CN115504943A - Phenolic hydroxyl group-containing targeted nucleolus and RNA fluorescent probe, preparation method and application thereof - Google Patents
Phenolic hydroxyl group-containing targeted nucleolus and RNA fluorescent probe, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a fluorescent probe containing phenolic hydroxyl targeted nucleolus and RNA, a preparation method and application thereof, belonging to the technical field of fluorescent probes. The structural formula of the fluorescent probe is shown as
The fluorescent probe is changed from a positive charge state to an uncharged state in a weakly alkaline environment of cell mitochondria, so that the fluorescent probe can leave the mitochondria, and the specific recognition of RNA and nucleolus in living cells is realized. The fluorescent probe has the characteristics of low price, quick dyeing time, no washing and good biocompatibility.
Description
Technical Field
The invention relates to a fluorescent probe containing phenolic hydroxyl targeted nucleolus and RNA, a preparation method and application thereof, belonging to the technical field of fluorescent probes.
Background
RNA is an important biological macromolecule, playing an important role in various vital activities. According to the central rule, RNA mediates transcription of DNA and translation of proteins. During protein synthesis, messenger RNA molecules transmit genetic information to the ribosome, transfer RNA transmits amino acids to the ribosome, and ribosomal RNA further links the amino acids together to form a specific encoded protein. In addition, RNA is also involved in signal transduction of biological processes, and is closely related to various diseases such as cardiovascular diseases, cancers and the like. Studies have shown significant differences in the shape, size and number of nucleoli in tumor cells. Therefore, observing the distribution of RNA in cells is an important research context in life science research.
Fluorescence imaging technology is a powerful tool for real-time, in-situ observation of biomacromolecules in common use at present. In order to specifically label RNA in cells, researchers have developed protein recognition-based labeled RNA technology, aptamer-based labeled RNA technology, and molecular beacon technology. Wherein the labeled RNA technology based on protein recognition is that a fluorescent protein is encoded on a protein that specifically recognizes RNA, and the labeled RNA technology based on aptamer is similar that a fluorophore is linked to an aptamer that specifically recognizes RNA. Both techniques achieve specific recognition of RNA in living cells, but are complex and time and labor consuming to design and manipulate. Molecular beacon technology can be used to target endogenous RNA, but molecular beacons can only enter living cells by microinjection or electroporation techniques, which can cause damage to living cells to some extent. Compared with the three technologies, the small-molecule fluorescent probe capable of recognizing the RNA has obvious advantages, can enter living cells only in a simple incubation mode, and can specifically recognize the RNA. However, the design of these small molecule fluorescent probes is very difficult due to the lack of an efficient design strategy.
The design of the current small-molecule RNA probe is mainly built on the unique single-helix structure containing a large amount of negative charges of RNA. Generally, most of the reported RNA probes are organic cation salts that target RNA through electrostatic interactions and assemble into the groove of the single-helix structure of RNA. For example, yu et al reported that a cationic fluorescent probe V-P1 was able to bind to the groove structure of G4 RNA (Yu L.et al.2020, CCS chem.2, 2725-2739); wang et al constructed a near-infrared fluorescent probe with quinoline salts that also bound to the groove of RNA (Wang Z.et al.2022, dyes Pigm.200, 110126); elhussin and colleagues developed a water-soluble cationic quinoline-indole derivative IM-3 for useRNA imaging in living cells (Elhussin I.E.H.et al.2020, chem.Commun.56, 1859-1862). Although these probes are capable of successfully illuminating RNA in living cells, a common problem is that in addition to illuminating RNA, these probes also illuminate mitochondria in the cell. As organelles with higher negative membrane potentials in living cells (Δ Ψ) m 180 mV), mitochondria can have a strong enrichment of cationic salts through electrostatic interactions, which greatly interferes with the targeting ability of RNA probes. Several studies have also explored the competitive effect of mitochondria and RNA on cationic salts. Tian et al reported two cationic salt-type probes that stain mitochondria when mitochondrial membrane potential is high in living cells and RNA when mitochondrial membrane potential is significantly reduced in apoptotic cells (Tian m.et al.2019, anal.chem.91, 10056-10063. Our group of subjects also reported a series of cationic salt-type molecules, IVP, in which IVP molecules with shorter alkyl chains preferentially concentrate on mitochondria in normal living cells and transfer to RNA once mitochondrial membrane potential is reduced (Zhang R.et al.2020, chem. Sci.11, 7676-7684). Therefore, it remains a great challenge to design a fluorescent probe that can be labeled specifically for RNA in living cells without mitochondrial interference.
Disclosure of Invention
In view of the above, the present invention aims to provide a fluorescent probe containing phenolic hydroxyl groups and targeting nucleolus and RNA, a preparation method and applications thereof. The fluorescent probe is changed from a positive charge state to an uncharged state in a weakly alkaline environment (pH 7.4-8.0) of cell mitochondria so as to leave the mitochondria, thereby realizing the specific recognition of RNA and nucleolus in living cells and solving the problem that the targeting property of the existing RNA probe of the living cells is often interfered by the mitochondria.
In order to realize the purpose, the technical scheme of the invention is as follows:
a fluorescent probe containing phenolic hydroxyl groups and targeting nucleolus and RNA, the structural formula of the fluorescent probe is as follows:
wherein, X - Halogen ions, perchlorate ions or hexafluorophosphate ions.
Preferably, the structural formula of the fluorescent probe is as follows:
the invention relates to a preparation method of a phenolic hydroxyl group-containing targeted nucleolar and RNA fluorescent probe, which comprises the following steps:
(1) Reacting 2-methylbenzothiazole with methyl iodide to generate 2,3-dimethyl benzothiazole-3-iodide;
(2) P-hydroxybenzaldehyde and 2,3-dimethylbenzothiazole-3-iodonium salt are reacted by Wen Ge (Knoevenagel reaction) to obtain (E) -2- (4-hydroxystyryl) -3-methylbenzo [ d ] thiazole-3-iodonium salt, namely X-is a fluorescent probe of a targeted nucleolus and RNA of iodide ions;
(3) And (E) -2- (4-hydroxystyryl) -3-methylbenzo [ d ] thiazole-3-iodide salt and other halogen ions, perchlorate ions or hexafluorophosphate ions are subjected to displacement reaction to obtain the fluorescent probe containing phenolic hydroxyl targeted nucleolus and RNA.
The invention relates to the application of the fluorescent probe containing phenolic hydroxyl group and targeting nucleolus and RNA in marking or displaying the nucleolus and RNA distribution in cells for non-disease diagnosis and treatment.
The invention relates to application of a phenolic hydroxyl group-containing fluorescent probe for targeting nucleolus and RNA in preparation of products for detecting nucleolus and RNA in cells.
The invention discloses application of a phenolic hydroxyl group-containing fluorescent probe targeting nucleolus and RNA in preparation of RNA and nucleolus staining imaging products in cells.
The invention discloses application of a phenolic hydroxyl group-containing fluorescent probe targeting nucleolus and RNA in preparation of a product for distinguishing cancer cells/tissues from normal cells/tissues.
Advantageous effects
The invention provides a fluorescent probe containing phenolic hydroxyl and targeting nucleolus and RNA, which can specifically mark RNA in cancer cells and normal cells, is not interfered by mitochondrial membrane potential, and provides a simple and visual biological detection reagent for RNA related research. The mechanism of the RNA-specific marker and no interference by mitochondrial membrane potential is shown in FIG. 1: since mitochondria and RNA are negatively charged in living cells, the fluorescent probes tend to stain both mitochondria and RNA simultaneously when staining living cells. However, after the fluorescent probe enters mitochondria, the mitochondria have a weak alkaline environment with the pH value of 7.4-8.0, the fluorescent probe is changed from a positive charge state to an uncharged state, and the uncharged structure no longer has electrostatic interaction with the mitochondrial membrane potential, so the fluorescent probe can be separated from the mitochondria and return to the cytoplasmic matrix. In a neutral cytoplasmic matrix, the fluorescent probes revert to a positively charged state, which targets the RNA through electrostatic interactions. Therefore, the fluorescent probe can specifically mark RNA when staining living cells, and cannot mark mitochondria.
The invention provides application of a phenolic hydroxyl group-containing fluorescent probe targeting nucleolus and RNA, and the fluorescent probe can be used for washing-free and fast imaging of RNA in living cells and giving a clear picture. The fluorescent probe can also track the dynamic change of nucleolus and RNA in the process of living cell injury in real time. Compared with commercially available RNA fluorescent probes with similar functions, the fluorescent probe provided by the invention has the characteristics of low price, quick dyeing time, no washing and good biocompatibility. The fluorescent probe also has high RNA positioning capability and low toxicity, has good biocompatibility with the currently marketed fluorescent probe Mito Tracker Deep Red, is particularly suitable for RNA fluorescence imaging under a confocal fluorescent microscope and a universal fluorescent microscope, and has wide application prospect in the field of fluorescence biomarker.
Drawings
FIG. 1 is a diagram showing the mechanism of staining live cell RNA but not staining mitochondria by the fluorescent probe of the present invention.
FIG. 2 is a confocal fluorescence micrograph obtained by 488nm laser irradiation after staining active and fixed normal cells AC16 and cancer cells A549 by H-SMBT described in example 3, wherein the collection wavelength band is 570-670nm red channel, and the scale is 20 μm.
FIG. 3 is a confocal fluorescence image of the cancer cells A549 fixed in example 4 treated with DNase and RNase respectively, the excitation wavelength is 488nm, and the collection band is 570-670nm.
FIG. 4 shows the binding pattern of H-SMBT to RNA as described in example 5.
FIG. 5 is a fluorescent titration of RNA with H-SMBT as described in example 5 at a concentration of 10. Mu.M and a curve was fitted according to the Scatchard equation.
FIG. 6 shows staining of active AC16 cells with H-SMBT as described in example 6 with 20mM H 2 O 2 And processing the 60min confocal fluorescence picture, wherein the excitation wavelength is 488nm, the collection wave band is 570-670nm, and the ruler is 20 microns.
FIG. 7 is a confocal fluorescence micrograph of the M-SMBT staining active AC16 cells described in comparative example 1, with an acquisition wavelength band of 435-545nm green channels; confocal fluorescence micrographs of active AC16 cells stained with M-SMBT and MTR, with the M-SMBT collection band being a 435-545nm green channel, the MTR collection band being a 570-670nm red channel, and the scale being 20 μ M.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
EXAMPLE 1 preparation of nucleolar and RNA-targeting fluorescent probes containing phenolic hydroxyl groups
Parahydroxybenzaldehyde (0.12g, 1mmol) and 2,3-dimethylbenzothiazole-3-iodonium salt (0.29g, 1mmol) were dissolved in 3.5mL ethanol, and 50. Mu.L tetrahydropyrrole was added, and the solution quickly turned red. After stirring at room temperature for 6h, the mixture was poured into n-hexane and an orange solid precipitated, filtered and rinsed with dichloromethane. The orange solid was then dissolved in 10mL acetone and KPF was added 6 (0.55g, 3mmol), stirring at 50 ℃ for 4h, removing acetone by distillation under reduced pressure, and purifying the remaining mixture by means of silica gel column chromatography to give an orange solid in 33% yield.
The preparation reaction formula is as follows:
the nuclear magnetic hydrogen spectrum results of the orange solid are as follows:
1 H NMR(400MHz,DMSO-d 6 ),δ(ppm):10.62(s,1H),8.38(dd,J＝8.1Hz,1.2Hz,1H),8.21-8.12(m,2H),7.96-7.94(m,2H),7.87-7.76(m,3H),6.96-6.93(m,2H),4.31(s,3H). 13 C NMR(100MHz,DMSO-d 6 ),δ(ppm):172.60,162.62,149.76,142.46,133.03,129.66,128.56,127.89,125.86,124.56,117.03,116.78,110.55,39.65,36.52.
the results indicated that the orange solid was (E) -2- (4-hydroxystyryl) -3-methylbenzo [ d ] thiazole-3-hexafluorophosphate salt, which was reported as H-SMBT.
Example 2 cancer cell (A549) and Normal cell (AC 16) culture
All cell lines were at 37 ℃ C. And 5% CO 2 The saturated humidity incubator. A549 cell strain and AC16 cell strain are cultured in culture solution containing 10% fetal bovine serum H-DMEM (containing 1% double antibody) in an adherent way. After the cells had grown to log phase, the cells in the 100mL cell vial were washed three times with PBS, digested with 1mL 0.25% trypsin for 3-5min, the medium was carefully decanted, a small amount of fresh medium was added and the cells were blown up evenly, after counting the cells, the cells were left at the appropriate density and the medium was added to the desired volume (final cell concentration was controlled at 1X 10) 4 ) Inoculating to confocal glass-bottom culture dish, adding CO 2 Culturing in an incubator to ensure that the cells grow adherent to the bottom of the glass.
Example 3H-SMBT staining of cancer cells (A549) and Normal cell (AC 16) experiments
The confocal glass-bottom plates seeded with A549 cells and AC16 cells were divided into two groups. One group was directly stained with 4. Mu. M H-SMBT for 5min. The other group was fixed with 4% paraformaldehyde solution for 30min and then stained with 4. Mu. M H-SMBT for 5min.
After staining, the stained sites of the cells were observed by confocal laser scanning microscopy, and as shown in fig. 2, H-SMBT was distributed similarly in active, fixed a549 and AC16 cells, mainly in the nucleolus and in the cytoplasm, consistent with the distribution of RNA in the cells.
Example 4 digestion experiments with DNase and RNAse
A549 cells seeded on a glass-bottom plate were fixed with 4% paraformaldehyde solution for 45min, and then treated with Triton X-100 (0.5%) at room temperature for 10min. For DNase digestion experiments, treated cells were incubated with 1 × reaction buffer for 5min at room temperature, and then treated with DNase (10U/mL) for 30min at 37 ℃. For RNase digestion experiments, cells were treated with RNase (100. Mu.g/mL) for 120min at 37 ℃.
After treating the cells with DNase and RNase respectively, staining the treated cells with H-SMBT for 5min, and observing the stained parts of the cells by using a laser scanning confocal microscope after staining; as shown in FIG. 3, the H-SMBT distribution did not change significantly after the cells were treated with DNase, and the nucleolar fluorescence decreased significantly after the cells were treated with RNase.
Example 5 binding mode of H-SMBT to RNA, RNA titration experiment
The binding mode of H-SMBT to RNA was simulated by Gaussian View and AutoDock 4.2, and the results are shown in FIG. 4, which indicates that H-SMBT binds to small grooves of RNA.
Mixing solutions of RNA and H-SMBT at different molar ratios were prepared, fluorescence spectra thereof were measured, curves were fitted according to the Scatchard equation, and binding constants of H-SMBT and RNA were calculated to be 1.82X 10, as shown in FIG. 5 6 。
Example 6H-SMBT assay for real-time tracking of the dynamic changes of nucleoli and RNA during the injury of living cells
The glass culture dish inoculated with the cells was stained with 4. Mu. M H-SMBT and placed on a confocal microscope, and 20mM H was added along the edge of the dish 2 O 2 Then, the process of morphological change of nucleolus and RNA in the cells was observed for 60min, and the result is shown in FIG. 6.
Comparative example 1
In order to verify that phenolic hydroxyl in H-SMBT determines the specificity of H-SMBT on RNA and prevents the specificity from being interfered by mitochondria, M-SMBT molecules are obtained after hydroxyl is replaced by methoxyl; and staining viable cells with M-SMBT molecules.
As shown in FIG. 7, AC16 cells were seeded into confocal glass-bottom plates and stained with 4. Mu. M M-SMBT for 5min. After staining, the stained parts of the cells were observed by laser scanning confocal microscopy. As a result, M-SMBT was found to be distributed mainly in the cytoplasm in active AC16 cells, and the staining pattern was consistent with the morphology of mitochondria. Further co-localized staining was performed with the commercial mitochondrial probe Mitochondrial Tracing Red (MTR) and M-SMBT, and cells were observed with a laser scanning confocal microscope. As a result, it was found that M-SMBT and MTR were stained at overlapping positions, confirming that M-SMBT stains mitochondria of living cells, and does not stain nucleoli and RNA.
Thus, the unique structure of H-SMBT, which contains phenolic hydroxyl groups, was confirmed to enable it to specifically stain RNA and nucleoli in living cells, but not mitochondria.
In summary, the invention includes but is not limited to the above embodiments, and any equivalent replacement or local modification made under the spirit and principle of the invention should be considered as being within the protection scope of the invention.
Claims (7)
1. A fluorescent probe containing phenolic hydroxyl groups and targeting nucleolus and RNA, characterized in that: the structural formula of the fluorescent probe is as follows:
wherein X - Is halogen ion, perchlorate ion or hexafluorophosphate ion.
2. A phenolic hydroxyl group-containing nucleolar and RNA targeting fluorescent probe according to claim 1, wherein: the structural formula of the fluorescent probe is as follows:
3. a method for preparing a nucleolar and RNA-targeting fluorescent probe according to claim 1 or 2, comprising: the method comprises the following steps:
(1) Reacting 2-methylbenzothiazole with methyl iodide to generate 2,3-dimethylbenzothiazole-3-iodonium salt;
(2) P-hydroxybenzaldehyde and 2,3-dimethylbenzothiazole-3-iodonium salt are reacted by Knoevenagel to obtain (E) -2- (4-hydroxystyryl) -3-methylbenzo [ d]Thiazole-3-iodonium salts, i.e. X - A fluorescent probe that targets nucleoli and RNA for iodide ions;
(3) And (E) -2- (4-hydroxystyryl) -3-methylbenzo [ d ] thiazole-3-iodonium salt and other halogen ions, perchlorate ions or hexafluorophosphate ions are subjected to displacement reaction to obtain the fluorescent probe containing phenolic hydroxyl targeted nucleolus and RNA.
4. Use of a fluorescent probe comprising phenolic hydroxyl groups targeting nucleoli and RNA according to claim 1 or 2 for labeling or displaying non-disease diagnostic and therapeutic purposes in the distribution of nucleoli and RNA in cells.
5. Use of the phenolic hydroxyl group-containing nucleolar and RNA targeting fluorescent probe of claim 1 or 2 in the preparation of a product for detecting nucleolar and RNA in cells.
6. Use of a phenolic hydroxyl group-containing nucleolar and RNA targeting fluorescent probe according to claim 1 or 2 in the preparation of a product for RNA and nucleolar staining imaging in cells.
7. Use of a phenolic hydroxyl group-containing nucleolar and RNA targeting fluorescent probe according to claim 1 or 2 in the preparation of a product for differentiating between cancer cells/tissues and normal cells/tissues.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109912532A (en) * | 2019-03-05 | 2019-06-21 | 暨南大学 | Benzothiazole compound and its application in preparation bacterial biof iotalm inhibitor |
| CN111116573A (en) * | 2019-12-31 | 2020-05-08 | 中山大学 | Near-infrared fluorescent probe for simultaneously detecting DNA and RNA under dual channels and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109912532A (en) * | 2019-03-05 | 2019-06-21 | 暨南大学 | Benzothiazole compound and its application in preparation bacterial biof iotalm inhibitor |
| CN111116573A (en) * | 2019-12-31 | 2020-05-08 | 中山大学 | Near-infrared fluorescent probe for simultaneously detecting DNA and RNA under dual channels and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| KANG-NAN WANG等: "Red fluorescent probes for real-time imaging of the cell cycle by dynamic monitoring of the nucleolus and chromosome", CHEM. COMMUN., vol. 54, pages 2635 - 2638 * |
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