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Optical click probe for intracellular fluorescent-responsive labeled DNA, preparation method and application thereof

An internal fluorescence and optical click technology, which is applied to the optical click probe of intracellular fluorescence response labeled DNA and its preparation field, can solve the problems of weak fluorescence, no light, and unclear fluorescence response behavior of the product, and achieves fast reaction speed, Small reactive group molecules, easy to obtain effects

Inactive Publication Date: 2020-05-22
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some literatures report that the products of tetrazole compounds labeled nucleic acid by optical click reaction have weak or no light fluorescence, and some literatures use tetrazole compounds to label RNA with fluorescence response [Li, J.; Huang, L.; Xiao, X.; Chen, Y.; Wang, X.; Zhou, Z.; Zhang, Y.J.Am.Chem.Soc.2016,138,15943-15949]
This ambiguous fluorescent response behavior becomes a primary concern during the photoclick reaction for labeling nucleic acids

Method used

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  • Optical click probe for intracellular fluorescent-responsive labeled DNA, preparation method and application thereof
  • Optical click probe for intracellular fluorescent-responsive labeled DNA, preparation method and application thereof
  • Optical click probe for intracellular fluorescent-responsive labeled DNA, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Preparation of a series of DNA probes based on optical click reaction and fluorescent response

[0079] (1) Add coumarin derivative 1' (1mmol) into 6mL of ethanol / water (EtOH / H2O, volume ratio 1:1), then add 1mL of concentrated hydrochloric acid with a concentration of 37% (v / v) and stir, and Slowly add to 1.5mL NaNO at 5°C 2 (1mmol) in an aqueous solution and stirred; then these mixtures were added dropwise to 4-methyl ((2-(benzenesulfonyl) hydrazino) methyl) benzoate at -15°C ((E)-methyl 4-((2-(phenylsulfonyl)hydrazono)methyl)benzoate, 0.9mmol) in dry pyridine (8ml) returned to room temperature and stirred overnight, after the reaction was complete, the reaction mixture was extracted with DCM (dichloromethane) , distilled under reduced pressure, and then further purified with a silica gel column (eluent is methanol: methylene chloride, volume ratio 1:5; the silica gel column specification is 300 mesh), to obtain pure product (coumarin tetrazolium compound 1...

Embodiment 2

[0102] Example 2: Photodynamic properties of coumarin tetrazolium 1-7 and labeled DNA products

[0103] The coumarin tetrazolium compound 1-7 obtained in Example 1 was reacted with the DNA structure of the modified alkene such as formula III, the concentration of the coumarin tetrazolium compound 1-7 was 100 μM, and the concentration of the DNA of the modified alkene was 10 μM , irradiated for 10 minutes under a 350nm ultraviolet lamp, and purified the product with an ultrafiltration tube after the reaction. The fluorescence of seven reaction products was measured and the fluorescence quantum yield of the reaction products was calculated. The results are shown in Table 1. figure 2 is the E of the coumarin derivative calculated in Example 1 HOMO (The energy value of the highest occupied molecular orbital, calculated according to the wB97XD / 6-311+G(d,p) method,) and the relationship diagram of the fluorescence quantum yield. From Table 1 and figure 2 Except for coumarin tet...

Embodiment 3

[0107] Example 3: Exploration of the principle of luminescence after coumarin tetrazole-labeled nucleic acid

[0108]Take coumarin tetrazolium 1 as the sample in the implementation case, with its activated tetrazolium compound 9 (the method is the same as in Example 1), get a tube of 5'-terminal amino-labeled nucleic acid sequence and centrifuge (12000rpm, 20min); Open the cap of the centrifuge tube, quickly add 55 μL of 0.1M sodium borate buffer solution (pH8.0) and quickly cover the tube cap; shake fully on the vortex for 10 min; add 30 μL of 50 mM tetrazole compound 9 (formulated in advance with dimethyl sulfoxide) 50mM solution); wrap the centrifuge tube with tinfoil, put it in the centrifuge tube and incubate with shaking for 14 hours; add 1ml of pre-cooled absolute ethanol to the centrifuge tube, shake up and down for 1min; then put it in an ultra-low temperature refrigerator and let it stand for 2 hours; centrifuge at 4°C, 12000rpm for 20min; absorb the upper layer of c...

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Abstract

The invention discloses a light click probe for intracellular fluorescence response labeled DNA, and a preparation method and an application thereof. The DNA light click probe with fluorescence response is at least one of tetrazolylcoumarin compounds W1 and W2; and the structural formula of the W1 is represented by formula I, and the structural formula of the W2 is represented by formula II. The probe has nucleus targeting property, a reaction group has a small volume and does not interfere the nucleic acid metabolism process, and the probe has the advantages of fluorescence response, real-time controllable photoinduction, fast reaction speed, high specificity, few byproducts, simple and mild reaction conditions, and easiness in operation; and the DNA light click probe with fluorescence response can be applied to the field of living cell nucleic acid labeling.

Description

technical field [0001] The invention belongs to the field of nucleic acid labeling, and in particular relates to an optical click probe for intracellular fluorescence response labeling DNA, a preparation method and application thereof. Background technique [0002] In the field of life science research, nucleic acid labeling technology with fluorescent response has very important value and significance in the study of nucleic acid molecular structure and its synthesis process in cells. In particular, fluorescence-responsive labeling methods triggered by bioorthogonal reactions have become an important tool for biomolecular labeling. This labeling method can selectively label and track biomolecules under mild conditions, and perform sensitive and visualized fluorescence imaging. The initial labeling methods are fluorescence-responsive post-synthetic labeling, template-mediated ligation and metabolic labeling of DNA or RNA, which are based on the cycloaddition reaction of azid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/04C09K11/06C12Q1/68C12N15/10
CPCC07D405/04C09K11/06C09K2211/1007C09K2211/1074C09K2211/1088C12Q1/6806C12Q2563/107
Inventor 张涛邢达吴云霞
Owner SOUTH CHINA NORMAL UNIVERSITY
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