阅读说明：本技术 一种基于G-四链体的检测Dnmt1活性的荧光传感器 (G-quadruplex-based fluorescence sensor for detecting activity of Dnmt1 ) 是由 邓惠敏 范子彦 刘珊珊 王颖 杨飞 边照阳 唐纲岭 刘洋 于 2019-09-30 设计创作，主要内容包括：一种基于G-四链体的检测Dnmt1活性的荧光传感器,属于生化分析领域。首先,以含有G-四链体序列、经半甲基化修饰的Hairpin DNA为底物,在S-腺苷甲硫氨酸存在下,于37℃条件下,以Dnmt1催化底物发生甲基化反应；然后加入对甲基化敏感的限制性内切酶BssHII,于60℃下进行剪切反应,被完全甲基化的底物不能被BssHII识别,从而不能被剪切,而未被完全甲基化的底物能被剪切,释放出G-四链体片段；最后,向体系中加入NMM,其与G-四链体结合后,产生强荧光信号,可根据荧光信号的强弱判断底物DNA的甲基化水平,从而进一步反映甲基化反应中Dnmt1的活性。该方法在均相反应体系中进行,操作简单。(A fluorescent sensor for detecting Dnmt1 activity based on G-quadruplex belongs to the field of biochemical analysis. Firstly, taking a half-methylation modified Hairpin DNA containing a G-quadruplex sequence as a substrate, and catalyzing the substrate to carry out methylation reaction by using Dnmt1 at 37 ℃ in the presence of S-adenosylmethionine; then adding a restriction enzyme BssHII sensitive to methylation, carrying out a shearing reaction at 60 ℃, wherein the completely methylated substrate cannot be identified by BssHII and thus cannot be sheared, and the incompletely methylated substrate can be sheared to release a G-quadruplex fragment; finally, NMM is added into the system, and after the NMM is combined with the G-quadruplex, a strong fluorescence signal is generated, and the methylation level of the substrate DNA can be judged according to the strength of the fluorescence signal, so that the activity of Dnmt1 in the methylation reaction is further reflected. The method is carried out in a homogeneous reaction system, and is simple to operate.)

1. A fluorescent sensor for detecting Dnmt1 activity based on G-quadruplexes, which is characterized in that: is constructed by the following method:

(1) dissolving substrate DNA containing a G-quadruplex sequence and modified by hemimethylation in TE buffer solution to prepare 100 mu M substrate DNA, heating in a water bath, and slowly cooling to room temperature to obtain 100 mu M Hairpin DNA substrate;

(2) adding the DNA substrate of the Hairpin prepared in the step (1) into a Dnmt1 buffer solution, then adding S-adenosylmethionine and Dnmt1, diluting with water, and then placing the mixed solution into a water bath at 37 ℃ to perform a methylation reaction of the DNA substrate of the Hairpin catalyzed by Dnmt 1;

(3) after the methylation reaction is finished, adding a CutSmart buffer solution and BssHII, diluting with ultrapure water, and carrying out a BssHII catalyzed shearing reaction in a water bath at 60 ℃;

(4) after the shearing reaction is finished, adding N-methylporphyrindipropionic acid IX (NMM) into the system, vortexing for 1min, measuring the fluorescence intensity, and exciting at the wavelength of 399 nm.

2. The G-quadruplex based fluorescence sensor for detecting Dnmt1 activity according to claim 1, wherein: the method comprises the following specific steps:

(1) dissolving substrate DNA containing a G-quadruplex sequence and modified by hemimethylation in TE buffer solution to prepare 100 mu M substrate DNA, heating to 90 ℃ in a water bath, keeping for 5min, and slowly cooling to room temperature to obtain 100 mu M Hairpin DNA substrate;

(2) adding 1 mu L of 100 mu M Hairpin DNA substrate into a 200 mu L centrifuge tube, adding 2.5 mu L of 10 XDnmt 1 buffer solution, adding 0.5 mu L of 32mM S-adenosylmethionine, adding 1 mu L of Dnmt1, finally diluting with water to the total volume of 25 mu L, placing the mixed solution into a 37 ℃ water bath to carry out Dnmt1 catalytic methylation reaction on the Hairpin DNA substrate for 2 h;

(3) after the methylation reaction is finished, adding 10 mu L of 10 XCutSmart buffer solution into a reaction system, adding 3 mu L of 1000U/mL BssHII, diluting with ultrapure water to the final volume of 100 mu L, and carrying out BssHII catalyzed shearing reaction in a water bath at 60 ℃ for 2 h;

(4) after the cleavage reaction was completed, 1. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM) was added to the system, vortexed for 1min, and the fluorescence intensity and excitation wavelength were measured at 399 nm.

3. The G-quadruplex based fluorescence sensor for detecting Dnmt1 activity according to claim 1 or 2, wherein: the TE buffer had a composition of 10mM Tris-HCl,1mM EDTA, pH 8.0.

4. The G-quadruplex based fluorescence sensor for detecting Dnmt1 activity according to claim 1 or 2, wherein: 1 XDnmt 1 component: 15% Glycerol,1mM DTT,1mM EDTA,50mM Tris-HCl, 100. mu.g/ml BSA, pH 7.8.

5. The G-quadruplex based fluorescence sensor for detecting Dnmt1 activity according to claim 1 or 2, wherein: 1 XCutSmart ingredient 50mM Potashium Acetate,20mM Tris-Acetate,10mM Magnesiucetate Acetate, 100. mu.g/ml BSA, pH 7.9.

6. The G-quadruplex based fluorescence sensor for detecting Dnmt1 activity according to claim 1 or 2, wherein: the N-methylporphyrin dipropionic acid IX (NMM) is dissolved in DMSO.

Technical Field

The invention relates to a G-quadruplex-based fluorescence sensor for detecting activity of Dnmt1, belonging to the field of biochemical analysis.

Background

DNA methylation is the most well-known epigenetic marker and is also the earliest characterized epigenetic modification at the molecular level. In mammals, DNA methylation refers to the biological process of transferring a methyl group to the 5-carbon atom of cytosine in CpG dinucleotide with S-adenosyl-L-methionine (SAM) as a methyl donor under the catalytic action of methyltransferase. DNA methyltransferases play an important role in the initial methylation of DNA and in the maintenance of methylation. There are two classes of methyltransferases in the human body: heavy-end methyltransferases (Dnmt3a, Dnmt3b and Dnmt3L) and maintenance methyltransferases (Dnmt 1). Among them, Dnmt1 is the most abundant methyltransferase, responsible for transferring the methylation state of the parent strand DNA to the newly synthesized daughter strand DNA during DNA replication, and is essential for maintaining the methylation state. Research proves that the phenomenon of over-expression of Dnmt1 is found in various cancer cells, and then hypermethylation of an oncogene suppressor promoter is caused, so that the oncogene is transcriptionally silenced and functionally inactivated, and finally, the cells grow and differentiate uncontrollably and become cancerous. Therefore, Dnmt1 is considered as a potential tumor marker, and the detection of Dnmt1 activity is of great significance for the early diagnosis of cancer.

The traditional analysis method of the activity of the methyltransferase mainly comprises gel electrophoresis-immunoblotting method and S-adenosylmethionine based on the super-heavy hydrogen label (³H-SAM), liquid chromatography-mass spectrometry, and the like. However, these methods generally rely on cumbersome procedures, radioisotope labeling, and/or expensive instrumentation. In view of this, many more flexibilities have been developed in recent yearsA variable electrochemical, ultraviolet absorption and fluorescence based sensing platform detects methyltransferase activity. It is worth mentioning that most of the reported sensing platforms for DNA methyltransferase activity are simulated based on bacteria-derived methyltransferases, such as DNA adenine methyltransferase (Dam) from e.coli, CpG methyltransferase (m.sssi) from spiropla sp, etc., and are relatively few for Dnmt 1. The methylation sites, recognition sequences and the like of the methylation transferase and the human methylation transferase are different to a certain extent, and the size of the human methylation transferase is far larger than that of the methylation transferase of microbial sources, so that great steric hindrance difference exists in recognition of substrate DNA, most of the currently reported sensing platforms are only conceptual researches and are not suitable for detection of actual samples, and the practicability of the sensing platforms is greatly reduced.

The invention aims to establish a G-quadruplex-based fluorescence sensor for detecting the activity of Dnmt1, and realizes monitoring of the activity of Dnmt1 by using a homogeneous reaction system by reasonably designing a substrate DNA sequence and using strong fluorescence generated by combination of N-methylporphyrindipropionic acid IX (NMM) and G-quadruplex as a signal source.

Disclosure of Invention

The invention aims to provide a fluorescent sensor for detecting Dnmt1 activity based on G-quadruplex, which takes Hairpin DNA containing a G-quadruplex sequence and modified by hemimethylation as a substrate and takes Dnmt1 as a catalytic substrate to carry out methylation reaction in the presence of a methyl donor; then, adding a restriction enzyme sensitive to methylation for carrying out a shearing reaction, shearing the incompletely methylated substrate to release a G-quadruplex fragment, combining with N-methylporphyrindipropionic acid IX (NMM) to generate a strong fluorescence signal, and judging the methylation level of the substrate DNA according to the strength of the fluorescence signal so as to further react the activity of Dnmt1 in the methylation reaction. The method is carried out in a homogeneous reaction system, is simple to operate and is suitable for detecting the activity of Dnmt 1.

The purpose of the invention is realized by the following technical scheme:

a fluorescent sensor for detecting Dnmt1 activity based on G-quadruplex comprises the following steps:

(1) the substrate DNA containing the G-quadruplex sequence and modified by hemimethylation is dissolved in TE buffer (10mM Tris-HCl,1mM EDTA, pH 8.0) to prepare 100 mu M substrate DNA, the substrate DNA is heated to 90 ℃ in a water bath, kept for 5min and slowly cooled to room temperature to obtain 100 mu M Hairpin DNA substrate.

(2) mu.L of 100. mu.M Hairpin DNA substrate was added to 200. mu.L centrifuge tubes, 2.5. mu.L of 10 XDnmt 1 buffer (5% Glycerol,1mM DTT,1mM EDTA,50mM Tris-HCl, 100. mu.g/ml BSA, pH 7.8) was added, 0.5. mu.L of 32mM S-adenosylmethionine was added, 1. mu.L of Dnmt1 was added, and finally diluted with water to a total volume of 25. mu.L, and the mixture was placed in a 37 ℃ water bath for Dnmt1 to catalyze methylation of the Hairpin DNA substrate for a reaction time of 2 h.

(3) After the methylation reaction was completed, 10. mu.L of 10 XCutSmart buffer (1 XCutSmart: 50mM Potashium Acetate,20mM Tris-Acetate,10mM Magnesium Acetate, 100. mu.g/mL BSA, pH7.9) was added to the reaction system, 3. mu.L of 10000U/mL BssHII was added, diluted with ultrapure water to a final volume of 100. mu.L, and BssHII-catalyzed cleavage reaction was carried out in a water bath at 60 ℃ for 2 hours.

(4) After the cleavage reaction was completed, 1. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM, dissolved in DMSO) was added to the system, vortexed for 1min, and the fluorescence intensity and excitation wavelength were measured at 399 nm.

The sensor prepared by the invention can be applied to the determination of Dnmt1 in the actual samples of cell lysate and the like, and has potential application value in the diagnosis of diseases related to methylation abnormality.

The invention has the advantages of

The invention establishes a fluorescent sensor for detecting Dnmt1 activity based on G-quadruplex. The method is carried out in a homogeneous reaction system, and is simple to operate. The detection method has detection limit of Dnmt1 of 0.12U/mL, quantitative limit of 0.40U/mL, and good linearity in the range of 0.2-60U/mL (R)²＝0.9907)。

Drawings

FIG. 1 is a schematic diagram of the detection principle;

FIG. 2 shows the results of fluorescence analysis for feasibility verification;

FIG. 3 shows the results of linear regression analysis of Dnmt1 activity assay.

Detailed Description

The invention is further described with reference to examples, but not limited thereto.

Example 1:

(1) reagent and apparatus

Substrate DNA purified by HPLC (5 '-GGG TAG GGC GGG TTG GG/i5MedC/GCG CGA GAA TGT TTT CAT TCT CGC GCG CCC AAC-3') purchased from Biotechnology engineering Co., Ltd (Shanghai), dissolved in TE buffer solution, and annealed at 90 ℃ to obtain 100. mu.M of Hairpin DNA substrate stock solution; 2000U/mL Dnmt1, 10000U/mL BssHII, 10 XDnmt 1 buffer, 10 XCutSmart buffer, 32mM SAM were purchased from New England Biolabs (Epstein, England); N-Methylporphyrin dipropionic acid IX was purchased from Frontier Scientific (USA).

Fluorescence analysis was carried out using a HORIBA Fluoromax-4 fluorometer (Japan), with an excitation wavelength of 399nm and a slit width of 5 nm.

(2) Feasibility analysis

The DNA sequence of the substrate adopted by the method is as follows: 5' -GGG TAG GGC GGG TTG GG/i5MedC/GCG CGA GAA TGT TTT CAT TCT CGC GCG CCC AAC-3 ', wherein the underline part is able to form G-quadruplex, the dotted underline part is half methylated CpG (5 ' -CG-3 ') containing Dnmt1 recognition sequence and BssHII recognition sequence (5 ' -GCGCGC-3 '), and the substrate DNA is annealed at 90 ℃ to obtain Hairpin DNA, on one hand, the G-quadruplex is blocked and protected, on the other hand, a double-stranded DNA region required for recognition of Dnmt1 and BssHII is formed.

The detection principle of the method is shown in figure 1, firstly, hemimethylated Hairpin DNA is taken as a substrate, dotted circles represent fragments capable of forming G-quadruplexes, and thick solid lines represent commonly recognized regions of Dnmt1 and BssHII. When SAM methyl donor and Dnmt1 were added, methylation of the substrate DNA occurred, followed by the addition of the methylation sensitive restriction enzyme BssHII, completely methylated substrate DNA could not be recognized by BssHII and thus could not be cleaved, while incompletely methylated substrate DNA could be recognized by BssHII and thus cleaved, releasing a DNA fragment that could form a G-quadruplex that could bind to NMM, producing enhanced fluorescence much stronger than NMM autofluorescence. The stronger the Dnmt1 activity in the system, the more fully methylated substrate DNA, the less G-quadruplex fragment released and the weaker the fluorescence, so the measured fluorescence intensity is inversely proportional to the Dnmt1 activity. Thus, a correlation of Dnmt1 activity with fluorescence intensity can be established, reflecting Dnmt1 activity in terms of fluorescence intensity.

Based on the above principle, to verify the feasibility of the substrate, a control experiment was first performed.

Wherein, the experimental group is that 1 μ L of 100 μ M Hairpin DNA substrate is added into 200 μ L centrifuge tube, 2.5 μ L10 XDnmt 1 buffer solution is added, 0.5 μ L32mM S-adenosylmethionine is added, 1 μ L Dnmt1 is added, finally, the mixture is diluted with water to the total volume of 25 μ L, the mixture is placed in 37 ℃ water bath for Dnmt1 to catalyze the methylation reaction of the Hairpin DNA substrate, and the reaction time is 2 h. After the methylation reaction was completed, 10. mu.L of 10 XCutSmart buffer (1 XCutSmart: 50mM Potassiium Acetate,20mM Tris-Acetate,10mM Magnesium Acetate, 100. mu.g/mL BSA, pH7.9) was added to the reaction system, 3. mu.L of 10000U/mL BssHII was added, diluted with ultrapure water to a final volume of 100. mu.L, and BssHII-catalyzed cleavage reaction was carried out in a water bath at 60 ℃ for 2 hours. After the cleavage reaction was complete, 1. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM, dissolved in DMSO) was added to the system and vortexed for 1 min.

The control group, except that Dnmt1 was not added, followed the same treatment as the experimental group.

The mixed solutions obtained in the laboratory and the control were subjected to fluorescence measurement, and the results are shown in fig. 2. In the presence of NMM alone, its autofluorescence is weak; when the substrate DNA was co-treated with Dnmt1 and BssHII (Experimental group), only a small amount of G-quadruplex fragment was released due to the higher methylation level, and thus the fluorescence intensity was only slightly increased; when the substrate DNA was treated with BssHII alone (control group), more G-quadruplex fragments could be released, and thus the fluorescence intensity was significantly increased. This result demonstrates the feasibility of the process.

By varying the Dnmt1 concentration, a correlation between fluorescence intensity and Dnmt1 activity was establishedThe results of the linear equations are shown in FIG. 3. It can be seen that the linearity is good in the range of 0.2-60U/mL (R²＝0.9907)

(3) Sample analysis

To verify the ability of the method to detect actual samples, Dnmt1 was added to human serum and the recovery was determined, with the results shown in table 1. Therefore, the recovery rate of the method is satisfactory.

TABLE 1 recovery of human serum supplemented with Dnmt1 of different activities

Addition level (U/mL)	Recovery (%)	Relative standard deviation (%)
			0.5	102.7	9.7
2.0	99.3	9.2
			10.0	98.9	8.4