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Visual analysis method and system for intracellular 5-aldehyde uracil

A technology of aldehyde uracil and analysis method, applied in the visual analysis method and system field of 5-formyl uracil in cells, can solve the problem of inability to specifically detect 5-formyl uracil, and achieve the solution of inability to specificity The effect of detecting intracellular 5-formyl uracil, accurate imaging, and mild reaction conditions

Active Publication Date: 2021-04-27
XI AN JIAOTONG UNIV
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  • Claims
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Problems solved by technology

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a visual analysis method and system for intracellular 5-formyl uracil, which solves the problem of the inability to specifically detect intracellular 5-formyl uracil in the prior art technical difficulties

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  • Visual analysis method and system for intracellular 5-aldehyde uracil
  • Visual analysis method and system for intracellular 5-aldehyde uracil
  • Visual analysis method and system for intracellular 5-aldehyde uracil

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Embodiment 1

[0046] Example 1 The single-molecule visual analysis of intracellular 5-formyluracil is realized by using the method of the present invention.

[0047] First, the bioorthogonal ATP reagent ATP-γ-alkynyl was synthesized, including the following operations:

[0048] Synthesis of ATP-γ-alkynyl using ATP and propylamine. Specifically, ATP disodium salt (4.9mM) and propylamine (245mM) were dissolved in water, and the pH of the solution was adjusted to 6.0 with dilute hydrochloric acid, then EDC-HCl (490mM) was added to the above solution, and stirred at room temperature for 24h Afterwards, the pH of the solution was quickly adjusted to 8.5 with sodium hydroxide. After rotary evaporation, a pale yellow viscous product was obtained, which was then purified by silica gel column chromatography to obtain a white solid product ATP-γ-alkynyl, which was stored until use.

[0049] Prepare circularized 5fU padlock probes: 10 μM padlock probe-5fU and 30 μM ligation probe-5fU are hybridized in...

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Abstract

The invention discloses a visual analysis method and system for intracellular 5-aldehyde uracil, and belongs to the fields of organic synthesis, analytical chemistry and cell imaging. The method comprises the following steps of: synthesizing a bioorthogonal ATP reagent ATP-gamma-alkynyl; completely closing 5-hydroxymethyl uracil existing in cells through an enzymatic chemical reaction; chemically reducing the intracellular 5-aldehyde uracil into 5-hydroxymethyl uracil; connecting a DNA primer to the 5-aldehyde uracil through specific enzyme recognition and click chemistry reaction; and amplifying a fluorescence signal through roll-change amplification to realize single-molecule visual analysis of the intracellular 5-aldehyde uracil. The technical defect that the intracellular 5-aldehyde uracil cannot be specifically detected in the prior art is overcome.

Description

technical field [0001] The invention belongs to the technical field of cell imaging, and relates to a visual analysis method and system for intracellular 5-formyl uracil. Background technique [0002] The epigenetic modification of nucleic acids plays an important role in life activities, and a comprehensive understanding of natural base modification is crucial for dissecting epigenetic regulation. In recent years, prior art literature has reported a new type of epigenetic marker 5-formyl uracil (abbreviated as 5fU). 5-formyl uracil exists in many cells and tissues and can be produced by ultraviolet exposure and ionizing radiation; The modification of 5-formyl uracil on DNA can lead to gene mismatch, change DNA structure, and cause disturbance of DNA function. Due to the low abundance of these modifications (5-formyluracil abundance in mouse embryonic stem cells is 2.5x10 -6 dN) and the similarity of the base structure, it is extremely difficult to establish a specific 5fU...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/125C12Q2563/107C12Q2525/117Y02P20/55
Inventor 陈锋赵永席白敏
Owner XI AN JIAOTONG UNIV