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Application of Tetraphenylethylene Derivatives in Detection of Methyltransferase Activity and Methyltransferase Inhibitor Concentration

A technology of methyltransferase and tetraphenylethylene, which is applied in the biological field to achieve the effects of high sensitivity, good water solubility and simple synthesis

Active Publication Date: 2015-11-18
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Tang Benzhong et al reported positively charged tetraphenylethylene derivatives and their preparation methods (Chem.Eur.J.2008,14,6428–6437; Chem.Eur.J.2010,16,1232–1245), now There is no report on the use of positively charged tetraphenylethylene derivatives in the detection of methyltransferase activity and methyltransferase inhibitors in the prior art

Method used

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  • Application of Tetraphenylethylene Derivatives in Detection of Methyltransferase Activity and Methyltransferase Inhibitor Concentration
  • Application of Tetraphenylethylene Derivatives in Detection of Methyltransferase Activity and Methyltransferase Inhibitor Concentration
  • Application of Tetraphenylethylene Derivatives in Detection of Methyltransferase Activity and Methyltransferase Inhibitor Concentration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] A short-chain nucleic acid modified with the quencher molecule dimethylaminoazobenzoyl at both ends, as shown in SEQ ID No: 1 (5'-GTTGGGATCGAGAG-3'), and a long-chain nucleic acid, such as SEQ ID No: 4 ( 5'-AGTGACATGATTTCCTCTCGATCCCAACCGCCGTATAGATAG-3'), mixed in an aqueous solution to obtain a quenched molecularly modified double-stranded nucleic acid (dsDNA3-2Q);

[0055] In a total reaction system of 50 μL, 2 μM dsDNA3-2Q, 1 μL enzyme reaction buffer solution [10×buffer: 200 mMTris-HAc, 500 mMKAc, 100 mMMg(Ac)2, 10 mMDTT, pH7.9], 80 μS-adenosylmethionine Acid (SAM), 400U / mL restriction endonuclease (DpnI) and different concentrations of methyltransferase Dam (concentrations were 0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 40, 80 and 160U / mL), placed at 37°C for 5 hours, and then placed in a 90°C water bath for 10 minutes to inactivate Dam and DpnI, and finally cooled to room temperature to obtain a mixed solution;

[0056] The above mixed solution was mixed evenly with 50 μL ...

Embodiment 2

[0061] A short-chain nucleic acid modified with a quencher molecule dimethylaminoazobenzoyl at both ends, as shown in SEQ ID No: 2 (5'-GTTGGCCGGGAGAG-3'), and a long-chain nucleic acid, such as SEQ ID No: 5 ( 5'-AGTGACATGATTTCCTCTCCCGGCCAACCGCCGTATAGATAG-3'), mixed in an aqueous solution to obtain a double-stranded nucleic acid (dsDNA4-2Q) modified by quenching molecules;

[0062] In a total reaction system of 50 μL, mix 2 μM dsDNA4-2Q, 1 μL enzyme reaction buffer solution [10×buffer: 200 mMTris-HAc, 500 mMKAc, 100 mMMg(Ac)2, 10 mMDTT, pH7.9], 80 μS-adenosylmethionine Acid (SAM), 400U / mL restriction endonuclease (HpaII) and different concentrations of methyltransferase HpaIIMTase (concentrations were 0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 40, 80 and 160U / mL), placed at 37°C for 5 hours, and then placed in a 90°C water bath for 10 minutes to inactivate HpaIIMTase and HpaII, and finally cooled to room temperature to obtain a mixed solution;

[0063] The above mixed solution was unif...

Embodiment 3

[0068] A short-chain nucleic acid with a quencher molecule dimethylaminoazobenzoyl modified at both ends, as shown in SEQ ID No: 3 (5'-GTTGGCGCGGAGAG-3'), and a long-chain nucleic acid such as SEQ ID No: 6 (5 '-AGTGACATGATTTCCTCTCCGCGCCAACCGCCGTATAGATAG-3'), mixed in an aqueous solution to obtain a double-stranded nucleic acid (dsDNA5-2Q) modified by quenching molecules;

[0069] In a total reaction system of 50 μL, 2 μM dsDNA5-2Q, 1 μL enzyme reaction buffer solution [10×buffer: 200 mMTris-HAc, 500 mMKAc, 100 mMMg(Ac)2, 10 mMDTT, pH7.9], 80 μS-adenosylmethionine acid (SAM), 400U / mL restriction endonuclease (BstUI) and different concentrations of methyltransferase M.SssI (concentrations were 0, 0.25, 0.5, 1, 2.5, 5, 10, 20, 40, and 160U / mL), placed at 37°C for 5 hours, and then placed in a 90°C water bath for 10 minutes to inactivate M.SssI and BstUI, and finally cooled to room temperature to obtain a mixed solution;

[0070]Mix the above mixed solution with 50 μL of an aqueo...

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Abstract

The invention provides application of tetraphenyl ethylene derivatives in detecting activity of transmethylase, belonging to the technical field of biology. The detection method comprises the following steps: preparing quenching-molecule-modified double-chain nucleic acid; reacting the quenching-molecule-modified double-chain nucleic acid with S-adenosylmethionine, different concentrations of transmethylase and an enzyme reaction buffer solution to obtain a mixed solution; and finally, reacting the obtained mixed solution with a tetraphenyl ethylene derivative water solution, and detecting the activity of the transmethylase. The invention also provides application of the tetraphenyl ethylene derivatives in detecting the concentration of the transmethylase inhibitor. The detection method is simple and quick, and has high sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of tetraphenylethylene derivatives in detecting the activity of methyltransferase and the concentration of methyltransferase inhibitors. Background technique [0002] A charged fluorescent conjugated polymer that interacts with an oppositely charged fluorescent quencher molecule. The excited state that contributes to quenching can be continuously transferred in a large number of repeating units of the conjugated polymer through charge (or energy) transfer, so that a small amount of quencher molecules can efficiently quench the fluorescence of the polymer. Based on this principle, researchers have developed many related biological analysis and new methods of biosensing. For example, a molecular beacon with superquenching properties can be obtained by covalently modifying a fluorescent conjugated polymer and a quencher molecule to both ends of a nucleic aci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/48C12Q1/44
CPCC12Q1/44C12Q1/48C12Q1/68C12Q2525/10
Inventor 于聪陈健王燕李文英周会鹏李永新
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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