Perylene excimer-based detection method for methylase activity and screening method of methylase inhibitor

A technology of methylase activity and methylase, applied in the field of screening of methylase inhibitors, can solve the problems of high cost, time-consuming, low sensitivity, etc. Effect

Active Publication Date: 2014-07-09
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of time-consuming, high cost and low sensitivity of the existing detection methods of methylase activity and screening methods of methylase inhibitors, and to provide a perylene excimer-based association Method for detecting methylase activity and screening method for methylase inhibitors thereof

Method used

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  • Perylene excimer-based detection method for methylase activity and screening method of methylase inhibitor
  • Perylene excimer-based detection method for methylase activity and screening method of methylase inhibitor
  • Perylene excimer-based detection method for methylase activity and screening method of methylase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Mix equal amounts of nucleic acid strands DNA-a (5'-GTTGGGATCGAGAAGddC-3') and DNA-b (5'-CTTCTCGATCCCAACddC-3') in buffer solution [2mMTris-HAc, 5mMKAc, 1mMMg(Ac) 2 ,pH7.9] in a water bath at 90°C for 10 minutes, then cooled to room temperature to obtain double-stranded DNA (dsDNA-1);

[0067] In a 50μL enzyme reaction system, 400nM dsDNA-1, 2.5μL reaction buffer [10×buffer: 200mM Tris-HAc, 500mMKAc, 100mMMg(Ac) 2,10mMDTT,pH7.9], 160μS-adenosylmethionine (SAM), 200U / mL restriction endonuclease (DpnI) and different concentrations of methylase Dam (concentrations were 0, 0.2, 0.5, 1, 2, 2.5, 10, 20, 40, and 80 U / mL), placed at 37°C for 2 hours, and then placed in a 90°C water bath for 10 minutes to inactivate methylases and restriction enzymes, Finally cooled to room temperature to obtain a mixed solution;

[0068] Add 8 U of terminal deoxynucleotidyl transferase (TdT), 200 μM deoxyribonucleoside triphosphate (dNTP), and 5 μL of TdT reaction buffer [5 μL, 10× buffer: 12...

Embodiment 2

[0077] Mix equal amounts of nucleic acid strands DNA-a (5'-GTTGGCCGGGAGAAGddC-3') and DNA-b (5'-CTTCTCCCGGCCAACddC-3') in buffer solution [2mMTris-HAc, 5mM KAc, 1mMMg(Ac) 2 ,pH7.9] in a water bath at 90°C for 10 minutes, then cooled to room temperature to obtain double-stranded DNA (dsDNA-1);

[0078] In a 50μL enzyme reaction system, 400nM dsDNA-1, 2.5μL reaction buffer [10×buffer: 200mM Tris-HAc, 500mMKAc, 100mMMg(Ac) 2 ,10mMDTT,pH7.9], 160μS-adenosylmethionine (SAM), 200U / mL restriction endonuclease (HpaII) and different concentrations of methylase HpaII (concentrations were 0, 0.2, 0.5, 1, 2, 2.5, 10, 20, 40, and 80 U / mL), placed at 37°C for 2 hours, and then placed in a 90°C water bath for 10 minutes to inactivate methylases and restriction enzymes, Finally cooled to room temperature to obtain a mixed solution;

[0079] Add 8 U of terminal deoxynucleotidyl transferase (TdT), 200 μM deoxyribonucleoside triphosphate (dNTP), and 5 μL of TdT reaction buffer [5 μL, 10× buffe...

Embodiment 3

[0084] Mix equal amounts of nucleic acid strands DNA-a (5'-GTTGGCGGAGAAGddC-3') and DNA-b (5'-CTTCTCCGCCAACddC-3') in buffer solution [2mMTris-HAc, 5mMKAc, 1mMMg(Ac) 2 ,pH7.9] in a water bath at 90°C for 10 minutes, then cooled to room temperature to obtain double-stranded DNA (dsDNA-1);

[0085] In a 50μL enzyme reaction system, 400nM dsDNA-1, 2.5μL reaction buffer [10×buffer: 200mM Tris-HAc, 500mMKAc, 100mMMg(Ac) 2 , 10mMDTT, pH7.9], 160μS-adenosylmethionine (SAM), 200U / mL restriction endonuclease (BstUI) and different concentrations of methylase M.SssI (concentrations were 0, 0.2, 0.5, 1, 2, 2.5, 10, 20, 40 and 80 U / mL), placed at 37°C for 2 hours, and then placed in a 90°C water bath for 10 minutes to deplete methylases and restriction enzymes live, and finally cooled to room temperature to obtain a mixed solution;

[0086] Add 8 U of terminal deoxynucleotidyl transferase (TdT), 200 μM deoxyribonucleoside triphosphate (dNTP), and 5 μL of TdT reaction buffer [5 μL, 10× bu...

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Abstract

The invention provides a perylene excimer-based detection method for methylase activity and a screening method of a methylase inhibitor, and belongs to the field of biotechnology. The method comprises the following steps: firstly, preparing double-chain DNA, and then enabling the double-chain DNA to react with S-adenosylmethionine, restriction enzyme and different concentration of methylase, so as to obtain a mixed solution; enabling terminal deoxyribonucleotidyl transferase, deoxyribonucleoside triphosphate and TdT reaction buffer to react with the mixed solution, so as to obtain a reaction solution; finally, enabling the mixed solution of a perylene derivative probe and polycation to react with the reaction solution, and carrying out fluorescence detection on the methylase activity. The invention also provides a screening method of the methylase inhibitor. The activity of the methylase and the inhibitor is detected by using the change of ratio of a small molecule probe monomer to the excimer fluorescence intensity, the ratio of two given fluorescence signals is provided from the test, the fluorescence signals are not easily interfered in comparison with a pure fluorescence-intensified or weakened signal, and the sensitivity is higher.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting methylase activity based on perylene excimer associations and a method for screening methylase inhibitors thereof. Background technique [0002] Excimer refers to the aggregation of two molecules and atoms of the same kind. In the excited state, the two molecules or atoms have a stronger effect, resulting in a new energy level, and the emission spectrum is different from that of a single species. structure. In the ground state, the effect is weak or no effect. Excimer fluorescence has a large Stokes shift and a long fluorescence lifetime, and has many applications in biological analysis and sensing. Many molecules with planar aromatic ring structures exhibit excimate fluorescence. Perylene derivatives are a class of condensed ring conjugated compounds, which have large π-π conjugated electronic structures, excellent fluorescence quantum efficienc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/48C12Q1/44
CPCC12Q1/48
Inventor 于聪王燕陈健
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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