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DNA double-stranded labelled compound and method for determining compound binding target protein by using DNA double-stranded labelled compound

A technology for targeting proteins and compounds, applied in the field of DNA double-stranded labeling compounds, can solve problems such as analysis interference, and achieve the effects of signal specific fidelity, high specificity, and mild reaction conditions

Inactive Publication Date: 2020-09-29
HITGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional heating dissociation method denatures the protein and separates the binding substance, but in this process, the irrelevant protein bound to the above-mentioned labeling medium will also break away and form analysis interference in the same system

Method used

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  • DNA double-stranded labelled compound and method for determining compound binding target protein by using DNA double-stranded labelled compound
  • DNA double-stranded labelled compound and method for determining compound binding target protein by using DNA double-stranded labelled compound
  • DNA double-stranded labelled compound and method for determining compound binding target protein by using DNA double-stranded labelled compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, the synthesis of biotin-DNA double-stranded labeling compound

[0063] Synthesis of step 1, compound 2

[0064]

[0065] Dissolve 5 μmol of HP-AOP in 0.25M BBs buffer (pH=9.4) to prepare an initial solution with a concentration of 1 mM; then compound 1 (ROCK2 protein inhibitory activity IC 50 About 50nM) DMA solution (250μmol, concentration 200mM / L), HATU DMA solution (250μmol, concentration 400mM / L) and DIPEA DMA solution (250μmol, concentration 400mM / L) were placed in -20℃ refrigerator and cooled To mix, the mixture was vortexed to mix thoroughly, and then stored for 10 minutes. Add the above mixed solution into the starting solution of the compound HP-AOP, vortex and oscillate to mix thoroughly, and then react with shaking at room temperature for 2-4 hours. Add 100 μL dd-H to 1 μL reaction solution 2 O dilution, LCMS to monitor the reaction. After the reaction was complete, 10% of the volume of the reaction solution in 5M NaCl aqueous solution an...

Embodiment 2

[0076] Example 2. Research on the enrichment effect of DNA marker compounds with different chain lengths on proteins

[0077] To culture Hela cells, mix 1*10 7 up to 5*10 7 The cells were suspended in HBSS buffer and centrifuged, the supernatant was discarded, and a small amount of HBSS buffer was added to resuspend the cells in a 1.5mL centrifuge tube, with a total volume of 200-1000μL, placed in liquid nitrogen for quick freezing, and then put it away after the sample was completely frozen. Slowly thaw on ice and repeat three times. Among them, HBSS buffer does not add detergent, reducing agent and inhibitor of protease or phosphatase, and the pH value is between 7.2-7.4.

[0078] The efficiency of protein enrichment of the DNA marker compounds with different chain lengths prepared in Example 1 was studied. 100 nmol of compound 5, compound 3, compound 6 and compound 4 were incubated with the cell lysate respectively. Incubate the resulting solution with 200 μL of Neutravi...

Embodiment 3

[0080] Example 3, DNA double-stranded labeling compound to determine the target protein

[0081] The cells were cultured and lysed according to the method described in Example 2, and 200 nmol of compound 4 (experimental group) and compound 3 (control group) were added respectively, and incubated in the same manner. Incubate the resulting solution with 500 μL of Neutravidin-coated magnetic beads (GE, #7815-2104-010350), and use the affinity interaction between biotin and Neutravidin to label the DNA double strands. The protein to which the compound binds is immobilized on neutravidin-coated magnetic beads, which are collected magnetically. The magnetic beads were further resuspended and eluted to remove weakly bound or non-specifically bound proteins. Add the same volume of endonuclease buffer and restriction endonuclease HindIII (Thermo, #FD0504) to each group, resuspend and incubate with the magnetic beads, and the restriction endonuclease cuts the corresponding restriction ...

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Abstract

The invention relates to a DNA double-stranded labelled compound and a method for determining a compound binding target protein by using the DNA double-stranded labelled compound, which can be used for discovering a compound binding target and a compound action mechanism. The method is especially suitable for researching a binding target and an action mechanism of a known compound which has a disease treatment effect or has a disease-related phenotype regulation effect in drug phenotype screening in clinical practice, and provides an action mechanism for new drug discovery.

Description

technical field [0001] The invention relates to a DNA double-strand marking compound and a method for using the DNA double-strand marking compound to determine the compound binding target protein, which can be used to discover the compound binding target and the compound action mechanism. It is especially suitable for studying the binding targets and mechanisms of action of known compounds that have been found to have therapeutic effects on diseases in clinical practice or have regulatory effects on disease-related phenotypes in drug phenotype screening, and provide mechanisms of action for new drug discovery. Background technique [0002] There are two classic paths for drug discovery. One is to find effective compounds through phenotypic screens or animal experiments, and then further study the binding targets and molecular mechanisms of action of the compounds; the other is to follow the modern molecular biology in the 1950s. The established target screening strategy is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11G01N27/62
CPCC12N15/11G01N33/6848
Inventor 李进巩晓明张毅陈秋霞窦登峰刘观赛王钊万金桥李萍罗华东
Owner HITGEN INC
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