阅读说明：本技术 一种dtns介导的检测8-og dna糖基化酶活性的方法 (DTNS (DTNS-mediated detection) method for detecting 8-OG DNA glycosylase activity ) 是由 吴玉姝 吴敏 刘敏 韩军 于 2021-09-15 设计创作，主要内容包括：本发明属于生物检测和分子生物学技术领域,具体涉及一种DTNS介导的检测8-OGDNA糖基化酶活性的方法。在8-OG DNA糖基化酶的作用下,DTNS的结构由开放态变为闭合态,使Cy3供体和Cy5受体之间的距离靠近,从而引发高效的FRET。利用本方法,不仅能够实现灵敏和选择性地检测细胞外的8-OG DNA糖基化酶活性,基于FRET信号输出模式,还能预防核酸酶降解产生的假阳性信号,有利于活细胞内的准确成像。(The invention belongs to the technical field of biological detection and molecular biology, and particularly relates to a DTNS (denaturing DTNS-mediated quantitative analysis) mediated method for detecting 8-OGDNA glycosylase activity. Under the action of 8-OG DNA glycosylase, the structure of the DTNS is changed from an open state to a closed state, so that the distance between a Cy3 donor and a Cy5 acceptor is close, and high-efficiency FRET is initiated. By utilizing the method, the activity of the extracellular 8-OG DNA glycosylase can be sensitively and selectively detected, a false positive signal generated by nuclease degradation can be prevented based on a FRET signal output mode, and accurate imaging in living cells is facilitated.)

1. A DNA tetrahedral nano-switch, comprising: the method comprises the following steps: a DNA tetrahedron, wherein one vertex of the DNA tetrahedron is connected with a double-stranded DNA probe;

the double-stranded DNA probe is formed by hybridizing a recognition strand containing 8-OG and a report strand marked with a donor/acceptor double fluorophore.

2. The method for preparing a DNA tetrahedron of claim 1, wherein:

1) preparation of DNA tetrahedron: mixing four DNA strands in equimolar amount, heating at 90-95 deg.C for 5-10min, rapidly transferring to ice water bath, cooling for 20-40min, and standing at 3-5 deg.C for 1-2 h;

2) preparation of double-stranded DNA Probe R-H: mixing the recognition chain R and the report chain H in equimolar amount, and incubating for 1-1.5H at 25 ℃;

3) preparing DTNS: the DNA tetrahedrons prepared above and the double-stranded DNA probes R-H were mixed in equimolar amounts and incubated at 25 ℃ for 1-1.5H.

3. The method of claim 2, wherein: the procedure for preparing the DNA tetrahedral double-stranded DNA probe R-H, DTNS was performed in 1 XPBS buffer solution.

4. A DTNS-mediated method for detecting 8-OG DNA glycosylase activity in an extracellular target, characterized by:

(1) constructing a DNA tetrahedral nano switch;

(2) incubating an extracellular target and a DNA tetrahedral nano switch together;

(3) fluorescence spectroscopy measurements were performed.

5. The method of claim 4, wherein: incubation is carried out at 37 ℃ for 60-100min, preferably 90 min.

6. The method of claim 4, wherein: in the fluorescence spectrum measurement process, the excitation wavelength is 525nm, and the emission wavelength is 550nm-750 nm; the excitation and emission slit width was 10nm and the photomultiplier voltage was 700V.

7. A method of DTNS-mediated imaging of the ratio of 8-oxoguanine DNA glycosylase activity in living cells, characterized by:

(1) constructing a DNA tetrahedral nano switch;

(2) incubating the target cell and the DNA tetrahedral nano switch together;

(3) and (5) carrying out confocal laser scanning microscope imaging.

8. The method of claim 7, wherein: target cells are cultured in a DMEM medium added with 10% FBS and 1% penicillin-streptomycin, then inoculated on a confocal culture dish, incubated for 20-25h, and then incubated with a DNA tetrahedral nano switch.

9. The method of claim 7, wherein: the incubation time is 2-4h, preferably 3 h.

10. The method of claim 7, wherein: in the imaging process of the confocal laser scanning microscope, Cy3 emission light in the range of 550nm-639nm and Cy5 emission light in the range of 640nm-700nm are collected under excitation light with the wavelength of 543 nm.

Technical Field

The invention belongs to the technical field of biological detection and molecular biology, and particularly relates to a DTNS (denaturing DTNS-mediated quantitative analysis) mediated method for detecting 8-OG DNA glycosylase activity.

Background

The information 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.

8-Oxoguanine (8-OG) DNA glycosylase is a DNA repair enzyme used to repair a common oxidative DNA damage 8-OG. The enzyme recognizes 8-OG in double-stranded DNA and cleaves glycosidic bonds to form an apurinic/Apyrimidinic (AP) site, which is then cleaved by the enzyme's inherent AP-cleaving activity. However, abnormalities in intracellular 8-OG DNA glucoamylase activity can lead to incorrect base pairing between 8-OG and adenine during DNA replication, causing gene mutations and increasing the incidence of several diseases, including digestive and pulmonary tumors. Therefore, accurate and in-situ detection of the activity of the intracellular 8-OG DNA glycosylase is of great significance for understanding the function of the 8-OG DNA glycosylase and further researching mutation-related diseases.

The method for detecting the activity of the 8-OG DNA glycosylase comprises a radioactive labeling method, a high performance liquid chromatography method, an electrochemical method, a colorimetric method and a fluorescence method. These methods typically detect 8-OG DNA glycosylase activity in buffered solutions or cell extracts and do not directly reflect the level of activity of the enzyme in the complex environment of living cells. To reveal the cellular processes in which 8-OG DNA glycosylase participates and to explore its mechanism of action, in situ fluorescence methods based on DNA functionalized nanomaterials have been developed for imaging intracellular 8-OG DNA glycosylase activity. These in situ fluorescence methods show information on the activity of intracellular 8-OG DNA glycosylase by only a single fluorescence signal intensity. Despite the significant advances that have been made, these in situ fluorescence methods that rely on the intensity of a single fluorescence signal are still subject to interference by false positive signals generated by enzymatic degradation in cells, thereby affecting the accuracy of the detection. Thus, accurate and in situ imaging of intracellular 8-OG DNA glycosylase activity remains a challenge.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a method for detecting the activity of 8-OG DNA glycosylase mediated by a DNA Tetrahedral Nano Switch (DTNS), wherein under the action of the 8-OG DNA glycosylase, the structure of the DTNS is changed from an open state to a closed state, and the distance between a Cy3 donor and a Cy5 acceptor is close, so that high-efficiency FRET is initiated. By utilizing the method, the activity of the extracellular 8-OG DNA glycosylase can be sensitively and selectively detected, a false positive signal generated by nuclease degradation can be prevented based on a FRET signal output mode, and accurate imaging in living cells is facilitated.

The invention specifically provides the following technical scheme:

in a first aspect, the present invention provides a DNA tetrahedral nano-switch, comprising: a DNA tetrahedron, wherein one vertex of the DNA tetrahedron is connected with a double-stranded DNA probe;

wherein the double-stranded DNA probe is formed by hybridizing a recognition strand containing 8-OG and a report strand marked with a donor/acceptor double fluorophore.

The invention provides a DTNS-mediated method for detecting 8-OG DNA glycosylase activity in an extracellular target, which comprises the following steps:

(1) constructing a DNA tetrahedral nano switch;

(2) incubating an extracellular target and a DNA tetrahedral nano switch together;

(3) fluorescence spectroscopy measurements were performed.

In a third aspect, the present invention provides a DTNS-mediated method for imaging 8-oxoguanine DNA glycosylase activity ratio in living cells, specifically comprising:

(1) constructing a DNA tetrahedral nano switch;

(2) and (3) incubating the target cells and the DNA tetrahedral nano switch together.

(3) And (5) carrying out confocal laser scanning microscope imaging.

One or more embodiments of the present invention have at least the following advantageous effects:

the invention provides a DTNS (DTNS) -mediated method for detecting 8-OG DNA glycosylase activity. Under the action of 8-OG DNA glycosylase, the structure of the DTNS is changed from an open state to a closed state, so that the distance between a Cy3 donor and a Cy5 acceptor is close, and high-efficiency FRET is initiated. By using the method, the activity of the extracellular 8-OG DNA glycosylase can be sensitively and selectively detected. In addition, based on the FRET signal output mode, the invention can prevent false positive signals generated by nuclease degradation and is beneficial to accurate imaging in living cells. In addition, in combination with the good cellular uptake capacity of DTNS, the invention has been successfully used for accurate and in situ imaging of 8-OG DNA glycosylase activity in living cells. The present invention provides a promising tool for accurate and in situ analysis of intracellular 8-OG DNA glycosylase activity, which helps to understand the function of 8-OG DNA glycosylase and to further study mutation-related diseases.

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 DTNS-mediated FRET strategy used for ratiometric imaging of 8-OG DNA glycosylase activity in living cells;

FIG. 2: (A) agarose gel electrophoresis characterization of DNA tetrahedrons and (B) DTNS; (C) fluorescence emission spectra of DTNS in the presence and absence of the target 8-OG DNA glycosylase.

FIG. 3(A) fluorescence emission spectra of DTNS in the presence of varying concentrations of the target 8-OG DNA glycosylase; (B) f_A/F_DA standard curve of the ratio relative to the concentration of the target 8-OG DNA glycosylase; (C) fluorescence emission spectra of DTNS and (D) F when a target or other DNA glycosylase is present_A/F_DA ratio; error bars are the standard deviation of the results of three replicates.

FIG. 4 shows F of DTNS when DNaseI is present and absent_A/F_DThe variation curve of the ratio with time; error bars are the standard deviation of the results of three replicates.

FIG. 5 imaging HeLa intracellular 8-OG DNA glycosylase activity using (A) DTNS and (B) control DTNS confocal fluorescence; a graduated scale: 20 μm.

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.

As described in the background, the prior art methods for detecting 8-OG DNA glycosylase activity generally only detect 8-OG DNA glycosylase activity in buffer solutions or cell extracts, and do not directly reflect the level of activity of the enzyme in the complex environment of living cells. Although the in-situ fluorescence method based on the DNA functionalized nano material can be used for imaging the activity of the intracellular 8-OG DNA glycosylase, the in-situ fluorescence methods only display the activity information of the intracellular 8-OG DNA glycosylase through single fluorescence signal intensity, and the in-situ fluorescence methods depending on the single fluorescence signal intensity can be interfered by false positive signals generated by degradation of the intracellular nuclease, so that the detection accuracy is influenced.

In order to solve the above technical problems, a first aspect of the present invention provides a DNA tetrahedral nano-switch comprising: a DNA tetrahedron, wherein one vertex of the DNA tetrahedron is connected with a double-stranded DNA probe;

wherein the double-stranded DNA probe is formed by hybridizing a recognition strand containing 8-OG and a report strand marked with a donor/acceptor double fluorophore.

The DNA tetrahedral nano switch provided by the invention has the advantages that the distance between the donor/acceptor double fluorophores is relatively long initially, and no FRET occurs. Under the action of 8-OG DNA glycosylase, 8-OG in the double-stranded DNA probe is removed and the resulting purine-free/pyrimidine-free (AP) site is also nicked, so that the reporter strand dissociates from the double-stranded DNA and forms a hairpin structure, bringing the donor/acceptor double fluorophores into close proximity to each other, thereby allowing efficient FRET.

The construction of the DNA tetrahedral nano switch can sensitively and selectively detect the activity of extracellular 8-OG DNA glycosylase. More importantly, false positive signals caused by nuclease degradation can be avoided based on the FRET signal output mode, so that the detection accuracy is improved. In addition, the activity of the intracellular 8-OG DNA glycosylase can be accurately imaged in situ by combining with the good cell uptake capacity of DTNS.

Further, the preparation method of the DNA tetrahedron comprises the following steps:

1) preparation of DNA tetrahedron: mixing four DNA strands in equimolar amount, heating at 90-95 deg.C for 5-10min, rapidly transferring to ice water bath, cooling for 20-40min, and standing at 3-5 deg.C for 1-2 h;

2) preparation of double-stranded DNA Probe R-H: mixing the recognition chain R and the report chain H in equimolar amount, and incubating for 1-1.5H at 25 ℃;

3) preparing DTNS: the DNA tetrahedrons prepared above and the double-stranded DNA probes R-H were mixed in equimolar amounts and incubated at 25 ℃ for 1-1.5H.

The procedure for preparing the DNA tetrahedral double-stranded DNA probe R-H, DTNS was performed in 1 XPBS buffer solution.

The invention provides a DTNS-mediated method for detecting 8-OG DNA glycosylase activity in an extracellular target, which comprises the following steps:

(1) constructing a DNA tetrahedral nano switch;

(2) incubating an extracellular target and a DNA tetrahedral nano switch together;

(3) fluorescence spectroscopy measurements were performed.

Further, incubation is carried out at 37 ℃ for 60-100min, preferably 90 min.

Furthermore, in the fluorescence spectrum measurement process, the excitation wavelength is 525nm, and the emission wavelength is 550nm-750 nm. The excitation and emission slit width was 10nm and the photomultiplier voltage was 700V.

In a third aspect, the present invention provides a DTNS-mediated method for imaging 8-oxoguanine DNA glycosylase activity ratio in living cells, specifically comprising:

(1) constructing a DNA tetrahedral nano switch;

(2) and (3) incubating the target cells and the DNA tetrahedral nano switch together.

(3) And (5) carrying out confocal laser scanning microscope imaging.

Furthermore, the target cells are firstly cultured in a DMEM medium added with 10% FBS and 1% penicillin-streptomycin, then inoculated on a confocal culture dish, incubated for 20-25h, and then incubated with a DNA tetrahedral nano switch.

Further, the incubation time is 2-4h, preferably 3 h.

Further, during the imaging process of the confocal laser scanning microscope, under the excitation light with the wavelength of 543nm, Cy3 emission light in the range of 550nm-639nm and Cy5 emission light in the range of 640nm-700nm are collected.

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.

1. Procedure of experiment

(1) Materials and instruments

The DNA oligonucleotides used in the present invention are synthesized and purified by the industry (China, Shanghai), and the specific sequences are shown in Table 1. 8-OG DNA saccharifying enzyme (Fpg), uracil DNA saccharifying enzyme (UDG), alkylated adenine DNA glycosylase (hAAG), DNase I (DNase I) were purchased from New England Biolabs (China, Beijing). Fetal Bovine Serum (FBS) was purchased from double wet organisms (china, shanghai). Cell culture media DMEM and RPMI-1640 were purchased from Prinoset (China, Wuhan). Penicillin-streptomycin solution and pancreatin were purchased from Biyun Tian (China, Shanghai).

Agarose gel imaging used the 4600SF gel imaging system (china, tianneng). The fluorescence spectrum measurement was carried out using an F-7000 spectrofluorometer. Confocal fluorescence imaging LSM-880 confocal laser scanning microscopy (zeiss, germany) was used.

(2) Preparation of DTNS

First, a DNA tetrahedron is prepared. Four DNA strands were mixed in equimolar amounts in 1 XPBS buffer (pH 7.4). The mixed sample was heated at 95 ℃ for 5min, then quickly transferred to an ice-water bath to cool for 30min, followed by standing at 4 ℃ for 1 h. The prepared samples were stored at 4 ℃ for further use.

Second, a double-stranded DNA probe R-H was prepared. Recognition strand R and reporter strand H were mixed in equimolar amounts in 1 XPBS buffer (pH 7.4) and incubated at 25 ℃ for 1H.

Finally, DTNS was prepared. The DNA tetrahedrons prepared above and the double-stranded DNA probe R-H were mixed in equimolar amounts in 1 XPBS buffer (pH 7.4) and incubated at 25 ℃ for 1H. The final DTNS concentration was 500 nM.

(3) Characterization of electrophoresis

DNA tetrahedra and unlabeled DTNS were characterized by agarose gel electrophoresis. Preparation of a 2.5% agarose gel: 1.5g agarose, 60mL 1 XTBE buffer, 8.0. mu.L nucleic acid dye GelRed. mu.L of DNA sample was mixed with 2.0. mu.L of 6 Xgel loading buffer, and 6.0. mu.L of the mixture was then sized. Electrophoresis was run in 1 × TBE buffer for about 1 hour at 100V.

(4) Detection of extracellular target 8-OG DNA glycosylase Activity

To test the extracellular target 8-OG DNA glycosylase activity, a 100. mu.L mixed solution containing 100nM DTNS, different concentrations of targets (0U/mL,1.0U/mL,2.0U/mL,5.0U/mL,8.0U/mL,10U/mL and 20U/mL),1 XNEBuffer 1 and 100. mu.g/mL Bovine Serum Albumin (BSA) was incubated at 37 ℃ for 90 min. Then, the mixed solution was subjected to fluorescence spectrum measurement. The excitation wavelength is 525nm, and the emission wavelength is 550nm-750 nm. The excitation and emission slit width was 10nm and the photomultiplier voltage was 700V. In addition, for the purpose of investigating selectivity, the procedure was identical to that described above except that the target 8-OG DNA glycosylase was changed to another DNA glycosylase.

(5) Study of the ability to prevent false Positive Signaling

To investigate the capacity of this strategy to prevent false positive signals caused by DNase I degradation, 100. mu.L of a mixed solution containing 100nM DTNS,0.5U/mL DNase I,1 XPBS buffer (pH 7.4) was incubated at 37 ℃ for 2 h. The control group was subjected to the same conditions as described above except that DNase I was not added. The spectral conditions when the fluorescence spectrum was measured were the same as in (4).

(6) Cell culture

HeLa cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. All cell lines were incubated at 37 ℃ with 5% CO₂The cell culture chamber of (2) for culturing.

(7) Confocal fluorescence imaging of 8-OG DNA glycosylase activity in living cells

To rate image 8-OG DNA glycosylase activity in living cells, HeLa cells were seeded on a confocal culture dish and incubated for 24 h. Then, HeLa cells were incubated with 100nM DTNS or control DTNS for 3 h. Subsequently, cells were washed three times with PBS for confocal laser scanning microscope imaging. Cy3 emission light in the range of 550nm-639nm and Cy5 emission light in the range of 640nm-700nm were collected under excitation light of 543nm wavelength.

2. Analysis of results

(1) Design principles of DTNS-mediated FRET strategies

The design principle of the invention is shown in figure 1, and the designed DTNS consists of two parts: DNA tetrahedral and double-stranded DNA probes. The DNA tetrahedron is assembled from four custom DNA strands (A, B, C, D, Table 1) with a short single-stranded DNA hanging at the apex. The double-stranded DNA probe is formed by hybridizing a recognition strand containing 8-OG damaged bases and a report strand marked with a Cy3/Cy5 double-fluorophore, and a short single-stranded DNA which is complementary to a DNA sequence hung on the top of a tetrahedron is hung at the tail end of the double-stranded DNA probe. Thus, the double-stranded DNA probe can be ligated to one vertex of a DNA tetrahedron by base complementary pairing to obtain DTNS.

Initially, DTNS is in an open state, and the labeled Cy3 donor and Cy5 acceptor are separated from each other, resulting in inefficient FRET. However, when 8-OG DNA glycosylase is present, the structure of DTNS changes from an open state to a closed state. Specifically, 8-OG DNA glycosylase removes the 8-OG from the double stranded DNA probe and excises the resulting AP site, causing the double stranded DNA to unwind and release the reporter strand. Subsequently, the released reporter strand forms a hairpin structure by base-complementary pairing, bringing the distance between the Cy3 donor and the Cy5 acceptor into close proximity, so that efficient FRET occurs. In addition, due to the good cell uptake capacity of the DNA tetrahedron, DTNS can easily enter living cells, and the fluorescence intensity of the Cy3 donor is weakened and the fluorescence intensity of the Cy5 acceptor is strengthened under the action of intracellular 8-OG DNA glycosylase.

(2) Characterization and detection feasibility study of DTNS

To verify the formation of DTNS, agarose gel electrophoresis analysis was performed. First, the electrophoretic mobility of the product bands gradually decreased with the addition of four DNA strands one by one (FIG. 2A). This is due to the increasing molecular weight of the hybridization product and the more complex spatial structure, indicating that the DNA tetrahedron has been successfully assembled. Next, the recognition strand R was mixed with the reporter strand H in equal amounts, and only one band was observed in lane 2 and the electrophoretic mobility of this band was lower than that of the reporter strand H in lane 1 (FIG. 2B), indicating that hybridization of R and H formed a DNA double strand R-H with a higher molecular weight. Finally, the DNA double strand R-H was mixed with the DNA tetrahedron in equal amounts, and it was observed in lane 4 that both the R-H band and the tetrahedral band disappeared, but a new band with lower mobility appeared (FIG. 2B), indicating that the DNA double strand R-H had ligated to the DNA tetrahedron, thereby forming DTNS.

In addition, the feasibility of DTNS-mediated FRET for detecting the target 8-OG DNA glycosylase activity was investigated extracellularly. As shown in FIG. 2C, the fluorescence intensity of the Cy3 donor at 565nm decreased, while the fluorescence intensity of the Cy5 acceptor at 665nm increased after addition of the target. This shows that DTNS can effectively recognize the target and generate a structural transition from an open state to a closed state, initiating efficient FRET, thereby verifying the feasibility of the present strategy for detecting the target extracellularly.

(3) Extracellular assay performance of DTNS-mediated FRET strategy

Subsequently, the ability of the DTNS-mediated FRET strategy to quantitatively detect the target 8-OG DNA glycosylase activity was investigated extracellularly. As shown in FIG. 3A, the fluorescence of the Cy3 donor gradually decreased with increasing concentration of the target,whereas Cy5 receptor fluorescence gradually increased. And, fluorescence ratio of Cy5 acceptor to Cy3 donor (F)_A/F_D) Shows a good linear relationship with the target concentration in the range of 0U/mL to 10U/mL (FIG. 3B). The detection limit of the strategy on the activity of 8-OG DNA glycosylase is 0.3653U/mL, which is superior to or equivalent to that of the previous research.

To examine the selectivity of this strategy, other DNA glycosylase activities were also examined extracellularly by this strategy. As shown in FIGS. 3C and 3D, only the target 8-OG DNA glycosylase could initiate high efficiency FRET and high F_A/F_DRatio, which confirms the good selectivity of the present strategy.

Therefore, the strategy can sensitively and selectively detect the activity of the target 8-OG DNA glycosylase outside cells, and also suggests that the strategy has potential application to cell imaging of the activity of the 8-OG DNA glycosylase.

(4) Examination of the ability of the DTNS-mediated FRET strategy to prevent false-positive signals

To investigate the prevention of false positive signals by this strategy, the background F by DNaseI was studied_A/F_DThe influence of the ratio. DNaseI is an endonuclease that can hydrolyze both single-stranded DNA and double-stranded DNA. As shown in FIG. 4, after adding DNaseI to DTNS, F of the background_A/F_DThe ratio is not only not increased, but slightly decreased. This is because after the DTNS is degraded by dnase i, the distance between the Cy3 donor and Cy5 acceptor will be further apart and no FRET will occur. This result indicates that this strategy can prevent false positive signals caused by nuclease degradation, which is critical for accurate intracellular imaging.

(5) Intracellular imaging of 8-OG DNA glycosylase Activity

Based on the above results, the feasibility of the DTNS-mediated FRET strategy for ratiometric imaging of 8-OG DNA glycosylase activity in living cells was further investigated. As shown in fig. 5A, after 3h incubation of DTNS with HeLa cells, a clear FRET signal was observed in the cytoplasm (red, Cy 5). This is because the structure of DTNS is changed from an open state to a closed state by the action of 8-OG DNA glycosylase endogenous to the cell, so that the distance between the Cy3 donor and the Cy5 acceptor is close to initiate high-efficiency FRET. This result demonstrates the feasibility of this strategy to ratiometrically image 8-OG DNA glycosylase activity in living cells. In addition, to confirm the specificity of this strategy to image endogenous 8-OG DNA glycosylase activity in living cells, a control group DTNS without 8-OG was designed. As shown in fig. 5B, there was no significant FRET signal in the cytoplasm after the control group DTNS was incubated with HeLa cells for 3h (red, Cy 5). These results demonstrate that the present strategy can be used for in situ and accurate imaging of intracellular 8-OG DNA glycosylase activity.

The DNA oligonucleotide sequences used in the examples are shown in table 1:

TABLE 1

Wherein the symbol "O" in SEQ ID NO.5 is a damaged base "8-OG".

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

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