Method for screening G-quadruplex ligands on basis of photoinduced electron transfer

An electron transfer, quadruplex technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to obtain effective data in the detection form, and achieve easy promotion, low toxicity and side effects, and strong biological activity. Effect

Active Publication Date: 2015-05-06
SHAANXI NORMAL UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0007] If the Z factor is less than 0.5, it indicates that the analysis conditio

Method used

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  • Method for screening G-quadruplex ligands on basis of photoinduced electron transfer
  • Method for screening G-quadruplex ligands on basis of photoinduced electron transfer
  • Method for screening G-quadruplex ligands on basis of photoinduced electron transfer

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Embodiment 1

[0028] With emodin and matrine as traditional Chinese medicine monomers, the method for screening G-quadruplex ligands based on photoinduced electron transfer (PIET) consists of the following steps:

[0029] (1) Add hairpin DNA (F-hpDNA, the sequence of F-hpDNA is 5'-FAM (carboxyfluorescein)-TAGCCCTTAGGGTTAGGGTTAGGGTTAGGGCTA-3') with 20mmol / L tris-hydrochloric acid at pH=7.4 Buffer solution to prepare a hairpin DNA solution with a concentration of 100 μmol / L, and put it in a refrigerator at 4°C overnight;

[0030] (2) Dilute the concentration of the hairpin DNA solution in step (1) from 100 μmol / L to 50 nmol / L with 20 mmol / L tris-hydrochloric acid buffer solution with pH=7.4, and take 400 μL of concentration as 50 nmol / L The hairpin type DNA solution is added in 16 wells of 96 well plates, and the initial fluorescence intensity value I in each well is measured with a fluorescence spectrophotometer 0 ;

[0031] (3) add 1 μ L 4mmol / L emodin solution again in 8 holes of step (2...

Embodiment 2

[0033] The method for screening apigenin based on photoinduced electron transfer (PIET) consists of the following steps:

[0034] (1) Add hairpin DNA (F-hpDNA, the sequence of F-hpDNA is 5'-FAM (carboxyfluorescein)-TAGCCCTTAGGGTTAGGGTTAGGGTTAGGGCTA-3') with 20mmol / L tris-hydrochloric acid at pH=7.4 Buffer solution to prepare a hairpin DNA solution with a concentration of 100 μmol / L, and store it in a -20°C refrigerator;

[0035] (2) Dilute the concentration of the hairpin DNA solution in step (1) from 100 μmol / L to 50 nmol / L with 20 mmol / L tris-hydrochloric acid buffer solution with pH=7.4, and take 200 μL of concentration as 50 nmol / L The hairpin type DNA solution is added in 8 wells of 96 well plates, and the initial fluorescence intensity value I in each well is measured with a fluorescence spectrophotometer 0 ;

[0036] (3) add 1 μ L 4mmol / L apigenin solution again in 8 wells of step (2), make its final concentration in the reaction system be 10 μ mol / L, measure the fluo...

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Abstract

The invention relates to a method for screening G-quadruplex ligands on the basis of photoinduced electron transfer. The method comprises the following steps: adding a certain amount of traditional Chinese medicine monomer solution into a diluted hairpin DNA solution, and detecting the change of the fluorescence strength of the diluted hairpin DNA solution before and after the traditional Chinese medicine monomer solution is into the diluted hairpin DNA solution, thus realizing the screening of G-quadruplex ligands. The method fully utilizes the mechanism that G-quadruplex ligands can induce a hairpin probe containing a human telomerase DNA fragment to form G-quadruplex, so that labeled carboxy fluorescein (FAM) on the hairpin probe piles on a G-tetrad plane consisting of four G-bases and is close to the G-bases to be subjected to photoinduced electron transfer, and the fluorescence strength is reduced. The method has the advantages of simplicity in operation, high response speed, high universality and easiness in popularization, can realize high-flux screening of G-quadruplex ligands, and provides a simple and effective way for screening antitumor drugs targeting G-quadruplex ligands.

Description

technical field [0001] The invention relates to a method for homogeneously screening G-quadruplex ligands in vitro based on light-induced electron transfer, and belongs to the technical field of antitumor drug screening. Background technique [0002] Telomerase uses its own RNA as a template to synthesize a telomere sequence at the end of the chromosome to keep the length of the telomere at the end of the chromosome stable, thereby making the cell immortal. Studies have found that telomerase is positively expressed in more than 85% of cancer cells, while there is almost no expression in normal somatic cells, and the degree of differentiation of tumor cells is directly related to the activity of telomerase, which shows that telomerase is closely related to tumor There may be a close relationship between malignant transformation and rapid proliferation of cells (Shay J.W., Wright W.E.Telomerase therapy for cancer: challenges and new directions[J].Nat.Rev.Drug Discov.2006,5:577...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 金燕时志路张琦
Owner SHAANXI NORMAL UNIV
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