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Photosensitive compound and preparation method and application thereof

A compound, light-sensitive technology, applied in the field of protein and nucleic acid co-immobilization, can solve the problems of data analysis and interpretation interference, difficult quantitative detection, low signal-to-noise ratio, etc., and achieve the effect of reducing sample loss, small sample volume and high sensitivity

Active Publication Date: 2021-06-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although single-cell accuracy can be achieved in terms of accuracy, it still requires a larger sample size and the operation is more complicated
[0010] 2. MNase-seq requires a large number of cells and strict enzymatic conditions
However, it is highly dependent on the fixation efficiency of formaldehyde
And the signal-to-noise ratio is too low, and the background signal that is too high will greatly interfere with the analysis and interpretation of the data
[0011] 3. Electrophoretic mobility migration analysis has a large amount of sample (2-20ug), and the label of the DNA probe used is radioactive, which is difficult to detect quantitatively, especially for the quantitative detection of proteins in the gel

Method used

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  • Photosensitive compound and preparation method and application thereof
  • Photosensitive compound and preparation method and application thereof
  • Photosensitive compound and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Principle of light-sensitive protein and nucleic acid co-immobilization gel

[0081] The principle of light-sensitive protein nucleic acid co-immobilization gel is as follows: figure 1 As shown, protein-nucleic acid co-immobilization includes the following steps:

[0082] 1.1 Material sedimentation: The purified protein of cells and nucleic acid probes is added dropwise to the gel containing microwells, allowing it to settle into the wells by gravity, and wash away the unsettled cells with PBS;

[0083] 1.2 Protein-nucleic acid binding: cells are cleaved in situ to release protein nucleic acid and incubated to combine protein and nucleic acid probe, nucleic acid and purified protein;

[0084] 1.3 Electrophoresis: Perform non-denaturing gel electrophoresis on a microchip to separate the polymers bound to each other and free monomers;

[0085]1.4 Photosensitive immobilization: the photosensitizer is activated by ultraviolet irradiation, thereby immobilizing p...

Embodiment 2

[0087] Example 2: The principle of light-sensitive protein-nucleic acid co-immobilization gel and protein and nucleic acid cross-linking after ultraviolet excitation

[0088] The novel photosensitizer MAP-FTC contained in the light-sensitive protein-nucleic acid co-immobilization gel provided by the present invention is a polyacrylamide compound with both a tetrazole group and a furan group. The principle of light-sensitive protein-nucleic acid co-immobilization gel cross-linking with protein and nucleic acid after ultraviolet excitation is as follows: figure 2 As shown, the chemical formula on the left indicates that the tetrazolyl group of the new photosensitizer MAP-FTC can undergo nucleophilic addition to the protein by the carbon-nitrogen triple bond generated by the tetrazole group of the new photosensitizer MAP-FTC under ultraviolet irradiation at about 346 nm; the chemical formula on the right indicates the furan of the new photosensitizer MAP-FTC The group can underg...

Embodiment 3

[0089] Embodiment 3: Preparation of light-sensitive compound

[0090] Step 3.1: Preparation of N-[3-(2-methylprop-2-allylamino)propyl tert-butyl carbamate (tert-butyl(3-methacrylamidopropyl) carbamate):

[0091] Such as image 3 As shown in the synthetic route diagram, add methacryloyl chloride to the first compound, ie, tert-butyl N-(3-aminopropyl)carbamate in dichloromethane (DCM), and stir at 15-40°C for 13- 20h, the resulting reaction mixture was treated with NH 4 Cl and brine, washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo; the resulting residue is purified by column chromatography to give tert-butyl(3-methacrylamidopropyl)carbamate as a white solid, i.e. image 3 The second compound shown.

[0092] Step 3.2: Preparation of N-(3-aminopropyl)-2-methyl-prop-2-enamide (N-(3-aminopropyl)-2-methyl-prop-2-enamide):

[0093] Such as image 3 As shown in the synthetic route diagram, to the second compound, i.e., N-[3-(2-methylprop-2-allylamino) propyl...

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Abstract

The invention discloses a photosensitive compound and a preparation method and application thereof, and relates to the technical field of protein nucleic acid co-immobilization, the photosensitive compound is a polyacrylamide compound, and comprises a tetrazole group and a furan group; the compound is prepared from the following raw materials: N-(3-aminopropyl) tert-butyl carbamate, methacryloyl chloride furan-2-yl boric acid, [hydroxyl (phenyl)-lambda 3-iodo] 4-methyl benzene sulfonate and the like. Protein and nucleic acid co-immobilization electrophoresis gel can be prepared, and protein and nucleic acid in single cells or multiple cells can be quantitatively or semi-quantitatively detected; the compound can also be used for microchip electrophoresis mobility analysis, western blotting, nucleic acid blotting hybridization, single-cell western blotting, single-cell northern / source blotting hybridization or capillary electrophoresis western blotting. The protein and nucleic acid co-immobilization gel can be used for simultaneously immobilizing protein and nucleic acid in situ to research the interaction between the protein and DNA (Deoxyribose Nucleic Acid).

Description

technical field [0001] The invention relates to the technical field of co-immobilization of proteins and nucleic acids, in particular to a light-sensitive compound and its preparation method and application. Background technique [0002] In most organisms, genetic information is stored by DNA, which is transcribed and translated into proteins to undertake various life activities. As the most important biological macromolecules, nucleic acid and protein have always been the focus of research in the field of life sciences. At present, various nucleic acid sequencing technologies and protein analysis technologies for single-cell high-throughput are developing vigorously. It makes possible the further study of many key biological problems. In recent years, genomics and transcriptomics research, which studies nucleic acids, proteins and their interactions, is developing vigorously. Cells exhibit high variability in genome, transcriptome, and proteome expression, which is close...

Claims

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Application Information

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IPC IPC(8): C07D405/04G01N27/447
CPCC07D405/04G01N27/447
Inventor 丁显廷谢海洋郭文珂张婷
Owner SHANGHAI JIAO TONG UNIV
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