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Biological fluorescent probe for real-time quantification of RNA output in cell nucleus and preparation method thereof

A fluorescent probe, biological technology, applied in the field of biological fluorescent probes, can solve problems such as inability to determine the ratio

Active Publication Date: 2021-08-20
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the ratio of dye that enters the cytoplasm with RNA to that that remains in the nucleus cannot be determined

Method used

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  • Biological fluorescent probe for real-time quantification of RNA output in cell nucleus and preparation method thereof
  • Biological fluorescent probe for real-time quantification of RNA output in cell nucleus and preparation method thereof
  • Biological fluorescent probe for real-time quantification of RNA output in cell nucleus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Probe preparation:

[0031] (1) Add N-phenylanthranilic acid (30g, 0.138mol) and phosphorus oxychloride (170g, 1.108mol) into a 500mL round bottom flask, heat slowly to 85°C in a water bath, then transfer to an oil bath Warm up to 135°C and react for 2 hours, cool the reaction solution to room temperature, concentrate under reduced pressure to 5% of the original volume of the reaction solution, then add it to an aqueous ammonia solution, adjust the pH to 5, precipitate a solid, and filter to obtain a gray-green intermediate AD -Cl, yield 70%. 1 H-NMR (400MHz) δ8.34(d, J=8.7Hz), 8.22(d, J=8.8Hz), 7.81–7.73(m), 7.61–7.53(m).HR-MS(m / z, ESI) Calculated for C 13 h 8 ClNm / z=213.0345[M+H].Found m / z=214.0420.

[0032] (2) Add intermediate AD-Cl (1g, 0.047mol) to a 50mL dark round-bottomed flask, then add 10mL tetrahydrofuran, stir well, then add dropwise an aqueous solution of sodium azide (0.456g, 0.070mol) ( 1mL of water), heated up to 40°C for 3h, added the reaction sol...

Embodiment 2

[0034] Biological studies of target molecules:

[0035] 1. Use probes AD-N respectively3 and AD-N 3 with H 2 Product AD-NH after S reaction 2 The cells were cultured, and then co-localized with commercial dyes of nuclear RNA and lysosome respectively, indicating that the probe of the present invention can quickly combine with nuclear RNA and be detected by H 2 S activates, while AD-N 3 with H 2 Product AD-NH after S reaction 2 , due to its basicity and acidity of lysosomes, the affinity of acridine probes from nucleus to lysosomes is changed, which lays the foundation for observing the transfer of RNA in the nucleus to the cytoplasm.

[0036] 2. Culture cells with different hormones and apoptosis-stimulating reagents, and observe that during the process of cell apoptosis, a large amount of RNA in the nucleus is exported to the cytoplasm. Adding thyroxine and epinephrine also promotes the export of nuclear RNA, while other Five hormones (methyltestosterone, diethylstilbes...

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Abstract

The invention discloses a biological fluorescent probe for real-time quantification of RNA output in a cell nucleus and a preparation method thereof, which relate to the technical field of biological fluorescent probes. The probe has an acridine group, and the acridine group is similar to a common nuclear RNA dye to achieve the ability of cell nucleus positioning and specific RNA recognition through interaction of a hydrogen bond and a pi-pi bond. Once the AD-N3 encounters H2S and -N3 groups which are inherent in the nucleus and have the typical concentration range, the AD-N3 is immediately converted into primary amine, amino acridine (AD-NH2) is generated, the non-emissive AD-N3 is converted into a high-fluorescence mark of living cell nucleus RNA, a fluorescence probe is prevented from returning to the cell nucleus, and real-time quantitative nuclear RNA output becomes possible.

Description

Technical field: [0001] The invention relates to the technical field of biological fluorescent probes, in particular to a biological fluorescent probe for real-time quantification of RNA output in the nucleus and a preparation method thereof. The biological fluorescent probe is not only compatible with H 2 S has high specific responsiveness and can target the nucleus at the same time, so as to achieve the effect of real-time quantification of RNA output in the nucleus. Background technique: [0002] The export of nuclear RNA to the cytoplasm is one of the key steps in protein expression to achieve biological functions. Currently, nuclear RNA export analysis is performed by isolating nuclei and cytoplasm from cell lysates and quantifying cytoplasmic RNA levels. Another widely accepted approach is to measure the transcript's corresponding protein, an indirect measure. A general and fundamental limitation of these methods is the inability to track the rapid dynamics of RNA ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6439
Inventor 张瑞龙沈杰陈娟刘正杰韩光梅张忠平
Owner ANHUI UNIVERSITY
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