reaction, real-time fluorescence detection was performed in the presence of hOGG1, APE1 and UDG. As in fig. 2B, no fluorescence signal could be observed without chogg 1 or APE 1. In the presence of hOGG1 and APE1 but without the UDG enzyme, a relatively weak fluorescence signal was observed due to the polymerization reaction alone. When hOGG1, APE1 and UDG enzymes were present, a stronger fluorescence signal was observed and increased with longer reaction time, indicating that the cycling enzyme repair amplification reaction proceeded in an exponentially amplified manner.

As shown in FIG. 2C, the hOGG 1-induced amplification product was subjected to PAGE analysis using SYBR Gold as an indicator. The product positions of the circular enzymatic repair amplification are indicated by 20nt synthesized trigger strand-X (FIG. 2C, lane 6) and 21nt synthesized trigger strand-Y (FIG. 2C, lane 5). Only in the presence of hOGG1, APE1 and UDG enzymes, a clear 20-21nt band (containing trigger strand-X and trigger strand-Y) appeared (FIG. 2C, lane 3), indicating the occurrence of a cycling enzymatic repair amplification reaction. In contrast, in the absence of hOGG1 (FIG. 2C, lane 2) or APE1 (FIG. 2C, lane 1), the control group did not show a 20-21nt band; bands of reduced mobility were generated in the presence of hOGG1 and APE1 but in the absence of UDG enzyme (FIG. 2C, lane 4).

Fluorescence spectroscopy measurements of the amplified products confirmed the above results further (fig. 2D). In the absence of hOGG1 or APE1, no significant fluorescence intensity was detected. In the presence of hOGG1 and APE1 but without the UDG enzyme, the polymerization reaction alone detected relatively low fluorescence intensity. However, in the presence of hOGG1, APE1 and UDG enzymes, strong fluorescence intensity was detected due to the cycling enzyme repair amplification. With hOGG1, APE1 and UDG enzymes (F-F)₀)/F₀Values (F and F)₀Fluorescence intensity with hOGG1 and without hOGG1) was much higher than with hOGG1 and APE1, respectively, but without UDG enzyme (F-F)₀)/F₀Values indicate that the addition of UDG enzyme can induce the amplification of the enzymatic cycle repair. These results indicate that cycling enzymatic repair amplification can significantly improve the amplification efficiency of the assay of hOGG1 compared to polymerization alone.

The results clearly show that the method can be used for detecting the DNA glycosylase hOGG 1.

Research on one-step detection system template

In order to study the process of multiplex cycle enzyme repair amplification, other templates were designed. Compared with the design of 3 bases of adenine (A) in template-1 of FIG. 1, which can achieve quadruple amplification, the design of 2 bases of adenine (A) in template-2 of FIG. 3A can achieve triple amplification; as in FIG. 3B, template-3 designs 1 adenine (A) base, which can only achieve single-plex amplification. These templates can generate 3, 2, 1 uracil (U) damage sites in the extension product, and the damage sites are subjected to repeated extension, cleavage, and release of small trigger chains, so as to achieve cyclic enzyme repair amplification and obtain strong fluorescence signals (FIGS. 4A-C). Wherein template-1 achieved high amplification efficiency (FIG. 4D).

The number of embedded dNTPs/dUTPs was further measured to calculate the amplification efficiency. Corresponding to the quadruple cycle amplification of template-1, the amplification efficiency was 6531 fold, i.e. each individual 8-oxoG base could cause the insertion of 6531 dNTP/dUTP in the presence of hOGG 1. For triple amplification of template-2, the amplification efficiency was 5092-fold; for single-fold amplification of template-3, the amplification efficiency was 3664-fold.

Optimization of one-step detection system experiment conditions

To obtain the best experimental results, seven variables of template concentration, different buffer combinations, APE1 enzyme dosage, UDG enzyme dosage, KF enzyme dosage, dNTP/dUTP concentration and reaction time are optimized.

The concentration of template-1 plays an important role in the mix-read assay of hOGG 1. As shown in FIG. 5A, (F-F)₀)/F₀Value of (F and F)₀Fluorescence intensity in the presence and absence of hOGG1, respectively) increased with increasing concentration of template-1 from 10nM to 15nM, and decreased beyond 15 nM. Thus, 15nM template-1 was used in subsequent studies.

To obtain the ideal buffer, optimization studies were performed on a combination of four buffers, including buffer A (20mM Tris-HCl (pH 8.0),1mM EDTA,1mM DTT,0.1mg/ml BSA), buffer B (50mM potassium acetate,20mM Tris-acetate,10mM magnesium acetate,1mM DTT, pH 7.9), buffer C (50mM NaCl,10mM Tris-HCl,10mM MgCl. RTM.)₂1mM DTT, pH 7.9), and buffer D (20mM Tris-HCl,1mM DTT,1mM EDTA, pH 8.0). (F-F) of the combination of buffers A + B (FIG. 5B, a) as in FIG. 5B₀)/F₀Higher than the combination of buffer a + B + C (fig. 5B, B), the combination of buffer a + B + C + D (fig. 5B, C), and therefore the combination of buffer a + B was used in subsequent studies.

Since APE1 can stimulate the 8-oxoG base excision repair reaction catalyzed by hOGG1 and plays a key role in the cycling enzymatic repair amplification reaction, the role of APE1 was studied. In FIG. 5C, (F-F)₀)/F₀Increases as the amount of APE1 increases from 0.2 to 0.8U and then decreases above 0.8U, so 0.8U of APE1 was used in subsequent experiments.

The effect of the amount of UDG on the efficiency of the cyclic amplification was subsequently examined. (F-F)₀)/F₀The value of (c) first increases with increasing amount of UDG, and does not increase much after 2U (as shown in fig. 5D), so 2U is selected for subsequent analysis.

As shown in FIG. 5E, (F-F)₀)/F₀The value increased as the amount of Klenow large fragment polymerase added increased from 0.5 to 2U and gradually decreased beyond 2U, so 2U of Klenow large fragment polymerase was used in subsequent studies.

(F-F) of dNTP/dUTP mixture as shown in FIG. 5F₀)/F₀The value increased as the concentration of the dNTP/dUTP mixture increased from 10. mu.M to 60. mu.M, and decreased beyond 60. mu.M. Therefore, 60 μ M was chosen as the optimal dNTP/dUTP concentration.

Further, (F-F)₀)/F₀The value of (D) was gradually increased with the increase of the cycle enzyme repair amplification reaction time, and reached a plateau value after 40min (FIG. 5G), so 40min was selected as the optimal amplification time.

Sensitivity of detection

Under the best experimental conditions, the sensitivity detection was evaluated. FIG. 6A shows the fluorescence emission spectra as a function of different concentrations of hOGG 1. Fluorescence intensity was varied from 0 to 2X 10 with hOGG1^-2Increase in U/. mu.L concentration. To perform quantitative analysis and obtain detection limits, a fitting equation is further obtained. Fluorescence intensity and hOGG1 concentration were 1X 10 on a logarithmic scale (FIG. 6B inset)^-7-5×10^-5The linear relation exists in the U/mu L range, and the regression equation is that F is 3720.1+459.8log₁₀C(R²0.996), where F and C are fluorescence intensity and concentration of hgg 1 (U/. mu.l), respectively. According to the principle of triple standard deviation of blank signal, the detection limit of said method is calculated to be 2.97X 10^-8U/. mu.L. The detection limit ratio is based on the fluorescent method of rolling circle amplification (1X 10)^-3U/. mu.L) and colorimetry (1.6X 10)^-3U/μ L) is about 4 orders of magnitude higher.

Specificity of detection

To examine the selectivity of this method for detecting hOGG1, Bovine Serum Albumin (BSA), immunoglobulin G (IgG), alkyl adenine DNA glycosylase (AAG), and Thymine DNA Glycosylase (TDG) were used as controls. As shown in fig. 6C, a significant fluorescent signal was observed only when the specific DNA glycosylase chogg 1 was added. The results indicate that the method can distinguish hOGG1 from unrelated protein and other DNA glycosylase members, and has high specificity.

Inhibitor assay

The feasibility of this approach for inhibition assays was demonstrated using an O8 inhibitor. As shown in fig. 7, the relative activity of hrogg 1 decreased with increasing O8 concentration. Its median Inhibitory Concentration (IC)₅₀) Value of 0.54. mu.M, and IC of gel assay₅₀Value 0.22. mu.M and IC of fluorimetry₅₀The value was close to 0.35. mu.M. These results demonstrate that O8 is a potent inhibitor of ogg1 activity, suggesting that this method can be used for screening for an ogg1 inhibitor.

Analysis of actual samples

To verify the application of this method to actual samples, extracts of A549, HeLa, HL-7702, HEK-293T cells were used for detection. FIG. 8A, A549 and HeLa cell samples obtained fluorescence intensities much higher than HL-7702, HEK-293T cell samples and heat-inactivated A549 cell extracts. As shown in fig. 8B, the fluorescence intensity increased with the increase in the number of a549 cells. In the range of 1-10000 cells, the fluorescence intensity is linearly related to the logarithm of the number of cells. The regression equation is F-595.5 log₁₀N+712.1(R²0.999), where F is the fluorescence intensity, N is the number of a549 cells, and the detection limit is 1 cell. These results indicate that the method can be used to detect hOGG1 enzyme in cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of Shandong Master

<120> one-step fluorescence detection system, and detection method and application of DNA glycosylase activity

<130>

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 26

<212> DNA

<213> Artificial sequence

<400> 1

tacacagaca ggaaaagaag gtacac 26

<210> 2

<211> 81

<212> DNA

<213> Artificial sequence

<400> 2

ccttcttttc ctgtctgtgt attctctctt tctgtcgtgt accttctttt cctgtctgtg 60

tattctctct ttctgtcgtg t 81

<210> 3

<211> 62

<212> DNA

<213> Artificial sequence

<400> 3

ccttcttttc ctgtctgtgt attctctctt tctgtcgtgt accttctttt cctgtctgtg 60

ta 62

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence

<400> 4

ttctctcttt ctgtcgtgta ccttcttttc ctgtctgtgt a 41

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

uacacgacag aaagagagaa 20

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<400> 6

uacacagaca ggaaaagaag g 21

<210> 7

<211> 26

<212> DNA

<213> Artificial sequence

<400> 7

tacacagaca ggaaaagaag gtacac 26

Claims

1. A one-step fluorescence detection system is characterized by comprising,

the nucleotide sequence of the substrate is shown as SEQ ID NO.1, and the nucleotide sequence of the template is shown as SEQ ID NO. 2;

APE1 enzyme: for cleavage of the AP site;

fluorescent dye: for generating fluorescence with the amplification product.

2. The one-step fluorescence detection system of claim 1, wherein the DNA polymerase is Klenow large fragment polymerase;

or, the fluorescent dye is SYBR Green II.

3. The one-step fluorescence detection system according to claim 1, wherein the concentration of the template is 14 to 16nM when the concentration of the substrate is 10 nM;

or, the amount of substrate is 2X 10^-4When nmol, the dosage of APE1 enzyme is 0.75-0.85U;

or, the amount of substrate is 2X 10^-4When nmol, the dosage of UDG enzyme is not less than 2U;

or, the amount of substrate is 2X 10^-4At nmol, the amount of Klenow large fragment polymerase is 1.9 ℃ -2.1U；

Or, when the concentration of the substrate is 10nM, the concentration of the dNTP/dUTP mixture is 58-62. mu.M.

4. The one-step fluorescence detection system according to claim 3, wherein the amount of the UDG enzyme is 2.0 to 2.2U.

5. A kit for detecting the activity of DNA glycosylase, which comprises the one-step fluorescence detection system of any one of claims 1 to 4 and a buffer solution.

6. Use of the one-step fluorescence detection system of any one of claims 1-4 for screening for an inhibitor of hOGG 1.

7. Use of the one-step fluorescence detection system of any one of claims 1 to 4 in the preparation of a reagent for detecting cancer cells.

8. The use according to claim 7, wherein the cancer cells are A549 cells and/or HeLa cells.