WO2021109862A1 - Light-sensitive living cell ribonucleic acid fluorescent probe - Google Patents

Light-sensitive living cell ribonucleic acid fluorescent probe Download PDF

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WO2021109862A1
WO2021109862A1 PCT/CN2020/129180 CN2020129180W WO2021109862A1 WO 2021109862 A1 WO2021109862 A1 WO 2021109862A1 CN 2020129180 W CN2020129180 W CN 2020129180W WO 2021109862 A1 WO2021109862 A1 WO 2021109862A1
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light
ribonucleic acid
fluorescent probe
sensitive
alkyl
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宋国芬
李鹏辉
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

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  • the invention relates to the field of probes, in particular to a light-sensitive ribonucleic acid fluorescent probe and its application.
  • Ribonucleic acid plays an important role in the process of protein synthesis. Ribosomal ribonucleic acid is the main place for cells to synthesize protein, messenger ribonucleic acid is the template for protein synthesis, and the function of transfer ribonucleic acid is to carry and transfer activated amino acids. Light irradiation has important effects on living cells and other biological samples. For example, the expression of light-sensitive proteins in some light-responsive cells plays a key role in their light sensitivity. The development of light-responsive ribonucleic acid probes is useful for studying the life activities of light-sensitive cells. And protein expression, the monitoring of light irradiation of biological samples is of great significance.
  • the long-wavelength red fluorescence has better cell and tissue penetration.
  • the purpose of the present invention is to provide a light-sensitive ribonucleic acid fluorescent probe and a preparation method and application thereof, aiming to solve the problem that the ribonucleic acid fluorescent probe in the prior art does not have light-responsive properties and emits a single color.
  • a light-sensitive ribonucleic acid fluorescent probe the structure of the light-sensitive ribonucleic acid fluorescent probe is shown in the following formula I:
  • said R 1 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 2 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 3 Any one selected from hydrogen, alkyl, alkoxy, and ether groups; the X is selected from halogen elements.
  • an application of a light-sensitive ribonucleic acid fluorescent probe the application of the light-sensitive ribonucleic acid fluorescent probe in identifying whether living cells have been irradiated with light, and the imaging of ribose in living cells after light irradiation Application of nucleic acid distribution.
  • the indolequinoline salt fluorescent probe provided by the present invention is a new type of ribonucleic acid specific recognition fluorescent probe molecule in the cell, compared with the light-sensitive ribonucleic acid fluorescent probe with similar function, the probe of the present invention
  • the uniqueness of the needle is that it has good photosensitivity, specifically binds to ribonucleic acid in cells after light irradiation, and emits red fluorescence that is different from the commercial ribonucleic acid probe RNA-select green fluorescence after binding to ribonucleic acid; At the same time, the light stability is strong, the membrane permeability is good, and the counterstaining compatibility is good.
  • the light-sensitive ribonucleic acid fluorescent probe provided by the present invention can be used as a fluorescent probe to label the distribution of ribonucleic acid in living cells after light irradiation and the application of related life activities.
  • Nucleic acid-related physiological and pathological research provides simple and intuitive biological detection reagents, and can also be developed for light irradiation monitoring of live biological samples protected from light. It has a wide range of applications and better effects.
  • Figure 1 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide staining HeLa living cells before light irradiation and Confocal fluorescence micrographs under 488nm laser excitation after irradiation.
  • Figure 1A, Figure 1B, and Figure 1C are micrographs of incubation in the dark (without light irradiation).
  • Figure 1D, Figure 1E, and Figure 1F are mercury The photomicrograph after the lamp is irradiated for 5 minutes.
  • the left image (AD) is a fluorescence micrograph;
  • the middle image (BE) is a bright-field differential interference micrograph;
  • the right image (CF) is a merged image of the two images on the left (co-localization image).
  • Figure 2 shows the staining of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide on living HeLa cells in the dark for different times (10-120min) fluorescence micrograph
  • Figure 2A is the fluorescence micrograph of staining for 10min
  • Figure 2B is the fluorescence micrograph of staining for 30min
  • Figure 2C is the fluorescence micrograph of staining for 60min
  • Figure 2D is the fluorescence micrograph of staining for 120min Fluorescence micrograph.
  • Figure 3 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide salt after staining HeLa living cells in the dark Fluorescence micrographs of mercury lamp green light 510-560nm irradiated for different time
  • Figure 3A is the fluorescence micrograph of mercury lamp green light 510-560nm irradiated for 10s
  • Figure 3B is mercury lamp green light 510-560nm irradiated for 1 min
  • Figure 3C is a fluorescent photomicrograph of mercury lamp green light 510-560nm irradiated for 2min
  • Figure 3D is a mercury lamp green light 510-560nm irradiated for 3min fluorescence photomicrograph
  • Figure 3E is mercury lamp green Fluorescence micrographs of 510-560nm irradiated for 4 minutes
  • Figure 3F is a fluorescence microscopy of mercury lamp green light 510-560nm irradi
  • Figure 4 shows the HeLa cells fixed with paraformaldehyde solution after being treated with deoxyribonuclease (DNase) and ribonuclease (RNase), and then treated with (E)-4-(2-(5-methoxy-1H) -Indole-3-)vinyl)-1-methylquinoline iodide was used to stain HeLa cells.
  • Fluorescence micrographs obtained under 510-560nm mercury lamp excitation.
  • Figure 4A is the control group (untreated group).
  • Fig. 4B is the fluorescence micrograph of the cells after DNase treatment
  • Fig. 4C is the fluorescence micrograph of the cells after RNase treatment.
  • Figure 5 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt and ribonucleic acid (RNA), deoxyribonucleic acid ( The absorption and fluorescence spectra before and after DNA).
  • Figure 5A shows the UV-visible absorption spectra of the probe molecule itself and after mixing with ribonucleic acid and deoxyribonucleic acid;
  • Figure 5B shows the probe itself and ribonucleic acid and deoxyribonucleic acid respectively. Fluorescence emission spectrum after mixing.
  • Figure 6 is the co-staining of HeLa cells with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide and the nuclear probe Hoechst33342 Later, the fluorescence micrographs obtained under mercury lamp irradiation, Figure 6A is the red fluorescence micrograph of the probe; Figure 6B is the blue fluorescence micrograph of Hoechst 33342; Figure 6C is the superimposition of Figure 6A and Figure 6B Figure.
  • first and second are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with “first” and “second” may explicitly or implicitly include one or more of these features.
  • “plurality” means two or more than two, unless otherwise specifically defined.
  • An example of the present invention provides a light-sensitive ribonucleic acid fluorescent probe.
  • the structure of the light-sensitive ribonucleic acid fluorescent probe is shown in the following formula I:
  • said R 1 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 2 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 3 Any one selected from hydrogen, alkyl, alkoxy, and ether groups; the X is selected from halogen atoms.
  • the indolequinoline salt fluorescent probe provided by the present invention is a new type of ribonucleic acid specific recognition light-sensitive fluorescent probe molecule in the cell.
  • the probe of the present invention The uniqueness of the needle is that it has good photosensitivity.
  • the probe is released from the cytoplasmic mitochondria under light irradiation, and specifically binds to the ribonucleic acid in the cell, and after binding to the ribonucleic acid, it emits RNA as a commercial ribonucleic acid probe. -Select red fluorescence with different green fluorescence; at the same time, it has strong light stability, good membrane permeability and good counterstaining compatibility.
  • said R 1 is selected from any one of hydrogen, C1-C5 alkyl, C1-C5 alkoxy, and aliphatic ether group
  • said R 2 is selected from hydrogen, C1-C5 Any one of the alkyl group, C1-C5 alkoxy group, and fatty ether group
  • the R 3 is selected from any one of hydrogen, C1-C5 alkyl group, C1-C5 alkoxy group, and fatty ether group
  • the X is selected from iodine, bromine, and chlorine.
  • the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl;
  • the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl;
  • aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  • the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl;
  • the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl;
  • aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  • the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl;
  • the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl;
  • aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  • said R 1 is selected from hydrogen; said R 2 is selected from methoxy; said R 3 is selected from methyl; and said X is selected from iodine.
  • said R 1 is selected from hydrogen; said R 2 is selected from methoxy; said R 3 is selected from methyl; and said X is selected from iodine, the resulting light sensor
  • the type ribonucleic acid fluorescent probe is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt.
  • the light-sensitive ribonucleic acid fluorescent probe provided by the present invention is prepared by the following preparation method of light-sensitive ribonucleic acid fluorescent probe.
  • the preparation method of the light-sensitive ribonucleic acid fluorescent probe is as follows: Indole-3-formaldehyde salt and 4-methylquinoline salt are mixed and dissolved in methanol/ethanol at a molar ratio of 1:(1.0 ⁇ 2) to obtain light. Yellow transparent solution, drop a small amount of piperidine, heat and reflux for 6-12 hours to produce a deep red precipitate, cool to room temperature, filter and vacuum dry to obtain the light-sensitive ribonucleic acid imaging fluorescent probe.
  • the indole-3-carbaldehyde is selected from 5-methoxy-3-formyl indole;
  • the 4-methylquinoline salt is selected from N-methyl-4-methyl Quinoline iodide salt.
  • the red organic precipitate is filtered, washed with a small amount of dichloromethane, and dried to obtain a dark red powder, which is the light-sensitive ribonucleic acid fluorescent probe.
  • the light-sensitive ribonucleic acid fluorescent probe is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt.
  • the light-sensitive ribonucleic acid fluorescent probe provided by the present invention can be used as a fluorescent probe to label the distribution of ribonucleic acid in living cells and related life activities after light irradiation, and can be related to cell nucleolus and ribonucleic acid caused by light radiation.
  • Physiology and pathology research provides simple and intuitive biological detection reagents, and can also be developed for light irradiation monitoring of live biological samples protected from light. It has a wide range of applications and better effects.
  • HeLa cells were cultured adherently in a culture medium containing 10% fetal bovine serum, cultured in a 37°C, 5% CO 2 saturated humidity incubator, and passaged every 2 to 3 days.
  • splicing culture Wash the overgrown cells in the 100mL cell bottle with PBS three times, digest with 1mL 0.25% trypsin for 1 minute, remove the trypsin, add fresh medium, pipet evenly, and count the cells , Control the cell density with the amount of medium added to make the final cell concentration 1x10 5 , then inoculate it into a petri dish with a sterile cover glass, and place it in a 5% CO 2 incubator to grow the cells. After the cell climbing sheet grows and covers the cover glass, it is used in the experiment.
  • Example 2 The slides prepared in Example 2 that were overgrown with HeLa cells were washed three times with PBS, and then used to dilute the culture solution with (E)-4-(2-(5-methoxy-1H-indole) at a concentration of 10 ⁇ M.
  • Dole-3-)vinyl)-1-methylquinoline iodide fluorescent probe solution was placed in a CO 2 incubator, and the cells were stained for 10-120 minutes in the dark, and then irradiated with a mercury lamp for 0-5 minutes. Including the following three groups of tests:
  • the results of test one are shown in Figure 1, the results of test two are shown in Figure 2, and the results of test three are shown in Figure 3.
  • the fluorescence images of Figure 1, Figure 2 and Figure 3 show that living cells without light irradiation have weak red fluorescence in the cytoplasmic area, but after light irradiation, there is a strong red fluorescence distribution in the cytoplasm and nucleolus area. It is suggested that the probe of the present invention is sensitive to light induction and can specifically image cytoplasm and nucleolus in living cells after light irradiation.
  • Figure 1 shows the confocal fluorescence micrographs under 488nm laser excitation before and after dyeing in Experiment 1.
  • Fig. 1A, Fig. 1B, and Fig. 1C are the micrographs of hatching in the dark (without light irradiation), and
  • Fig. 1D, Fig. 1E, and Fig. 1F are the micrographs of mercury lamp irradiated for 5 minutes.
  • Fig. 1A and Fig. 1D are fluorescence micrographs;
  • Fig. 1B and Fig. 1E are bright-field differential interference micrographs;
  • Fig. 1C and Fig. 1F are the merged pictures of the two images in the left middle (co-localization picture). Living cells that are not irradiated with light have weak red fluorescence only in the cytoplasmic area, and after light irradiation, there is a strong red fluorescence distribution in the cytoplasm and nucleolus area.
  • Figure 2 is experiment two (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide to stain HeLa living cells in the dark Fluorescence micrographs after different time (10-120min).
  • Fig. 2A is a fluorescence micrograph of dyeing for 10 min;
  • Fig. 2B is a fluorescence micrograph of dyeing for 30 min;
  • Fig. 2C is a fluorescence micrograph of dyeing for 60 min;
  • Fig. 2D is a fluorescence micrograph of dyeing for 120 min.
  • the picture shows that live cells stained in the dark, as the staining time increases, only weak red fluorescence in the cytoplasmic area.
  • Figure 3 is experiment three (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt staining HeLa living cells in the dark Fluorescence micrographs after irradiating with mercury lamp green light 510-560nm for different time
  • Figure 3A is a fluorescence micrograph of mercury lamp irradiated with 510-560nm green light for 10s
  • Figure 3B is a fluorescence photomicrograph of mercury lamp irradiated with 510-560nm green light for 1 min
  • Figure 3C is a mercury lamp irradiated with 510-560nm green light 2min fluorescence micrograph
  • 3D is a mercury lamp green light 510-560nm irradiated for 3min
  • Fig. 3E is a mercury lamp green light 510-560nm irradiated 4min fluorescence photomicrograph
  • Fig. 3F is a mercury lamp Fluorescence micrograph of green light 510-560nm irradiated for 5 min. The picture shows that with the increase of light irradiation time, the fluorescence gradually increases, and it shifts from only in the cytoplasm to the cytoplasm and the nucleolus area with strong red fluorescence distribution.
  • Preparation of fixed cells First, soak the cover glass (climbing piece) full of HeLa cells prepared in Example 2 in 4% paraformaldehyde solution for 30 minutes, and then permeate with 0.5% Triton X-100 at room temperature for 2 minutes, before staining and fixing cell.
  • Figure 4A is a fluorescence micrograph of cells in the control group (untreated group)
  • Figure 4B is a fluorescence micrograph of cells after DNase treatment
  • Figure 4C is a fluorescence micrograph of cells after RNase treatment Fluorescence micrograph. It can be seen from Figure 4 that the fluorescence of the DNase-treated cell group under the microscope ( Figure 4B) is similar to that of the control group (untreated group) ( Figure 4A). The fluorescence is still concentrated in the cytoplasm and nucleolus area, and in the nuclear area. The fluorescence of the cells treated with RNase under the microscope ( Figure 4C) was greatly reduced compared with that of the control group (untreated group) ( Figure 4A), and the fluorescence was almost invisible. Since DNase and RNase can respectively digest and hydrolyze deoxyribonucleic acid and ribonucleic acid in cells, it can be confirmed and verified from this perspective that the probe of the present invention can specifically image ribonucleic acid in cells.
  • Fig. 5A is the UV-visible absorption spectrum of the probe molecule itself and mixed with ribonucleic acid and DNA
  • Fig. 5B is the fluorescence emission spectrum of the probe itself and mixed with ribonucleic acid and deoxyribonucleic acid.
  • the HeLa cell slides prepared in Example 2 were washed three times with PBS, and then used to dilute the culture solution with (E)-4-(2-(5-methoxy-1H-indole) at a concentration of 10 ⁇ M. -3-) Vinyl)-1-methylquinoline iodide fluorescent probe solution was stained for 30 minutes in a CO 2 incubator. Then use Hoechst33342 diluted with PBS at a concentration of 2 ⁇ g/mL to stain in a CO 2 incubator for 30 min.
  • Figure 6A is the red fluorescence image of the probe
  • Figure 6B is the blue fluorescence image of Hoechst 33342
  • Figure 6C is the superimposed image of Figure 6A and Figure 6B
  • Figure 6C can clearly see the red fluorescence and blue fluorescence The color and fluorescence do not affect each other, indicating that the fluorescent probe of the present invention does not interfere with each other when co-stained with Hoechst 33342, and has good counterstaining compatibility.

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Abstract

The present invention provides a light-sensitive ribonucleic acid fluorescent probe, and the structure of the light-sensitive ribonucleic acid fluorescent probe is as shown in formula I below. Wherein, said R1 is any one selected from hydrogen, alkyl, alkoxy, and ether groups; said R2 is any one selected from hydrogen, alkyl, alkoxy, and ether groups; said R3 is any one selected from hydrogen, alkyl, alkoxy, and ether groups; and said X is selected from halogen elements.

Description

一种光感应型活细胞核糖核酸荧光探针Light-sensitive living cell ribonucleic acid fluorescent probe 技术领域Technical field
本发明涉及探针领域，尤其涉及一种光感应型核糖核酸荧光探针及应用。The invention relates to the field of probes, in particular to a light-sensitive ribonucleic acid fluorescent probe and its application.
背景技术Background technique
核糖核酸在蛋白质合成过程中起着重要作用，其中核糖体核糖核酸是细胞合成蛋白质的主要场所，信使核糖核酸是合成蛋白质的模板，转移核糖核酸功能是携带和转移活化氨基酸。光辐照对活细胞等生物样品有重要影响，例如某些光响应型细胞中光敏蛋白的表达对其光感应性起着关键作用，开发光响应型核糖核酸探针对于研究光感应细胞生命活动与蛋白表达、对生物样品的光辐照监测具有重要意义。Ribonucleic acid plays an important role in the process of protein synthesis. Ribosomal ribonucleic acid is the main place for cells to synthesize protein, messenger ribonucleic acid is the template for protein synthesis, and the function of transfer ribonucleic acid is to carry and transfer activated amino acids. Light irradiation has important effects on living cells and other biological samples. For example, the expression of light-sensitive proteins in some light-responsive cells plays a key role in their light sensitivity. The development of light-responsive ribonucleic acid probes is useful for studying the life activities of light-sensitive cells. And protein expression, the monitoring of light irradiation of biological samples is of great significance.
目前已报道的核糖核酸探针不具有光响应性质，而且大多发绿色荧光。而长波长红色荧光具有更好的细胞和组织穿透性。The ribonucleic acid probes reported so far do not have light-responsive properties, and most of them emit green fluorescence. The long-wavelength red fluorescence has better cell and tissue penetration.
发明内容Summary of the invention
本发明的目的在于提供一种光感应型核糖核酸荧光探针及其制备方法和应用，旨在解决现有技术中的核糖核酸荧光探针不具备光响应性质且发光颜色单一的问题。The purpose of the present invention is to provide a light-sensitive ribonucleic acid fluorescent probe and a preparation method and application thereof, aiming to solve the problem that the ribonucleic acid fluorescent probe in the prior art does not have light-responsive properties and emits a single color.
为实现上述发明目的，本发明采用的技术方案如下：In order to achieve the above-mentioned purpose of the invention, the technical solutions adopted by the present invention are as follows:
一种光感应型核糖核酸荧光探针，所述光感应型核糖核酸荧光探针的结构如下式I所示：A light-sensitive ribonucleic acid fluorescent probe, the structure of the light-sensitive ribonucleic acid fluorescent probe is shown in the following formula I:
Figure PCTCN2020129180-appb-000001
Figure PCTCN2020129180-appb-000001
其中，所述R 1选自氢、烷基、烷氧基、醚基的任意一种；所述R 2选自氢、烷基、烷氧基、醚基的任意一种；所述R 3选自氢、烷基、烷氧基、醚基的任意一种；所述X选自卤元素。 Wherein, said R 1 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 2 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 3 Any one selected from hydrogen, alkyl, alkoxy, and ether groups; the X is selected from halogen elements.
以及，一种光感应型核糖核酸荧光探针的应用，所述光感应型核糖核酸荧光探针在鉴别活细胞是否经过光辐照中的应用，和在成像经过光辐照后活细胞中核糖核酸分布的应用。And, an application of a light-sensitive ribonucleic acid fluorescent probe, the application of the light-sensitive ribonucleic acid fluorescent probe in identifying whether living cells have been irradiated with light, and the imaging of ribose in living cells after light irradiation Application of nucleic acid distribution.
本发明提供的吲哚喹啉盐类荧光探针是一类新型的细胞中核糖核酸专一性识别荧光探针分子，与其功能相近的光感应型核糖核酸荧光探针比，本发明所述探针的独特之处在于具有良好的感光性，在光辐照后与细胞中的核糖核酸特异性结合，而且与核糖核酸结合后发射与商业核糖核酸探针RNA-select绿色荧光不同的红色荧光；同时光稳定性较强、膜通透性好、复染兼容性好。The indolequinoline salt fluorescent probe provided by the present invention is a new type of ribonucleic acid specific recognition fluorescent probe molecule in the cell, compared with the light-sensitive ribonucleic acid fluorescent probe with similar function, the probe of the present invention The uniqueness of the needle is that it has good photosensitivity, specifically binds to ribonucleic acid in cells after light irradiation, and emits red fluorescence that is different from the commercial ribonucleic acid probe RNA-select green fluorescence after binding to ribonucleic acid; At the same time, the light stability is strong, the membrane permeability is good, and the counterstaining compatibility is good.
本发明所提供的光感应型核糖核酸荧光探针，可作为荧光探针进行标记光辐照后的活细胞中核糖核酸的分布和相关生命活动的应用，能够为光辐照导致的细胞核仁和核糖核酸相关的生理和病理学研究提供简捷、直观的生物检测试剂，也可开发应用于避光活生物样本的光辐照监控，应用广泛，作用效果较佳。The light-sensitive ribonucleic acid fluorescent probe provided by the present invention can be used as a fluorescent probe to label the distribution of ribonucleic acid in living cells after light irradiation and the application of related life activities. Nucleic acid-related physiological and pathological research provides simple and intuitive biological detection reagents, and can also be developed for light irradiation monitoring of live biological samples protected from light. It has a wide range of applications and better effects.
附图说明Description of the drawings
图1是(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa活细胞染色后光辐照前和辐照后在488nm激光激发下的共聚焦荧光显微照片，图1A、图1B、图1C为黑暗中孵化(未经过光辐照)的显微图片，图1D、图1E、图1F为汞灯辐照5min后的显微图片。左图(AD)为荧光显微照片；中图(BE) 为明场微分干涉显微照片；右图(CF)为左中两图的合并图(共定位图)。Figure 1 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide staining HeLa living cells before light irradiation and Confocal fluorescence micrographs under 488nm laser excitation after irradiation. Figure 1A, Figure 1B, and Figure 1C are micrographs of incubation in the dark (without light irradiation). Figure 1D, Figure 1E, and Figure 1F are mercury The photomicrograph after the lamp is irradiated for 5 minutes. The left image (AD) is a fluorescence micrograph; the middle image (BE) is a bright-field differential interference micrograph; the right image (CF) is a merged image of the two images on the left (co-localization image).
图2是(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa活细胞在黑暗中进行染色不同时间(10-120min)后的荧光显微照片，图2A为染色10min的荧光显微照片；图2B为染色30min的荧光显微照片；图2C为染色60min的荧光显微照片；图2D为染色120min的荧光显微照片。Figure 2 shows the staining of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide on living HeLa cells in the dark for different times (10-120min) fluorescence micrograph, Figure 2A is the fluorescence micrograph of staining for 10min; Figure 2B is the fluorescence micrograph of staining for 30min; Figure 2C is the fluorescence micrograph of staining for 60min; Figure 2D is the fluorescence micrograph of staining for 120min Fluorescence micrograph.
图3是(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa活细胞在黑暗中进行染色后用汞灯绿光510-560nm辐照不同时间后的荧光显微照片，图3A为汞灯绿光510-560nm辐照10s的荧光显微照片；图3B为汞灯绿光510-560nm辐照1min的荧光显微照片；图3C为汞灯绿光510-560nm辐照2min的荧光显微照片；图3D为汞灯绿光510-560nm辐照3min的荧光显微照片；图3E为汞灯绿光510-560nm辐照4min的荧光显微照片；图3F为汞灯绿光510-560nm辐照5min的荧光显微照片。Figure 3 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide salt after staining HeLa living cells in the dark Fluorescence micrographs of mercury lamp green light 510-560nm irradiated for different time, Figure 3A is the fluorescence micrograph of mercury lamp green light 510-560nm irradiated for 10s; Figure 3B is mercury lamp green light 510-560nm irradiated for 1 min Figure 3C is a fluorescent photomicrograph of mercury lamp green light 510-560nm irradiated for 2min; Figure 3D is a mercury lamp green light 510-560nm irradiated for 3min fluorescence photomicrograph; Figure 3E is mercury lamp green Fluorescence micrographs of 510-560nm irradiated for 4 minutes; Figure 3F is a fluorescence microscopy of mercury lamp green light 510-560nm irradiated for 5 minutes.
图4是对用多聚甲醛溶液固定的HeLa细胞分别经脱氧核糖核酸酶(DNase)和核糖核酸酶(RNase)处理后，用(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa细胞进行染色，在510-560nm汞灯激发下得到的荧光显微照片，图4A为对照组(未经处理组)的细胞的荧光显微照片，图4B为经DNase处理后的细胞的荧光显微照片，图4C为经RNase处理后的细胞的荧光显微照片。Figure 4 shows the HeLa cells fixed with paraformaldehyde solution after being treated with deoxyribonuclease (DNase) and ribonuclease (RNase), and then treated with (E)-4-(2-(5-methoxy-1H) -Indole-3-)vinyl)-1-methylquinoline iodide was used to stain HeLa cells. Fluorescence micrographs obtained under 510-560nm mercury lamp excitation. Figure 4A is the control group (untreated group). ) The fluorescence micrograph of the cells, Fig. 4B is the fluorescence micrograph of the cells after DNase treatment, and Fig. 4C is the fluorescence micrograph of the cells after RNase treatment.
图5是(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐与核糖核酸(RNA)、脱氧核糖核酸(DNA)作用前后的吸收谱和荧光谱，图5A为探针分子本身和与核糖核酸、脱氧核糖核酸分别混合后的紫外可见吸收光谱；图5B为探针本身和与核糖核酸、脱氧核糖核酸分别混合后的荧光发射光谱。Figure 5 is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt and ribonucleic acid (RNA), deoxyribonucleic acid ( The absorption and fluorescence spectra before and after DNA). Figure 5A shows the UV-visible absorption spectra of the probe molecule itself and after mixing with ribonucleic acid and deoxyribonucleic acid; Figure 5B shows the probe itself and ribonucleic acid and deoxyribonucleic acid respectively. Fluorescence emission spectrum after mixing.
图6是(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐与细胞核探针Hoechst33342对HeLa细胞进行共染色后在汞灯辐照下得到的荧光显微照片，图6A为所述探针的红色荧光显微照片；图6B为Hoechst33342的蓝色荧光显微照片；图6C为图6A和图6B的叠加图。Figure 6 is the co-staining of HeLa cells with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide and the nuclear probe Hoechst33342 Later, the fluorescence micrographs obtained under mercury lamp irradiation, Figure 6A is the red fluorescence micrograph of the probe; Figure 6B is the blue fluorescence micrograph of Hoechst 33342; Figure 6C is the superimposition of Figure 6A and Figure 6B Figure.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和技术效果更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。结合本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the objectives, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not All examples. In combination with the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
在本发明的描述中，需要理解的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。In the description of the present invention, it should be understood that the terms "first" and "second" are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more than two, unless otherwise specifically defined.
本发明实例提供一种光感应型核糖核酸荧光探针，所述光感应型核糖核酸荧光探针的结构如下式I所示：An example of the present invention provides a light-sensitive ribonucleic acid fluorescent probe. The structure of the light-sensitive ribonucleic acid fluorescent probe is shown in the following formula I:
Figure PCTCN2020129180-appb-000002
Figure PCTCN2020129180-appb-000002
其中，所述R 1选自氢、烷基、烷氧基、醚基的任意一种；所述R 2选自氢、烷基、烷氧基、醚基的任意一种；所述R 3选自氢、烷基、烷氧基、醚基的任意一种；所述X选自卤素原子。 Wherein, said R 1 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 2 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 3 Any one selected from hydrogen, alkyl, alkoxy, and ether groups; the X is selected from halogen atoms.
本发明提供的吲哚喹啉盐类荧光探针是一类新型的细胞中核糖核酸专一性识别光感应型荧光探针分子，与其功能相近的核糖核酸荧光探针比，本发明所述探针的独特之处在于具有良好的感光性，在光辐照下探针从细胞质线粒体中释放出来，与细胞中的核糖核酸特异性结合，而且与核糖核酸结合后发射与商 业核糖核酸探针RNA-select绿色荧光不同的红色荧光；同时光稳定性较强、膜通透性好、复染兼容性好。The indolequinoline salt fluorescent probe provided by the present invention is a new type of ribonucleic acid specific recognition light-sensitive fluorescent probe molecule in the cell. Compared with the ribonucleic acid fluorescent probe with similar function, the probe of the present invention The uniqueness of the needle is that it has good photosensitivity. The probe is released from the cytoplasmic mitochondria under light irradiation, and specifically binds to the ribonucleic acid in the cell, and after binding to the ribonucleic acid, it emits RNA as a commercial ribonucleic acid probe. -Select red fluorescence with different green fluorescence; at the same time, it has strong light stability, good membrane permeability and good counterstaining compatibility.
上述结构式I中，优选的，所述R 1选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述R 2选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述R 3选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述X选自碘、溴、氯。 In the above structural formula I, preferably, said R 1 is selected from any one of hydrogen, C1-C5 alkyl, C1-C5 alkoxy, and aliphatic ether group; said R 2 is selected from hydrogen, C1-C5 Any one of the alkyl group, C1-C5 alkoxy group, and fatty ether group; the R 3 is selected from any one of hydrogen, C1-C5 alkyl group, C1-C5 alkoxy group, and fatty ether group ; The X is selected from iodine, bromine, and chlorine.
进一步优选的，所述R 1中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 Further preferably, in the R 1 , the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl; The C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; in the fatty ether group, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
进一步优选的，所述R 2中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 Further preferably, in the R 2 , the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl; The C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; in the fatty ether group, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
进一步优选的，所述R 3中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 Further preferably, in the R 3 , the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl; The C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; in the fatty ether group, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
优选的，所述R 1选自氢；所述R 2选自甲氧基；所述R 3选自甲基；所述X选自碘。在本发明优选实施例中，当所述R 1选自氢；所述R 2选自甲氧基；所述R 3选自甲基；所述X选自碘时，得到的所述光感应型核糖核酸荧光探针为(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐。 Preferably, said R 1 is selected from hydrogen; said R 2 is selected from methoxy; said R 3 is selected from methyl; and said X is selected from iodine. In a preferred embodiment of the present invention, when said R 1 is selected from hydrogen; said R 2 is selected from methoxy; said R 3 is selected from methyl; and said X is selected from iodine, the resulting light sensor The type ribonucleic acid fluorescent probe is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt.
本发明所提供的光感应型核糖核酸荧光探针由以下光感应型核糖核酸荧光探针的制备方法制备得到。The light-sensitive ribonucleic acid fluorescent probe provided by the present invention is prepared by the following preparation method of light-sensitive ribonucleic acid fluorescent probe.
所述光感应型核糖核酸荧光探针的制备方法如下：吲哚-3-甲醛盐与4-甲基喹啉盐按照摩尔比1：(1.0～2)混合溶解于甲醇/乙醇中，得淡黄色透明溶液， 滴加少量哌啶，加热回流反应6-12h，产生深红色沉淀，冷却至室温，过滤真空干燥得到所述光感应型核糖核酸成像的荧光探针。The preparation method of the light-sensitive ribonucleic acid fluorescent probe is as follows: Indole-3-formaldehyde salt and 4-methylquinoline salt are mixed and dissolved in methanol/ethanol at a molar ratio of 1:(1.0～2) to obtain light. Yellow transparent solution, drop a small amount of piperidine, heat and reflux for 6-12 hours to produce a deep red precipitate, cool to room temperature, filter and vacuum dry to obtain the light-sensitive ribonucleic acid imaging fluorescent probe.
Figure PCTCN2020129180-appb-000003
Figure PCTCN2020129180-appb-000003
在本发明优选实施例中，所述吲哚-3-甲醛选自5-甲氧基-3-甲酰基吲哚；所述4-甲基喹啉盐选自N-甲基-4-甲基喹啉碘盐。以所述5-甲氧基-3-甲酰基吲哚、所述N-甲基-4-甲基喹啉碘盐作为反应物，所述光感应型核糖核酸荧光探针的制备方法如下：In a preferred embodiment of the present invention, the indole-3-carbaldehyde is selected from 5-methoxy-3-formyl indole; the 4-methylquinoline salt is selected from N-methyl-4-methyl Quinoline iodide salt. Using the 5-methoxy-3-formylindole and the N-methyl-4-methylquinoline iodonium salt as reactants, the preparation method of the light-sensitive ribonucleic acid fluorescent probe is as follows:
配置5-甲氧基-2-甲酰基吲哚与N-甲基-4-甲基吡啶的乙醇溶液；Prepare the ethanol solution of 5-methoxy-2-formylindole and N-methyl-4-methylpyridine;
在所述乙醇混合溶液中加入催化剂哌啶，将所述加了哌啶的有机混合物进行85℃加热回流反应12小时，冷却至室温，得到红色有机沉淀物；Adding catalyst piperidine to the ethanol mixed solution, and subjecting the piperidine-added organic mixture to 85° C. heating and refluxing reaction for 12 hours, and cooling to room temperature to obtain a red organic precipitate;
将所述红色有机沉淀物进行过滤、用少量二氯甲烷进行洗涤，干燥得到深红色的粉末，所述深红色粉末为所述光感应型核糖核酸荧光探针。所述光感应型核糖核酸荧光探针为(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐。The red organic precipitate is filtered, washed with a small amount of dichloromethane, and dried to obtain a dark red powder, which is the light-sensitive ribonucleic acid fluorescent probe. The light-sensitive ribonucleic acid fluorescent probe is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt.
本发明所提供的光感应型核糖核酸荧光探针，可作为荧光探针进行标记光辐照后活细胞中核糖核酸的分布和相关生命活动的应用，能够为光辐射导致的细胞核仁和核糖核酸相关的生理和病理学研究提供简捷、直观的生物检测试剂，也可开发应用于避光活生物样本的光辐照监控，应用广泛，作用效果较佳。The light-sensitive ribonucleic acid fluorescent probe provided by the present invention can be used as a fluorescent probe to label the distribution of ribonucleic acid in living cells and related life activities after light irradiation, and can be related to cell nucleolus and ribonucleic acid caused by light radiation. Physiology and pathology research provides simple and intuitive biological detection reagents, and can also be developed for light irradiation monitoring of live biological samples protected from light. It has a wide range of applications and better effects.
下面以具体实施例进一步进行说明。Specific embodiments are used for further description below.
实施例1Example 1
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐的合成Synthesis of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt
将5-甲氧基-2-甲酰基吲哚与N-甲基-4-甲基吡啶溶于乙醇，得淡黄色透明溶液，加4～5滴哌啶，溶液逐渐变红色。回流反应12h，有红色沉淀析出。冷 却，过滤，少量二氯甲烷洗涤，得深红色粉末，产率41％。Dissolve 5-methoxy-2-formyl indole and N-methyl-4-methylpyridine in ethanol to obtain a light yellow transparent solution. Add 4 to 5 drops of piperidine and the solution gradually turns red. The reaction was refluxed for 12 hours, and a red precipitate was precipitated. After cooling, filtering, and washing with a small amount of dichloromethane, a dark red powder was obtained with a yield of 41%.
1H NMR(400MHz,DMSO-d6),δ(ppm):12.09(s,1H),9.10(d,J＝4.0Hz,1H),8.97(d,J＝8.0Hz,1H),8.61(d,J＝16.0Hz,1H),8.46(d,J＝4.0Hz,1H),8.42(s,1H)8.34(d,J＝8.0Hz,1H),8.23(d,J＝4.0Hz,1H),8.02(t,J＝8.0Hz,2H),7.71(d,J＝4.0Hz,1H),7.43(d,J＝8.0Hz,1H),6.92(dd,J＝4.0,1.7Hz,1H),4.44(s,3H),3.90(s,3H)。 13C NMR(400MHz,DMSO-d6),δ(ppm):155.71,154.06,146.87,139.31,138.81,135.01,132.45,128.92,127.25,126.62,125.82,119.48,114.98,113.79,112.96,112.79,102.70,56.08,44.30。 1 H NMR (400MHz, DMSO-d6), δ (ppm): 12.09 (s, 1H), 9.10 (d, J = 4.0 Hz, 1H), 8.97 (d, J = 8.0 Hz, 1H), 8.61 (d ,J=16.0Hz,1H),8.46(d,J=4.0Hz,1H),8.42(s,1H)8.34(d,J=8.0Hz,1H),8.23(d,J=4.0Hz,1H) ,8.02(t,J=8.0Hz,2H),7.71(d,J=4.0Hz,1H),7.43(d,J=8.0Hz,1H),6.92(dd,J=4.0,1.7Hz,1H) , 4.44 (s, 3H), 3.90 (s, 3H). 13 C NMR (400MHz, DMSO-d6), δ (ppm): 155.71,154.06,146.87,139.31,138.81,135.01,132.45,128.92,127.25,126.62,125.82,119.48,114.98,113.79,112.96,112.79,102.70, 56.08,44.30.
实施例2Example 2
HeLa细胞培养HeLa cell culture
将HeLa细胞贴壁培养于内含10％胎牛血清培养液中，在37℃，5％CO 2的饱和湿度孵箱中培养，每2～3天传代1次。 HeLa cells were cultured adherently in a culture medium containing 10% fetal bovine serum, cultured in a 37°C, 5% CO 2 saturated humidity incubator, and passaged every 2 to 3 days.
待细胞生长到对数期，接片培养：将100mL细胞瓶中长满的细胞用PBS洗三遍，用1mL 0.25％胰酶消化1分钟，去除胰酶，加入新鲜培养基吹打均匀并细胞计数，以培养基的添加量控制细胞密度，使细胞终浓度为1x10 5，然后接种至内无菌盖玻片的培养皿中，放入5％CO 2培养箱中培养，使细胞爬片生长。待细胞爬片生长并长满盖玻片后，用于实验。 After the cells have grown to the logarithmic phase, splicing culture: Wash the overgrown cells in the 100mL cell bottle with PBS three times, digest with 1mL 0.25% trypsin for 1 minute, remove the trypsin, add fresh medium, pipet evenly, and count the cells , Control the cell density with the amount of medium added to make the final cell concentration 1x10 5 , then inoculate it into a petri dish with a sterile cover glass, and place it in a 5% CO 2 incubator to grow the cells. After the cell climbing sheet grows and covers the cover glass, it is used in the experiment.
实施例3Example 3
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa细胞的染色观察Staining observation of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide on HeLa cells
将实施例2制备的分别长满HeLa细胞的爬片用PBS洗三遍，然后用以培养液稀释的浓度为10μM的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐荧光探针溶液在CO 2培养箱中，细胞分别避光染色10-120min，然后用汞灯分别辐照0-5min。包括以下三组试验： The slides prepared in Example 2 that were overgrown with HeLa cells were washed three times with PBS, and then used to dilute the culture solution with (E)-4-(2-(5-methoxy-1H-indole) at a concentration of 10 μM. Dole-3-)vinyl)-1-methylquinoline iodide fluorescent probe solution was placed in a CO 2 incubator, and the cells were stained for 10-120 minutes in the dark, and then irradiated with a mercury lamp for 0-5 minutes. Including the following three groups of tests:
试验一：Test 1:
(1)将细胞避光染色30分钟，再用汞灯白光进行光辐照5分钟；(1) Stain the cells in the dark for 30 minutes, and then irradiate the cells with white light of a mercury lamp for 5 minutes;
(2)将细胞避光染色30分钟，不进行光辐照。(2) The cells were stained for 30 minutes in the dark without light irradiation.
试验二：Test two:
(1)将细胞避光染色10分钟。(1) Stain the cells in the dark for 10 minutes.
(2)将细胞避光染色30分钟。(2) Stain the cells in the dark for 30 minutes.
(3)将细胞避光染色60分钟。(3) Stain the cells in the dark for 60 minutes.
(4)将细胞避光染色120分钟。(4) Stain the cells in the dark for 120 minutes.
试验三：Test three:
(1)将细胞染色30分钟，再用汞灯510-560nm进行光辐照10秒。(1) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 10 seconds.
(2)将细胞染色30分钟，再用汞灯510-560nm进行光辐照1分钟。(2) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 1 minute.
(3)将细胞染色30分钟，再用汞灯510-560nm进行光辐照2分钟。(3) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 2 minutes.
(4)将细胞染色30分钟，再用汞灯510-560nm进行光辐照3分钟。(4) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 3 minutes.
(5)将细胞染色30分钟，再用汞灯510-560nm进行光辐照4分钟。(5) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 4 minutes.
(6)将细胞染色30分钟，再用汞灯510-560nm进行光辐照5分钟。(6) Stain the cells for 30 minutes, and then irradiate the cells with light at 510-560nm for 5 minutes.
将染色后的爬片在荧光显微镜和激光扫描共聚焦镜下观察，记录细胞中着色部位，荧光分布及亮度变化等。Observe the stained slide under a fluorescence microscope and a laser scanning confocal microscope, and record the stained parts in the cells, fluorescence distribution and brightness changes.
试验一结果见图1、试验二结果见图2、试验三结果见图3。图1、图2和图3荧光图像显示不经过光辐照的活细胞，细胞质区域有较弱的红色荧光，而经过光辐照之后，在细胞质和核仁区有强的红色荧光分布，明确提示本发明的探针对光感应敏感，能够在光辐照后的活细胞中专一性的成像细胞质和核仁。The results of test one are shown in Figure 1, the results of test two are shown in Figure 2, and the results of test three are shown in Figure 3. The fluorescence images of Figure 1, Figure 2 and Figure 3 show that living cells without light irradiation have weak red fluorescence in the cytoplasmic area, but after light irradiation, there is a strong red fluorescence distribution in the cytoplasm and nucleolus area. It is suggested that the probe of the present invention is sensitive to light induction and can specifically image cytoplasm and nucleolus in living cells after light irradiation.
图1为试验一染色后光辐照前和辐照后在488nm激光激发下的共聚焦荧光显微照片。图1A、图1B、图1C为黑暗中孵化(未经过光辐照)的显微图片，图1D、图1E、图1F为汞灯辐照5min后的显微图片。图1A、图1D为荧光显微照片；图1B、图1E为明场微分干涉显微照片；图1C、图1F为左中两图的合并图(共定位图)。不经过光辐照的活细胞，仅在细胞质区域有较弱的红色荧光，而经过光辐照之后，在细胞质和核仁区有强的红色荧光分布，Figure 1 shows the confocal fluorescence micrographs under 488nm laser excitation before and after dyeing in Experiment 1. Fig. 1A, Fig. 1B, and Fig. 1C are the micrographs of hatching in the dark (without light irradiation), and Fig. 1D, Fig. 1E, and Fig. 1F are the micrographs of mercury lamp irradiated for 5 minutes. Fig. 1A and Fig. 1D are fluorescence micrographs; Fig. 1B and Fig. 1E are bright-field differential interference micrographs; Fig. 1C and Fig. 1F are the merged pictures of the two images in the left middle (co-localization picture). Living cells that are not irradiated with light have weak red fluorescence only in the cytoplasmic area, and after light irradiation, there is a strong red fluorescence distribution in the cytoplasm and nucleolus area.
图2为试验二(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa活细胞在黑暗中进行染色不同时间(10-120min)后的荧光显微照片。图2A为染色10min的荧光显微照片；图2B为染色30min的荧光显微照片；图2C为染色60min的荧光显微照片；图2D为染色120min的荧光显微照片。图片显示，黑暗中染色的活细胞，随着染色时间加长，仅仅在细胞质区域有较弱的红色荧光。Figure 2 is experiment two (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide to stain HeLa living cells in the dark Fluorescence micrographs after different time (10-120min). Fig. 2A is a fluorescence micrograph of dyeing for 10 min; Fig. 2B is a fluorescence micrograph of dyeing for 30 min; Fig. 2C is a fluorescence micrograph of dyeing for 60 min; Fig. 2D is a fluorescence micrograph of dyeing for 120 min. The picture shows that live cells stained in the dark, as the staining time increases, only weak red fluorescence in the cytoplasmic area.
图3为试验三(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐对HeLa活细胞在黑暗中进行染色后用汞灯绿光510-560nm辐照不同时间后的荧光显微照片。图3A为汞灯绿光510-560nm辐照10s的荧光显微照片；图3B为汞灯绿光510-560nm辐照1min的荧光显微照片；图3C为汞灯绿光510-560nm辐照2min的荧光显微照片；图3D为汞灯绿光510-560nm辐照3min的荧光显微照片；图3E为汞灯绿光510-560nm辐照4min的荧光显微照片；图3F为汞灯绿光510-560nm辐照5min的荧光显微照片。图片显示，随着光辐照时间的增加，荧光逐渐增强，而且从仅仅在细胞质中转移到细胞质和核仁区都有强的红色荧光分布。Figure 3 is experiment three (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodonium salt staining HeLa living cells in the dark Fluorescence micrographs after irradiating with mercury lamp green light 510-560nm for different time Figure 3A is a fluorescence micrograph of mercury lamp irradiated with 510-560nm green light for 10s; Figure 3B is a fluorescence photomicrograph of mercury lamp irradiated with 510-560nm green light for 1 min; Figure 3C is a mercury lamp irradiated with 510-560nm green light 2min fluorescence micrograph; Fig. 3D is a mercury lamp green light 510-560nm irradiated for 3min; Fig. 3E is a mercury lamp green light 510-560nm irradiated 4min fluorescence photomicrograph; Fig. 3F is a mercury lamp Fluorescence micrograph of green light 510-560nm irradiated for 5 min. The picture shows that with the increase of light irradiation time, the fluorescence gradually increases, and it shifts from only in the cytoplasm to the cytoplasm and the nucleolus area with strong red fluorescence distribution.
实施例4Example 4
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐荧光探针对HeLa细胞、和经DNase和RNase处理后的HeLa细胞(DNase和RNase消化实验)的染色观察。(E)-4-(2-(5-Methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide fluorescent probe on HeLa cells and after treatment with DNase and RNase Observation of staining of HeLa cells (DNase and RNase digestion experiments).
固定细胞制备：首先将实施例2制备的长满HeLa细胞的盖玻片(爬片)浸泡于4％多聚甲醛溶液30min，然后用0.5％Triton X-100室温通透2min，得待染色固定细胞。Preparation of fixed cells: First, soak the cover glass (climbing piece) full of HeLa cells prepared in Example 2 in 4% paraformaldehyde solution for 30 minutes, and then permeate with 0.5% Triton X-100 at room temperature for 2 minutes, before staining and fixing cell.
取上述三组固定细胞，其中一组中加入2U/mL不含RNase的DNase，一组加入50μg/mL不含DNase的RNase在培养箱中消化2h；然后对三组固定细胞用PBS清洗3遍，并同时都用浓度为10μM的(E)-4-(2-(5-甲氧基-1H-吲哚 -3-)乙烯基)-1-甲基喹啉碘盐荧光探针溶液在CO 2培养箱中孵化染色30min。 Take the above three groups of fixed cells, add 2U/mL DNase without RNase to one group, and add 50μg/mL RNase without DNase to digest for 2h in the incubator; then wash the three groups of fixed cells with PBS 3 times , And at the same time use (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide fluorescent probe solution with a concentration of 10μM in Incubate the staining in a CO 2 incubator for 30 min.
将染色后的爬片在宽场荧光显微镜下观察，记录细胞中着色部位，荧光分布及亮度变化等。Observe the stained slide under a wide-field fluorescence microscope to record the stained parts in the cells, fluorescence distribution and brightness changes.
结果如图4所示，图4A为对照组(未经处理组)的细胞的荧光显微照片，图4B为经DNase处理后的细胞的荧光显微照片，图4C为经RNase处理后的细胞的荧光显微照片。由图4中可发现，经DNase处理后的细胞组显微镜下的荧光(图4B)较对照组(未经处理组)(图4A)荧光类似，仍然集中在细胞质和核仁区域，在核区基本看不到荧光；经RNase处理后的细胞在显微镜下的荧光(图4C)较对照组(未经处理组)(图4A)荧光大大减弱，基本看不到荧光。由于DNase和RNase分别可以消化水解掉细胞中的脱氧核糖核酸和核糖核酸，因此，从此角度可证实并验证本发明的探针能够专一性成像细胞中的核糖核酸。The results are shown in Figure 4. Figure 4A is a fluorescence micrograph of cells in the control group (untreated group), Figure 4B is a fluorescence micrograph of cells after DNase treatment, and Figure 4C is a fluorescence micrograph of cells after RNase treatment Fluorescence micrograph. It can be seen from Figure 4 that the fluorescence of the DNase-treated cell group under the microscope (Figure 4B) is similar to that of the control group (untreated group) (Figure 4A). The fluorescence is still concentrated in the cytoplasm and nucleolus area, and in the nuclear area. The fluorescence of the cells treated with RNase under the microscope (Figure 4C) was greatly reduced compared with that of the control group (untreated group) (Figure 4A), and the fluorescence was almost invisible. Since DNase and RNase can respectively digest and hydrolyze deoxyribonucleic acid and ribonucleic acid in cells, it can be confirmed and verified from this perspective that the probe of the present invention can specifically image ribonucleic acid in cells.
实施例5Example 5
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐在溶液中对核糖核酸、脱氧核糖核酸的识别作用(E)-4-(2-(5-Methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide in solution for the recognition of ribonucleic acid and deoxyribonucleic acid
将10μM探针与120μg/mL的核糖核酸/脱氧核糖核酸的PBS溶液分别混合均匀，作用5分钟，测定紫外可见吸收光谱和488nm激发下荧光光谱。结果见图5，图5A为探针分子本身和与核糖核酸、DNA分别混合后的紫外可见吸收光谱；图5B为探针本身和与核糖核酸、脱氧核糖核酸分别混合后的荧光发射光谱，可发现，与核糖核酸作用后的荧光发射峰在600nm附近，与探针本身相比，荧光强度增加了～40倍，而且明显强于与脱氧核糖核酸结合后的荧光。The 10μM probe and the 120μg/mL ribonucleic acid/deoxyribonucleic acid PBS solution were mixed uniformly and reacted for 5 minutes to measure the ultraviolet-visible absorption spectrum and the fluorescence spectrum under 488nm excitation. The results are shown in Fig. 5. Fig. 5A is the UV-visible absorption spectrum of the probe molecule itself and mixed with ribonucleic acid and DNA; Fig. 5B is the fluorescence emission spectrum of the probe itself and mixed with ribonucleic acid and deoxyribonucleic acid. It was found that the fluorescence emission peak after interaction with ribonucleic acid was around 600nm, compared with the probe itself, the fluorescence intensity increased by ~40 times, and it was significantly stronger than the fluorescence after combining with deoxyribonucleic acid.
实施例6Example 6
(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐与Hoechst33342对HeLa细胞的共染色观察Co-staining observation of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide and Hoechst33342 on HeLa cells
将实施例2制备的分别长满HeLa细胞爬片用PBS洗三遍，然后用以培养液稀释的浓度为10μM的(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐荧光探针溶液在CO 2培养箱中，细胞染色30min。然后再用以PBS稀释的浓度为2μg/mL的Hoechst33342在CO 2培养箱中染色30min。 The HeLa cell slides prepared in Example 2 were washed three times with PBS, and then used to dilute the culture solution with (E)-4-(2-(5-methoxy-1H-indole) at a concentration of 10 μM. -3-) Vinyl)-1-methylquinoline iodide fluorescent probe solution was stained for 30 minutes in a CO 2 incubator. Then use Hoechst33342 diluted with PBS at a concentration of 2μg/mL to stain in a CO 2 incubator for 30 min.
将染色后的爬片洗去未结合的多余染液，汞灯白光下辐照5min后，在荧光显微镜下观察，记录细胞中着色部位，荧光分布及亮度变化等。Wash the stained slide to remove the unbound excess dye solution, irradiate it under the white light of a mercury lamp for 5 minutes, observe it under a fluorescence microscope, record the stained parts in the cells, fluorescence distribution and brightness changes.
结果见图6，图6A为所述探针的红色荧光图；图6B为Hoechst33342的蓝色荧光图；图6C为图6A和图6B的叠加图，图6C可以明显的看出红色荧光和蓝色荧光互不影响，说明本发明所述荧光探针与Hoechst33342共染色时互不干扰，具有良好的复染兼容性。The results are shown in Figure 6, Figure 6A is the red fluorescence image of the probe; Figure 6B is the blue fluorescence image of Hoechst 33342; Figure 6C is the superimposed image of Figure 6A and Figure 6B, Figure 6C can clearly see the red fluorescence and blue fluorescence The color and fluorescence do not affect each other, indicating that the fluorescent probe of the present invention does not interfere with each other when co-stained with Hoechst 33342, and has good counterstaining compatibility.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection of the present invention. Within range.

Claims (7)

  1. 一种光感应型核糖核酸荧光探针，其特征在于，所述光感应型核糖核酸荧光探针的结构如下式I所示：A light-sensitive ribonucleic acid fluorescent probe is characterized in that the structure of the light-sensitive ribonucleic acid fluorescent probe is shown in the following formula I:
    Figure PCTCN2020129180-appb-100001
    Figure PCTCN2020129180-appb-100001
    其中，所述R 1选自氢、烷基、烷氧基、醚基的任意一种；所述R 2选自氢、烷基、烷氧基、醚基的任意一种；所述R 3选自氢、烷基、烷氧基、醚基的任意一种；所述X选自卤元素。 Wherein, said R 1 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 2 is selected from any one of hydrogen, alkyl, alkoxy, and ether groups; said R 3 Any one selected from hydrogen, alkyl, alkoxy, and ether groups; the X is selected from halogen elements.
  2. 根据权利要求1所述的光感应型核糖核酸荧光探针，其特征在于，所述R 1选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述R 2选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述R 3选自氢、C1-C5的烷基、C1-C5的烷氧基、脂肪醚基的任意一种；所述X选自碘、溴、氯。 The light-sensitive ribonucleic acid fluorescent probe according to claim 1, wherein the R 1 is selected from any one of hydrogen, C1-C5 alkyl, C1-C5 alkoxy, and fatty ether group The R 2 is selected from any one of hydrogen, C1-C5 alkyl, C1-C5 alkoxy, and aliphatic ether group; the R 3 is selected from hydrogen, C1-C5 alkyl, C1-C5 Any one of the alkoxy group and aliphatic ether group; the X is selected from iodine, bromine, and chlorine.
  3. 根据权利要求2所述的光感应型核糖核酸荧光探针，其特征在于，所述R 1中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 The light-responsive RNA fluorescent probe according to claim 2, wherein said R 1 is a C1-C5 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n Butyl, isobutyl, sec-butyl, tert-butyl; the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; the fatty ether group Among them, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  4. 根据权利要求2所述的光感应型核糖核酸荧光探针，其特征在于，所述R 2中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 The light-responsive RNA fluorescent probe according to claim 2, wherein, in said R 2, said C1-C5 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n Butyl, isobutyl, sec-butyl, tert-butyl; the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; the fatty ether group Among them, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  5. 根据权利要求2所述的光感应型核糖核酸荧光探针，其特征在于，所述R 3中，所述C1-C5的烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基；所述C1-C5的烷氧基包括羟甲基、羟乙基、羟丙基、羟丁基、羟戊基；所述脂肪醚基中，脂肪烃为C1-C5的脂肪烃。 The light-sensitive ribonucleic acid fluorescent probe according to claim 2, wherein in the R 3 , the C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-propyl, Butyl, isobutyl, sec-butyl, tert-butyl; the C1-C5 alkoxy group includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and hydroxypentyl; the fatty ether group Among them, aliphatic hydrocarbons are C1-C5 aliphatic hydrocarbons.
  6. 根据权利要求1～5任一所述的光感应型核糖核酸荧光探针，其特征在于，所述光感应型核糖核酸荧光探针包括(E)-4-(2-(5-甲氧基-1H-吲哚-3-)乙烯基)-1-甲基喹啉碘盐。The light-sensitive ribonucleic acid fluorescent probe according to any one of claims 1 to 5, wherein the light-sensitive ribonucleic acid fluorescent probe comprises (E)-4-(2-(5-methoxy -1H-indole-3-)vinyl)-1-methylquinoline iodonium salt.
  7. 一种如权利要求1-6任一项所述光感应型核糖核酸荧光探针的应用，所述光感应型核糖核酸荧光探针作为荧光探针进行标记光辐照后活细胞中核糖核酸的分布和相关生命活动的应用。An application of the light-sensitive ribonucleic acid fluorescent probe according to any one of claims 1 to 6, which is used as a fluorescent probe to label the ribonucleic acid in living cells after light irradiation. Distribution and application of related life activities.
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WO2021110094A1 (en) * 2019-12-04 2021-06-10 深圳先进技术研究院 Mitochondrion/ribonucleic-acid-targeting transferable photoactive probe and use thereof
CN112402608B (en) * 2020-11-30 2021-09-07 深圳先进技术研究院 Application of 5-alkoxy indole-3-vinyl quinoline salt as targeted migratable photosensitizer
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050054006A1 (en) * 2002-09-09 2005-03-10 New York University Combinatorial fluorescent library based on the styryl scaffold
CN103265947A (en) * 2013-06-03 2013-08-28 山东大学 Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell
CN106833623A (en) * 2017-02-17 2017-06-13 广东工业大学 A kind of fluorescence probe and preparation method thereof
CN107311977A (en) * 2017-06-27 2017-11-03 广东工业大学 A kind of indoles ethylene compounds and its preparation method and application
CN108309977A (en) * 2018-05-15 2018-07-24 广东工业大学 Application of the indoles ethylene substituted chinoline derivative in preparing drug-resistance bacteria medicine

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011057229A2 (en) * 2009-11-09 2011-05-12 University Of Miami Fluorescent analogs of neurotransmitters, compositions containing the same and methods of using the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050054006A1 (en) * 2002-09-09 2005-03-10 New York University Combinatorial fluorescent library based on the styryl scaffold
CN103265947A (en) * 2013-06-03 2013-08-28 山东大学 Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell
CN106833623A (en) * 2017-02-17 2017-06-13 广东工业大学 A kind of fluorescence probe and preparation method thereof
CN107311977A (en) * 2017-06-27 2017-11-03 广东工业大学 A kind of indoles ethylene compounds and its preparation method and application
CN108309977A (en) * 2018-05-15 2018-07-24 广东工业大学 Application of the indoles ethylene substituted chinoline derivative in preparing drug-resistance bacteria medicine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"RN 1104911-13-8", REGISTRY, 12 February 2009 (2009-02-12) *
DATABASE REGISTRY 16 February 2009 (2009-02-16), ANONYMOUS: "Quinolinium, 1-ethyl-4-[2-(5-methoxy-1H-indol-3-yl)ethenyl]-, iodide (1:1) (CA INDEX NAME)", XP055818994, retrieved from STN Database accession no. 1107020-96-1 *
LU YU-JING, XIAO-LU GUO, MIAO-HAN XU, WEI-WU CHEN, WING-LEUNG WONG, KUN ZHANG, CHEUK-FAI CHOW: "Selective Visualization of DNA G-Quadruplex Structures in Live Cells with 1-Methylquinolinium-Based Molecular Probes: The Importance of Indolyl Moiety Position towards Specificity", DYES AND PIGMENTS, vol. 143, 20 April 2017 (2017-04-20), pages 331 - 341, XP055819003, DOI: 10.1016/j.dyepig.2017.04.038 *
R. KRIEG ET AL.: "N, N-Dialkylaminostyryl Dyes: Specific and Highly Fluorescent Substrates of Peroxidase and Their Application in Histochemistry", JOURNAL OF MOLECULAR HISTOLOGY, vol. 39, no. 2, 29 November 2007 (2007-11-29), pages 169 - 191, XP019573294 *
ROSANIA GUSTAVO R, LEE JAE WOOK, DING LIANG, YOON HAI-SHIN, CHANG YOUNG-TAE: "Combinatorial Approach to Organelle-Targeted Fluorescent Library Based on the Styryl Scaffold", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, US, vol. 125, no. 5, 5 February 2003 (2003-02-05), US, pages 1130 - 1131, XP009514702, ISSN: 0002-7863, DOI: 10.1021/ja027587x *
ZHANG JIANGHUA, L YING; JIA HONGLIANG; SONG YINYIN; SUN XIAOXIA; CHAI DUNXIAO; WANG LANYING: "Synthesis, Crystal Structure and Spectral Properties of Six Indole Dimethine Cyanines as Well as Their Biological Application", CHEMICAL JOURNAL OF CHINESE UNIVERSITIES, vol. 36, no. 10, 1 October 2015 (2015-10-01), pages 1924 - 1932, XP055819001, DOI: 10.7503/cjcu20150128 *

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