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Pyrrole pyridine salt fluorescent probe used for RNA (ribonucleic acid) and nucleolus imaging in living cell

A fluorescent probe, pyrrole pyridine technology, applied in the field of fluorescent probes, can solve the problems of unclear mechanism of interaction between small molecules and RNA, limitations of pathological research and drug development, and difficulties in RNA probe research, achieving strong photostability , good membrane permeability, strong color effect

Active Publication Date: 2013-09-04
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with many commercial DNA probes, there are very few RNA probes, because the existing nucleic acid probes generally have greater affinity with double-stranded DNA, and the mechanism of action between small molecules and RNA is not yet clear, so RNA probes research is more difficult
[0005] A search revealed that Molecular Probes Co. could only provide one typical commercial RNA probe "SYTO RNA-Select" that can be used for RNA imaging, but its chemical structure is still under wraps
Current scarcity of RNA visible probes has limited pathology research and drug development

Method used

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  • Pyrrole pyridine salt fluorescent probe used for RNA (ribonucleic acid) and nucleolus imaging in living cell
  • Pyrrole pyridine salt fluorescent probe used for RNA (ribonucleic acid) and nucleolus imaging in living cell
  • Pyrrole pyridine salt fluorescent probe used for RNA (ribonucleic acid) and nucleolus imaging in living cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Synthesis of (E)-1-(2-hydroxyethyl)-4-(2-(1-methyl-1H-pyrrole-2-)vinyl)pyridine iodide salt (1c)

[0027] Dissolve N-methyl-2-formylpyrrole and N-hydroxyethyl-4-picoline in methanol to obtain a light yellow transparent solution, add 3-4 drops of piperidine, and the solution turns red rapidly. After reflux reaction for 4h, a purple-red precipitate precipitated out. Cool, filter, and wash with dichloromethane to obtain small purple-red crystals with a yield of 90%.

[0028] 1H NMR(400MHz,DMSO-d6),δ(ppm):8.70(d,J=6.9Hz,2H),8.12(d,J=6.9Hz,2H),8.40(d,J=15.9Hz,1H) ,7.08(d,J=16.0Hz,2H),6.89(dd,J=3.96,1.44Hz,1H),6.22(dd,J=3.84,2.56Hz,1H),5.21(t,J=5.24Hz, 1H),4.47(t,J=5.04Hz,2H),3.84(t,J=6.0Hz,2H),3.81(s,3H).13C NMR(400MHz,DMSO-d6),δ(ppm):154.23 ,144.31,130.98,130.11,129.20,122.53,117.94,112.97,110.39,61.99,60.53,34.59.HRMS(m / z):[M-I]+.Calcd for C14H17IN2O,229.13;found,229.14.

Embodiment 2

[0029] Example 2: HeLa, SiHa and H17702 cell culture

[0030] HeLa, SiHa and H17702 cells were adherently cultured in culture medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultured in a saturated humidity incubator, and subcultured once every 2-3 days.

[0031]When the cells grow to the logarithmic phase, culture the slices: ① Soak the coverslips in absolute ethanol for 30 minutes, dry them with an alcohol lamp and place them in a disposable 35mm culture dish for later use; Wash the cells three times with PBS, digest with 1 mL of 0.25% trypsin for 3-5 minutes, pour out the trypsin carefully, add fresh culture medium and pipette evenly, and count the cells. Concentration is 1x10 5 , and then inoculated into the above-mentioned petri dish with coverslip in 5% CO 2 Cultured in an incubator to allow the cells to grow on the sheet.

[0032] After the cell slides grow and cover the coverslip, they are used for experiments.

Embodiment 3

[0033] Example 3: (E)-1-(2-hydroxyethyl)-4-(2-(1-methyl-1H-pyrrole-2-)vinyl) pyridinium iodide salt (1c) to HeLa, SiHa and Staining Observation of H17702 Cells

[0034] Wash the coverslips (climbing slides) covered with HeLa, SiHa and H17702 cells prepared in Example 2 three times with PBS, and then use (E)-1-(2-hydroxyl at a concentration of 20 μM diluted with culture medium Ethyl)-4-(2-(1-methyl-1H-pyrrole-2-)vinyl)pyridinium iodide fluorescent probe solution in CO 2 In the incubator, stain the various cells for 30 min respectively.

[0035] Take out the stained slides, wash away the unbound excess dye solution, cover the cell growth side down on the glass slide, observe under the fluorescence microscope and laser scanning confocal lens, and record the stained parts, fluorescence distribution and brightness in the cells change etc.

[0036] see results figure 1 , figure 2 . The fluorescence image shows obvious green light distribution in the cytoplasm and nucleolus, c...

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Abstract

The invention discloses a pyrrole pyridine salt fluorescent probe used for RNA (ribonucleic acid) and nucleolus imaging in a living cell. The structural general formula of the fluorescent probe is shown in a formula (I), wherein R<1> and R<2> represent an alkyl group, a hydroxyalkyl or an ether group. The invention also discloses application of marking or displaying distribution of RNA and nucleolus in the living cell and identifying life or death state of the cell of the fluorescent probe. The probe has the characteristics of wide application scope, good membrane permeability and low cytotoxicity and can be used for specific fluorescence imaging of RNA in the living cell.

Description

technical field [0001] The invention relates to a fluorescent probe, in particular to a pyrrolidine salt fluorescent probe for RNA and nucleolus imaging in living cells. Background technique [0002] Nucleic acid is not only the basic component of all biological cells, but also plays a dominant role in the growth, development, reproduction, inheritance and variation of organisms and other major life phenomena. For example, the oxidative decomposition product of nucleic acid - purine is the main cause of human uric acid increase and gout. Nucleic acid macromolecules are divided into two categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which play a role in storing and transmitting genetic information in the replication and synthesis of proteins. DNA is the main material basis for storing, replicating and transmitting genetic information. RNA plays an important role in the process of protein synthesis, in which transfer RNA (tRNA) carries and transfers activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D401/06G01N21/64
Inventor 于晓强孙渝明宋国芬
Owner SHANDONG UNIV
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