TET enzyme activity determination method and high-throughput screening method of TET enzyme activity micromolecule activator or inhibitor

A technology for enzyme activity determination and screening method, applied in the biological field, can solve problems such as time-consuming, high cost, complicated operation, etc., and achieve the effect of wide application range, high accuracy and low cost

Pending Publication Date: 2021-11-12
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can be used for quantitative analysis, the flux is larger than the first two, but the operation is complicated, the cost is high, and only the content of a reaction intermediate product of 5hmC can be analyzed, and the activity of TET enzyme cannot be accurately measured (highly active TET enzymes may be considered low activity by this method due to the increased proportion of 5caC and decreased proportion of 5hmC in the oxidation product)
These three methods have become a huge obstacle for the automatic detection of TET enzyme activity and the screening of high-throughput small molecule activator and inhibitor drugs due to their long time-consuming (more than 8 h) and complicated operations (hundreds of operations).

Method used

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  • TET enzyme activity determination method and high-throughput screening method of TET enzyme activity micromolecule activator or inhibitor
  • TET enzyme activity determination method and high-throughput screening method of TET enzyme activity micromolecule activator or inhibitor
  • TET enzyme activity determination method and high-throughput screening method of TET enzyme activity micromolecule activator or inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Determination of TET enzyme activity process and standard curve by chromogenic method.

specific Embodiment approach

[0041] In this example, we have published the measurement process of TET enzyme activity (such as figure 1 and figure 2 ), and the enzymatic activities of NgTET1(Active motif), mTET1(WiseGene), mTET2(Creative Biomart), hTET1(ActiveMotif), hTET2(Active Motif) and hTET3(Active Motif) were determined according to this process (such as image 3 ). The specific implementation is as follows:

[0042] Table 1

[0043] components Dosage 5mC DNA Standard 10ng TET enzyme reaction buffer 3 μL TET enzyme to be tested 0.1-10μg Add ddH2O to 30μL

[0044] React at 37°C for 30 min.

[0045]Table 2

[0046] components Dosage above reaction 30μL Succinate Dehydrogenase Reaction Buffer 4 μL SDHA 2 μg SDHB 2 μg Add ddH2O to 40μL

[0047] React at 37°C for 30 min. After the reaction, 5 uL of 35% trichloroacetic acid was added to terminate the reaction.

[0048] The peak absorption of the reactio...

Embodiment 2

[0050] Example 2: Comparison of three TET enzyme assay methods.

[0051] In this example, we compared the effect of three enzyme activity assay methods on TET enzyme activity: chromogenic method, LC-MS method and ELISA method.

[0052] Determination of TET enzyme activity by LC-MS method: phenol-chloroform extraction and ethanol precipitation were used to recover DNA, and LC-MS was used to measure the content ratio of 5mC, 5hmC, 5fC and 5caC in DNA (Hashimoto H , Pais J E , et al. Structure of aNaegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA. Nature, 2013, 506(7488):391-395.).

[0053] Determination of TET enzyme activity by ELISA: Use Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method) to determine the enzyme specific activity of each TET protein according to the procedure in the manual.

[0054] The result is as Figure 4-Figure 9 As shown, compared with the LC-MS method and the ELISA method, the chromogenic met...

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PUM

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Abstract

The invention provides a TET enzyme activity determination method. The TET enzyme activity determination method is characterized by comprising the following steps: mixing 5mC DNA with a TET enzyme to be detected, and incubating; adding SDH and DCPIP into a reaction system for reaction; adding trichloroacetic acid to terminate the reaction; measuring the absorption peak value of the reaction liquid at 600nm by a microplate reader, wherein the higher the peak value is, the stronger the enzyme activity is. The invention also discloses an application of the combination of SDH and DCPIP in determination of TET enzyme activity, and a high-throughput screening method of a TET enzyme activity small molecule activator or inhibitor. The invention provides a simple and rapid TET enzyme activity determination method, a TET enzyme oxidation reaction product succinate releases 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 process of the method only needs four-step operation and one-tube reaction, can be completed within 1 hour, and has the advantages of low cost, large flux, wide application range and high accuracy.

Description

technical field [0001] The patent of the invention relates to a method for measuring TET enzyme activity and a high-throughput screening method for small molecule activators or inhibitors of TET enzyme activity, belonging to the field of biotechnology. Background technique [0002] Cytosine methylation (5mC) is the most common modified base on DNA, accounting for 1%-8% of all cytosines, and is the main form of DNA methylation. Some specific cytosine sites, such as Dam methylation sites in bacteria or CpG islands in eukaryotic organisms, have a methylation ratio of nearly 100%, so 5mC is also called "the first Five bases". DNA methylation is an important form of regulating gene expression, mainly mediating gene silencing. This regulatory mode of gene silencing mediated by DNA methylation occurs all the time in the living body. It is the molecular basis of differential expression of genes and a key way to determine embryonic development, cell functional differentiation, indi...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/31
CPCG01N21/78G01N21/3103
Inventor 江翱刘倩马海玲孙睿陈晶晶侯策曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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