阅读说明：本技术 Tet酶活性测定方法及tet酶活性小分子激活剂或抑制剂的高通量筛选方法 (TET enzyme activity determination method and high-throughput screening method of TET enzyme activity small molecule activator or inhibitor ) 是由 江翱 刘倩 马海玲 孙睿 陈晶晶 侯策 曹振 宋东亮 于 2021-08-23 设计创作，主要内容包括：本发明提供了一种TET酶活性测定方法,其特征在于其步骤包括：将5mC DNA与待测TET酶混合后孵育；在反应体系中加入SDH和DCPIP进行反应；加入三氯乙酸终止反应；酶标仪测量反应液在600nm处的吸收峰值,峰值越高则代表酶活性越强。还公开了SDH和DCPIP联合在测定TET酶活性中的应用,以及TET酶活性小分子激活剂或抑制剂的高通量筛选方法。本发明提供了一种简便快速的TET酶活性测定方法,利用TET酶氧化反应产物琥珀酸盐在琥珀酸脱氢酶的催化下释放出还原氢,还原氢被DCPIP捕获生成DCPIPH2(蓝色),在波长600 nm处出现特异性的吸收峰。整个方法流程只需要4步操作、一管式反应、耗时1 h即可完成,具有成本低、通量大、适用范围广和准确性高的优点。(The invention provides a TET enzyme activity determination method, which is characterized by comprising the following steps: mixing 5mC DNA with TET enzyme to be detected and then incubating; adding SDH and DCPIP into a reaction system for reaction; adding trichloroacetic acid to terminate the reaction; the microplate reader measures the absorption peak value of the reaction solution at 600nm, and the higher the peak value is, the stronger the enzyme activity is. Also discloses application of the combination of SDH and DCPIP in the determination of TET enzyme activity, and a high-throughput screening method of a small molecule activator or inhibitor of the TET enzyme activity. The invention provides a simple and rapid TET enzyme activity determination method, which utilizes the TET enzyme oxidation reaction product succinate to release reducing hydrogen under the catalysis of succinate dehydrogenase, the reducing hydrogen is captured by DCPIP to generate DCPIPH2 (blue), and a specific absorption peak appears at the wavelength of 600 nm. The whole method flow can be completed only by 4 steps of operation, one-tube reaction and 1 h of consumed time, and has the advantages of low cost, high flux, wide application range and high accuracy.)

1. A method for measuring TET enzyme activity, which is characterized by comprising the following steps:

(1) mixing DNA containing 5mC with TET enzyme to be detected and then incubating;

(2) adding SDH and DCPIP into a reaction system for reaction;

(3) adding trichloroacetic acid to terminate the reaction;

(4) the microplate reader measures the absorption peak value of the reaction solution at 600nm, and the higher the peak value is, the stronger the enzyme activity is.

2. The method for measuring TET enzyme activity according to claim 1, wherein: the DNA containing 5mC in the step (1) is a 5mC DNA standard product, and the sequence of the DNA is AAAAAAAm5 CGAAAAATTTTTTTTM 5 CGTTTTTTT.

3. The method for measuring TET enzyme activity according to claim 1, wherein: and (2) incubating 5mC DNA and TET enzyme in a TET enzyme reaction buffer solution for 10-30min in the step (1).

4. The method for measuring TET enzyme activity according to claim 3, wherein: phenazine dimethyl ester sulfate is also added in the step (2).

5. The method for measuring TET enzyme activity according to claim 4, wherein: the SDH in the step (2) at least contains human SDHA and SDHB.

Use of a combination of SDH and DCPIP for the determination of TET enzyme activity.

7. A high-throughput screening method for a small molecule activator or inhibitor of TET enzyme activity is characterized by comprising the following steps:

A. adding TET enzyme reaction buffer solution and TET enzyme into a reaction container;

B. adding a small molecule drug into a reaction container, and incubating for a period of time;

C. adding 5mC DNA into a reaction container, and incubating for a period of time;

D. adding SDH and DCPIP into a reaction container for reaction;

E. adding trichloroacetic acid to terminate the reaction;

F. and measuring the absorption peak value of the reaction liquid at 600nm by using an enzyme-labeling instrument, comparing the activity change of the TET enzyme by using a sample without the addition of the small-molecule drug as a control, wherein the increase of the absorption peak value represents that the small-molecule drug is an activity promoting small molecule of the TET enzyme, and the decrease of the absorption peak value represents that the small-molecule drug is an activity inhibiting small molecule drug of the TET enzyme.

8. High throughput screening method according to claim 7, characterized in that: the reaction container is a 96-well plate or a 384-well plate, and the screening of a plurality of small molecule medicines is carried out simultaneously.

9. High throughput screening method according to claim 8, characterized in that: incubating at room temperature for 5-10min in step (2), and incubating at 37 deg.C for 30-60min in step (3).

Technical Field

The invention relates to a TET enzyme activity determination method and a high-throughput screening method of a TET enzyme activity small molecule activator or inhibitor, belonging to the technical field of biology.

Background

Cytosine methylation (5 mC) is the most common modified base on DNA, accounting for 1% -8% of all cytosines, and is the predominant form of DNA methylation. Some specific cytosine sites, such as bacterial Dam methylation sites or cytosine sites in CpG islands of eukaryotes, may be methylated up to approximately 100%, and thus 5mC is also referred to as "fifth base". DNA methylation is an important form of regulation of gene expression, primarily mediating gene silencing. This regulation mode of gene silencing mediated by DNA methylation occurs at a time in vivo, is the molecular basis for differential expression of genes, and is also a key pathway for determining embryonic development, functional differentiation of cells, individual growth, senescence and pathology. For example, genes that maintain cell sternness during cell differentiation are highly methylated to inhibit expression, while tissue-specific functional genes that promote cell differentiation are demethylated to enhance expression activity. This ordered methylation/demethylation homeostasis is the basis for ensuring the normal physiological state of cells. The DNA 5mC dioxygenase family TET (also known as DNA 5mC hydroxylase) is the major protein for DNA demethylation and is capable of mediating the oxidation of 5mC-5hmC-5fC-5 caC. The human TET enzyme family has 3 members, TET1, TET2, and TET3, which function to demethylate DNA in different life cycles. In the course of many diseases, the phenomenon of disorder of gene expression related to diseases, such as tumor, leukemia, autoimmune disease, hereditary disease, etc., occurs due to disorder of TET enzyme activity (factors such as change in expression level, gene rearrangement, gene mutation, change in protein localization, etc.). Therefore, TET enzyme activity is an important target for diagnosis and treatment of many diseases, and small molecule enhancers and inhibitors against TET enzyme are also key breakthrough in clinical treatment of relevant diseases. However, the determination of TET enzyme activity is a difficult problem in clinical diagnosis and scientific research of DNA methylation.

Due to the existence of complex triple catalytic reaction (5 mC-5hmC-5fC-5 caC) in TET enzyme mediated 5mC oxidation, the activity of TET enzyme is difficult to accurately and effectively determine by the existing enzyme activity determination method. The existing TET enzyme activity determination methods are mainly divided into three types. The first method is a detection method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), m5C oligo oxidized by TET enzyme is recovered and digested by nuclease, and the ratio of a substrate m5C and oxidation products 5hmC, 5fC and 5caC is analyzed by LC-MS, so that the activity of the TET enzyme is judged. The method has the advantages of capability of distinguishing oxidation products of various stages of TET enzyme, but has the disadvantages of heavy background, complex operation, high cost, poor accuracy and difficulty in high-throughput analysis. The second method is dot hybridization detection technology, in which 5mC DNA oligo is cross-linked to nylon membrane after TET oxidation, 5mC, 5hmC, 5fC and 5caC antibodies are respectively used for hybridization, and finally, a special labeled secondary antibody is used for signal amplification and color development. This method is widely used because it can detect a plurality of samples at the same time, but it is small in throughput, complicated in operation, high in cost, and difficult to perform quantitative analysis. The third method uses enzyme-linked immunosorbent assay (ELISA) for activity determination. TET enzyme oxidized 5mC DNA oligo was fixed to a 96-well plate, and 5hmC antibody was added for binding, and a color reaction was performed by a labeled secondary antibody. Although this method can be used for quantitative analysis, the throughput is higher than the former two methods, the operation is complicated, the cost is high, and only the content of 5hmC of one reaction intermediate product can be analyzed, and the activity of the TET enzyme cannot be accurately measured (the high-activity TET enzyme may be measured as low activity by this method because the 5caC ratio is increased and the 5hmC ratio is decreased in the oxidation product). All three methods become great obstacles for the automatic detection of TET enzyme activity and the screening of high-flux small molecule activators and inhibitors due to long time consumption (more than 8 h) and complex operation (hundreds of steps).

Disclosure of Invention

The invention aims to provide a simple and rapid TET enzyme activity determination method, which has the reaction principle that three steps of oxidizing 5mC by TET enzyme are accompanied by consumption of alpha-ketoglutaric acid and generation of succinate, the succinate releases reducing hydrogen under the catalysis of Succinate Dehydrogenase (SDH), the reducing hydrogen is captured by 2, 6-dichlorophenol indophenol (DCPIP) to generate blue DCPIPH2, and a specific absorption peak appears at the wavelength of 600 nm.

The technical scheme adopted by the invention is as follows: a method for measuring TET enzyme activity, which is characterized by comprising the following steps:

(1) mixing 5mC DNA with TET enzyme to be detected, and then incubating, wherein the incubation aims at carrying out oxidation reaction;

(2) adding SDH and DCPIP into a reaction system for reaction;

(3) adding trichloroacetic acid to terminate the reaction;

(4) the microplate reader measures the absorption peak value of the reaction solution at 600nm, and the higher the peak value is, the stronger the enzyme activity is.

Preferably, the 5mC DNA in step (1) has the sequence AAAAAAAm5 CGAAAAAAATTTTTTTtm 5 CGTTTTTTT.

Preferably, 5mC DNA is incubated with TET enzyme in TET enzyme reaction buffer for 10-30min in step (1).

Preferably, phenazine dimethyl sulfate is also added in the step (2), and the phenazine dimethyl sulfate can be used as reducing hydrogen to be transferred to the intermediate product of DCPIP to promote the efficiency of the reaction.

Preferably, the SDH of step (2) is human-derived SDHA or SDHB.

The invention also discloses the application of the combination of SDH and DCPIP in the determination of TET enzyme activity.

A high-throughput screening method for a small molecule activator or inhibitor of TET enzyme activity is characterized by comprising the following steps:

A. adding TET enzyme reaction buffer solution and TET enzyme into a reaction container;

B. adding a small molecule drug into a reaction container, and incubating for a period of time;

C. adding 5mC DNA into a reaction container, and incubating for a period of time;

D. adding SDH and DCPIP into a reaction container for reaction;

E. adding trichloroacetic acid to terminate the reaction;

F. and measuring the absorption peak value of the reaction liquid at 600nm by using an enzyme-labeling instrument, comparing the activity change of the TET enzyme by using a sample without the addition of the small-molecule drug as a control, wherein the increase of the absorption peak value represents that the small-molecule drug is an activity promoting small molecule of the TET enzyme, and the decrease of the absorption peak value represents that the small-molecule drug is an activity inhibiting small molecule drug of the TET enzyme.

Preferably, the reaction vessel is a 96-well plate or a 384-well plate, and the screening of a plurality of small molecule drugs is performed simultaneously.

Preferably, the incubation is performed at room temperature for 5-10min in step (2) and at 37 ℃ for 30-60min in step (3).

The invention has the beneficial effects that:

the invention provides a simple and rapid TET enzyme activity determination method (a color development method), which utilizes the TET enzyme oxidation reaction product succinate to release reducing hydrogen under the catalysis of succinate dehydrogenase, the reducing hydrogen is captured by 2, 6-dichlorophenol indophenol (DCPIP) to generate DCPIPH2 (blue), and a specific absorption peak appears at the wavelength of 600 nm. The whole method flow only needs 4 steps of operation, one-tube reaction and 1 hour of time consumption to complete, has the advantages of low cost, large flux, wide application range, high accuracy and the like, and is very suitable for industrial automatic detection of TET enzyme activity and high-throughput screening of small molecule activators and inhibitors.

Drawings

FIG. 1 is a schematic diagram of the principle of the color-developing TET enzyme activity measurement.

FIG. 2 is a schematic diagram of a process for determining TET enzyme activity by a color-developing method.

FIG. 3 is a standard curve for determining the respective TET enzymes by the chromogenic method.

FIG. 4 compares the three TET enzyme activity assays (NgTET 1).

FIG. 5 compares the three TET enzyme activity assays (mTET 1).

FIG. 6 compares the three TET enzyme activity assays (mTET 2).

FIG. 7 compares the three TET enzyme activity assays (hTET 1).

FIG. 8 compares the three TET enzyme activity assays (hTET 2).

FIG. 9 compares the three TET enzyme activity assays (hTET 3).

FIG. 10 is a schematic diagram of a high-throughput TET enzyme small molecule drug screening and identification process.

FIG. 11384 shows the screening results of drug molecule libraries.

Figure 12 validation results of potential TET enzyme inhibitor compound 127 #.

Figure 13 validation results of potential TET enzyme activator compound # 35.

Detailed Description

In order to further describe the concrete contents of the present invention, the present invention will be described in detail with reference to the following examples. The methods and reagents involved in the examples are well known to those skilled in the art, and it should be understood that the specific examples described below are merely illustrative of the invention and that the embodiments of the invention are not limited by the examples described below.

Example 1: and (3) measuring a TET enzyme activity flow and a standard curve by a color development method.

In this example, we published a process for measuring TET enzyme activity (see fig. 1 and 2), and measured the enzyme activities of NgTET1(Active motion), mTET1 (wisegen), mTET2(Creative Biomart), hTET1(Active motion), hTET2(Active motion), and hTET3(Active motion) (see fig. 3). The specific implementation mode is as follows:

TABLE 1

Components	Dosage of
		5mC DNA standard	10 ng
TET enzyme reaction buffer	3 μL
		Test TET enzyme	0.1-10 μg
Supplement of ddH2O to	30 μL

The reaction was carried out at 37 ℃ for 30 min.

TABLE 2

Components	Dosage of
		The above reaction	30 μL
Succinate dehydrogenase reaction buffer	4 μL
		SDHA	2 μg
SDHB	2 μg
		Supplement of ddH2O to	40 μL

The reaction was carried out at 37 ℃ for 30 min. After the reaction was completed, 5 uL of 35% trichloroacetic acid was added to terminate the reaction.

And measuring the absorption peak value of the reaction hole at 600nm on a microplate reader, and measuring an enzyme activity measuring curve.

As shown in FIG. 3, the activity of each TET enzyme (NgTET 1, mTET1, mTET2, hTET1, hTET2 and hTET 3) was measured by a color development method, and the absorption value at a wavelength of 600nm was significantly correlated with the amount of the TET enzyme added, the linear correlation coefficient was 0.95 or more, and the range of use was wide (no significant variation occurred in the range of 0.1-10 ug of the amount added). This suggests that the chromogenic method may be effective in accurately measuring the TET enzyme activity.

Example 2: three TET enzyme assay methods were compared.

In this example, the results of the enzyme activity measurement of TET by the chromogenic method, LC-MS method and ELISA method were compared.

The LC-MS method is used for determining the TET enzyme activity: DNA was recovered by phenol chloroform extraction and ethanol precipitation, and the ratio of the content of 5mC, 5hmC, 5fC and 5caC in the DNA was measured by LC-MS (Hashimoto H, Pais J E, et al. Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylsaccharopine DNA. Nature, 2013, 506(7488):391 395.).

The enzyme activity of TET is determined by ELISA: the specific enzyme activity of each TET protein was determined using the Epigenase 5 mC-hydroxylase TET activity/inhibition assay kit (fluorescence method) from Epigentek following the protocol of the instructions.

As shown in FIGS. 4-9, compared with LC-MS and ELISA, the chromogenic method has the advantages of simple operation, low cost, high flux, and obvious linear relationship of the measured TET enzyme activity. This shows that the color development method has the obvious advantages of higher accuracy, wider application range and the like in the TET enzyme activity determination.

Example 3: and (3) screening the high-throughput TET enzyme small-molecule targeted drug by using a color development method.

In this example, we performed screening of high-throughput small molecule drug libraries using the developed TET enzyme activity assay (color development). The schematic view is shown in fig. 10. The specific implementation mode is as follows:

design and preparation of small molecule drug libraries: we screened 382 small molecule clinical drugs from MCE clinical compound library.

Pre-binding of TET enzyme and small molecule drug library: add 1 uL TET enzyme buffer and 0.3 ug TET enzyme to 384-well plates, add small molecule drug to each well at a final concentration of 1 uM, and incubate for 10min at room temperature.

TET enzymatic oxidation: 2 ng of 5mC DNA was added and the reaction volume was 10 uL. The reaction was started and carried out at 37 ℃ for 30 min.

And (3) color development reaction: 1.3 uL of succinate dehydrogenase reaction buffer and 700 ng of SDHA and SDHB were added, reaction volume 13 uL. React at 37 ℃ for 30 min.

And (3) terminating the reaction: 7 ul of 10% trichloroacetic acid was added.

Measurement: the absorbance at a wavelength of 600nm was measured on a microplate reader.

As shown in FIG. 11, the developed TET enzyme activity assay system can be effectively used for screening high-throughput TET enzyme inhibitors and activators. We picked compound 127 (potential inhibitor) and compound 35 (potential activator) for activity validation, and the results showed that high throughput drug screening using chromogenic method could effectively screen out activators and inhibitors of TET enzyme (fig. 12 and 13).

In conclusion, a simple and rapid TET enzyme activity determination method (a color development method) is developed, and the TET enzyme oxidation reaction product succinate releases reduced hydrogen under the catalysis of succinate dehydrogenase, the reduced hydrogen is captured by 2, 6-dichlorophenol indophenol (DCPIP) to generate DCPIPH2 (blue), and a specific absorption peak appears at the wavelength of 600 nm. The whole method flow only needs 4 steps of operation, one-tube reaction and 1 hour of time consumption to complete, has the advantages of low cost, large flux, wide application range, high accuracy and the like, and is very suitable for industrial automatic detection of TET enzyme activity and high-throughput screening of small molecule activators and inhibitors.