Method for efficiently screening out DNA chain scission injury protective drugs

A protective and chain-breaking technology, applied in the field of pharmacy, can solve the problems of large signal difference, chain-like structure breakage, false positives, etc., and achieve the effect of simplifying the screening process.

Active Publication Date: 2015-04-22
SHANGHAI YANGPU CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Because this technology uses a DNA base damage as a detection index, it has the following shortcomings: First, according to literature reports, DNA is attacked by oxygen free radicals not only produces base damage including 8-OH-dG, Its chain structure will also break, that is, broken chain damage
This means that in the above-mentioned technical system, the reaction situation shown in the figure below actually exists. The two situations shown in the figure actually produce the same amount of 8-OH-dG, but due to the simultaneous occurrence of DNA chain scission , and according to the third step of the above technique, the magnetic microspheres are washed to separate the remaining 8-OH-dG to be detected from impurities such as Chinese herbal medicines, and also from the part of the DNA strand that has been broken, and the resulting 8-OH-dG is uncertain. Is it concentrated on the broken part or the reserved part, so even if the same amount of 8-OHdG is produced on the DNA, the detected signal may vary greatly, figure 1 The Chinese herbal medicine added in the test did not actually inhibit the effect of 8-OH-dG, but the signal was much lower than that of the control group, so it is easy to produce "false positive" results
The second point is that the detection of DNA damage by this technology requires a two-step immune response, which is not simple enough

Method used

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  • Method for efficiently screening out DNA chain scission injury protective drugs
  • Method for efficiently screening out DNA chain scission injury protective drugs
  • Method for efficiently screening out DNA chain scission injury protective drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Detection of DNA chain scission damage caused by Fenton reagent

[0037] 1.1 Reagents and instruments

[0038] All reagents were of analytical grade. N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) was purchased from Sigma; HRP-labeled rabbit anti-FAM was from Invitrogen.Molecular probe; HRP CL kit Purchased from Milipore Company; Plus carboxylated magnetic microspheres (MB, 1.5 μm, 20 mg / mL) were purchased from Polyscience; BSA was purchased from Huamei Biotechnology Company; DNA (5′-FAM-AAAGGGGAAA-NH2-3′) was purchased from Shanghai Sangon Biotechnology Ltd; Fenton's reagent: (NH 4 ) 2 Fe(SO 4 ) 2 Prepare a fresh aqueous solution stock solution with EDTA at a molar ratio of 1:3 every day, dilute before use, and use 30% H2 o 2 The solution is stored in the refrigerator in the refrigerated layer, protected from light, and diluted before use.

[0039] The ultrapure water used in the test was prepared by a Milli-XQ instrument, and al...

Embodiment 2

[0048] The selection of embodiment 2 DNA charging amount

[0049] The amount of DNA input determines the density of the DNA immobilized on the magnetic microspheres and the probability of subsequent chain scission reactions. Therefore, it is necessary to investigate the influence of the amount of DNA input on the detection signal in order to select the appropriate amount of DNA input while saving costs. figure 2 The detection signal of negative control and positive control is shown by the amount of DNA input

[0050] Intensity of influence. The experimental conditions are: Fe 2+ The concentration is 0.9Mm, H 2 o 2 54mM, HRP-labeled rabbit anti-

[0051] The FAM antibody was diluted 1 / 10000. Such as figure 2 It can be seen that along with the gradual increase of the amount of DNA feeding, the signals of the negative control and the positive control are all gradually enhanced and tend to be saturated, and the degree of DNA chain scission represented by the difference bet...

Embodiment 3

[0052] The influence of the concentration of embodiment 3 Fenton's reagents on the intensity of chemiluminescence

[0053] Fenton's reagent that can generate hydroxyl radicals (ie Fe 2+ Mediated H 2 o 2 Reaction :) It is a relatively classic poisoning system that can cause DNA strand breakage damage. The Fenton reaction system that the present invention adopts: (NH 4 ) 2 Fe(SO 4 ) 2 / EDTA / H 2 o 2 The molar ratio is 1:3:60. Of which (NH 4 ) 2 Fe(SO 4 ) 2 Configured with EDTA, it can significantly reduce Fe 2+ The rate of autoxidation in solution increases with H 2 o 2 Reaction efficiency of post-exposure DNA infection step. image 3 The effect of Fenton's reagent concentration on the chemiluminescent signal produced after exposure is shown. As the concentration of Fenton's reagent increases, the number of broken chains of immobilized DNA gradually increases, resulting in a gradual decrease in the signal detected by chemiluminescence enzyme-linked immunoassay of...

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Abstract

The invention relates to a method for efficiently screening out DNA chain scission injury protective drugs. The method is characterized by comprising the steps that DNA with one end modified by FAM and the other end modified by amino is fixed to a magnetic microsphere surface through a covalent bond, and immobilized DNA is obtained; a drug solution is added into the immobilized DNA, and DNA with drugs is obtained; in vitro exposure is carried out on the DNA with the drugs, and exposure DNA is obtained; magnetic separation is carried out on the exposure DNA, exposure ingredients and reaction impurities are separated away, and DNA free of chain scission is retained; an enzyme-labeled FAM antibody is added into the DNA free of chain scission, the enzyme-labeled FAM antibody and the FAM at the free end of the DNA free of chain scission have an immunoreaction, and DNA is obtained after the immunoreaction; an enhanced chemiluminescent is added into the DNA obtained after the immunoreaction, the high-sensitivity chemiluminiscence method is adopted for detecting the DNA obtained after the immunoreaction, and detection signals are obtained; the detection signals are compared with comparison group signals, and whether the drugs protect the DNA or not is judged. The method can reduce false positives, and has the practical value.

Description

technical field [0001] The present invention relates to the field of pharmaceutical methods, in particular to drug screening, in particular to a method for efficiently screening DNA chain-breaking damage-protective drugs. Background technique [0002] With the rapid development of life sciences, people not only pay attention to the treatment of some common diseases, but also begin to dig out the secrets of genetic diseases and aging, hoping to fundamentally eliminate hidden dangers and resist aging. DNA is an important biomolecule that carries and transmits genetic information. The maintenance of its integrity has become a key research field in biological sciences. Various techniques for evaluating DNA damage continue to evolve, and reports on finding efficient and safe DNA protective drugs are also increasing. [0003] Although traditional DNA evaluation techniques such as radioactive element labeling, high performance liquid chromatography-electrochemical measurement, gas ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/551
CPCG01N33/551
Inventor 陈红君余自成曹志娟卢建忠
Owner SHANGHAI YANGPU CENT HOSPITAL
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