RNA (Ribonucleic Acid) fluorescent probe as well as preparation method and application thereof in cell nucleolus staining

Abstract

The invention discloses an RNA (Ribonucleic Acid) fluorescent probe as well as a preparation method and application thereof in cell nucleolus staining, and belongs to the technical field of biomedical detection. The probe is a water-soluble pyridine salt compound, the chemical name of the probe is 1-(2-(2-(2, 2-dicyanovinyl))-5-(dimethylamino phenoxy) ethyl) pyridine-1-ammonium bromide, and the preparation method of the probe is characterized in that 2-(2-bromoethoxy)-4-(dimethylamino) benzaldehyde and malononitrile are used as raw materials, and the probe is synthesized through two-step reaction. The probe has the characteristics of large Stokes shift, low cytotoxicity and fluorescence enhancement after being combined with RNA (Ribonucleic Acid). Experiments prove that the probe can specifically carry out efficient fluorescence labeling on nucleolus and cytoplasm RNA in fixed cells, and a high-contrast imaging effect is obtained. The probe disclosed by the invention is simple and convenient to synthesize and excellent in performance, and has important application value in the fields of cell nucleolus research, tumor diagnosis and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically an RNA fluorescent probe, its preparation method, and its application in cell nucleolar staining. Background Technology

[0002] The nucleolus is one of the most important subcellular organelles in the cell, playing a crucial role in the synthesis and storage of deoxyribonucleic acid (RNA) and the assembly of ribosomes. It is also the most prominent structure in the eukaryotic cell nucleus. Since the discovery of the nucleolus's important functions in the 1960s, the academic and clinical medical communities have significantly increased their research attention on the structure, dynamics, and function of the nucleolus, especially in cases of abnormally enlarged tumor cell nuclei and an abnormally increased number of nucleoli. For tumor diagnosis, traditional hematoxylin / eosin (HE) staining requires the preparation of ultrathin sections of pathological tissue in advance and heavily relies on years of experience by physicians, resulting in a significant need to improve diagnostic efficiency and accuracy. Therefore, fluorescence imaging of the nucleolus can not only provide intuitive and detailed evidence for nucleolus research but also offer important technical references for the identification of tumor cells.

[0003] Live cells collected in clinical settings typically have a relatively short lifespan and may rapidly denature within minutes. To effectively prevent cell autolysis and bacterial degradation, and to ensure the integrity of cellular components and their microstructure, researchers need to fix the samples immediately before storage, transfer, and staining. This process is crucial for preserving the cells' original state. Therefore, it is essential to research and develop methods suitable for imaging fixation of nucleoli in samples.

[0004] Given the established fact that RNA is a major component of the nucleolus in cell biology research, researchers have long been dedicated to developing various RNA fluorescent probes to conduct in-depth, dynamic visualization studies of the nucleolus. Furthermore, since RNA is also widely distributed in the cytoplasm, it is anticipated that RNA probes will simultaneously stain both the nucleolus and the cytoplasm during practical applications. In fact, to date, only one commercially available RNA fluorescent probe, SYTO RNA-Select, is used for nucleolus imaging, and its chemical structure remains under patent protection. Therefore, the development of RNA fluorescent probes with independent intellectual property rights is urgently needed.

[0005] Water-soluble organic small molecule ionic salts have shown great potential in the development of fluorescent probes due to their controllable structure, simple synthesis, and the ability to achieve targeted binding to biological targets by introducing specific functional groups. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, the present invention aims to design and provide an RNA fluorescent probe, its preparation method, and its application in cell nucleolar staining. This probe has the advantages of simple synthesis route, good water solubility, large Stokes shift after binding with RNA, low cytotoxicity, and excellent staining performance on cell nucleoli.

[0007] The RNA fluorescent probe described above is characterized in that the fluorescent probe is a compound having the following chemical structural formula: ; Its chemical name is 1-(2-(2-(2,2-dicyanovinyl))-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as QDCNPy, and its chemical formula is C 19 H 19 BrN4O.

[0008] The method for preparing an RNA fluorescent probe is characterized by comprising the following steps: 1) Mix 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde and malononitrile in toluene solvent, add alumina, and react at room temperature for 2-4 hours. After post-treatment, the intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethylene)malononitrile, abbreviated as QDCN, has the chemical formula C 14 H 14 BrN3O; 2) The obtained intermediate was reacted in pyridine solvent at 70-90℃ for 16-20 hours. After cooling, precipitation, filtration and washing, the target product RNA fluorescent probe was obtained.

[0009] The method for preparing an RNA fluorescent probe, as described above, is characterized by the following synthetic route: .

[0010] The method for preparing an RNA fluorescent probe is characterized in that, in step 1), the molar ratio of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde to malononitrile is 1:1.5-2.5, and the amount of alumina used is 8-12 equivalents of the molar amount of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde.

[0011] The method for preparing an RNA fluorescent probe is characterized in that, in step 1), the post-processing includes filtration and washing the filter cake with dichloromethane, removing the solvent from the filtrate by vacuum rotary evaporation, recrystallizing the filtrate with dichloromethane / n-hexane, and filtration to obtain a solid intermediate.

[0012] The method for preparing an RNA fluorescent probe is characterized in that, in step 2), the reaction temperature is 80°C and the reaction time is 18 hours.

[0013] The application of the RNA fluorescent probe in cell nucleolar staining.

[0014] The application is characterized in that the application object is fixed cells, the concentration of the RNA fluorescent probe is 1-20 μM, preferably 5-10 μM, and the staining incubation time is 30-60 minutes.

[0015] Compared with the prior art, the present invention has the following significant advantages: 1. The QDCNPy probe provided by this invention exhibits a large Stokes shift (41 nm) in aqueous solution, effectively avoiding interference between excitation and emission light and reducing background noise. Its fluorescence intensity significantly increases after binding to RNA, with a red shift in the emission peak and a Stokes shift increasing to 51 nm. This indicates that QDCNPy has a strong binding affinity to RNA, and this binding helps to avoid biological autofluorescence and enhance the brightness of the labeled region, resulting in high-quality fluorescence imaging images.

[0016] 2. Good biocompatibility: When the concentration used does not exceed 60 μM, QDCNPy has low cytotoxicity and is suitable for live cell labeling.

[0017] 3. High staining specificity: QDCNPy can specifically label the nucleolus and cytoplasm of fixed cells at low concentrations (the nucleus is not labeled) and the laser confocal imaging is clear, indicating that this small organic molecule ion salt is an effective RNA fluorescent probe. It is expected to break through technical barriers and has broad application prospects in basic research related to nucleoli and clinical application research closely related to tumor diagnosis.

[0018] 4. Simple preparation method: The synthesis route of this probe is simple, requiring only two steps. The raw materials are readily available, the reaction conditions are mild, and it is easy to achieve large-scale preparation, thus having high industrial application value. Attached Figure Description

[0019] Figure 1a The UV-Vis absorption spectrum and fluorescence emission spectrum of the QDCNPy probe prepared in Example 1 of this invention in aqueous solution; Figure 1b The luminescence spectra of the QDCNPy probe aqueous solution after adding different equivalents of RNA; Figure 2 The effect of the QDCNPy probe prepared in Example 1 of the present invention on cell viability at different concentrations (cytotoxicity experiment). Figure 3Laser confocal microscopy images of HeLa live cells (top) and fixed cells (bottom) after staining with the QDCNPy probe prepared in Example 1 of this invention. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] The RNA fluorescent probe described in this invention is a compound having the following chemical structural formula:

[0022] Its chemical name is 1-(2-(2-(2,2-dicyanovinyl))-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as QDCNPy, and its chemical formula is C 19 H 19 BrN4O.

[0023] The synthetic route of the compound QDCNPy of this invention is as follows: .

[0024] Example 1: The preparation method of the RNA fluorescent probe of the present invention includes the following steps: 1) Mix an appropriate amount of QBr, 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde (refer to authorized patent CN119661424B), and two equivalents of malononitrile in toluene. Stir at room temperature, add ten equivalents of aluminum oxide to the reaction system, react for 2 hours, filter, and wash the filter cake with dichloromethane. After removing the solvent by vacuum rotary evaporation, recrystallize the filtrate from dichloromethane / n-hexane and filter to obtain a red solid intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethylene)malononitrile, abbreviated as QDCN, with the chemical formula C 14 H 14 BrN3O.

[0025] The molecular structure of QDCN with atomic numbers marked and its precise NMR data are as follows: ; 1 H NMR (400 MHz, Methanol- d4) δ 8.22 (dd, J = 9.3, 0.6 Hz, 1H, 3), 8.06 (d, J = 0.6 Hz, 1H, 1), 6.55 (ddd, J = 9.4, 2.5, 0.7 Hz, 1H, 4), 6.29 (d, J = 2.4 Hz, 1H, 6), 4.63 (dq, J = 7.1, 2.6 Hz, 2H, 12), 3.99–3.93 (m, 2H,13), 3.19 (s, 6H, 9, 10). Mass spectrum m / z: 321.02 [M] + (Theoretical value: 320.19). 2) Add an appropriate amount of QDCN to pyridine and stir evenly. React at 80℃ for 18 hours. Cool the system to room temperature, and an orange-red solid precipitates. Filter the solid and wash the filter cake with dichloromethane to obtain a dark red solid, which is the target product 1-(2-(2-(2,2-dicyanovinyl))-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as QDCNPy, with the chemical formula C 19 H 19 BrN4O.

[0026] The molecular structure of QDCNPy with atomic numbers marked and its precise NMR data are as follows: ; 1 H NMR (400 MHz, Methanol- d 4) δ 9.21–9.15 (m, 2H, 16, 20), 8.69 (tt, J= 7.9, 1.3 Hz, 1H, 18), 8.20 (dd, J = 7.8, 6.6 Hz, 2H, 17, 19), 8.14 (dd, J =9.4, 0.5 Hz, 1H, 3), 7.79 (d, J = 0.6 Hz, 1H, 1), 6.56–6.49 (m, 1H, 4), 6.24 (d, J = 2.4 Hz, 1H, 6), 5.19–5.12 (m, 2H, 12), 4.72–4.65 (m, 2H, 13), 3.16(s, 6H, 9, 10). Mass spectrometry m / z: 320.31 [M–Br] + 338.18 [(M–Br)*H2O] +(Theoretical value: 399.29). Elemental analysis: C, 57.27; H, 4.93; N, 13.97; Theoretical value: C, 57.15; H, 4.80; N, 14.03. In Example 1, 1) Mix 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde and malononitrile in toluene solvent, add alumina, and react at room temperature for 2-4 hours. After post-treatment, the intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethylene)malononitrile, abbreviated as QDCN, has the chemical formula C 14 H 14 BrN3O; 2) The obtained intermediate was reacted in pyridine solvent at 70-90℃ for 16-20 hours. After cooling, precipitation, filtration and washing, the target product RNA fluorescent probe was obtained.

[0027] In step 1) of this invention: the molar ratio of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde to malononitrile is 1:1.5 or 1:2.5, the reaction time is 3 hours or 4 hours, and the amount of alumina used is 8 or 12 equivalents of the molar amount of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde; in step 2): the reaction temperature is 70℃ or 90℃, and the reaction time is 16 hours or 20 hours; other aspects are the same as in Example 1, and the beneficial effects described in this invention can also be obtained.

[0028] Experiment 1: Photophysical properties and RNA binding ability test results are shown below. Figure 1a and Figure 1b .

[0029] The solubility of QDCNPy in water at room temperature was previously measured to be 1.8 mg / mL. From... Figure 1a It can be seen that QDCNPy is at 1'10 -5 The maximum UV-Vis absorption peak of M in aqueous solution is located at 445 nm. Using this value as the excitation wavelength, the emission peak of the emission spectrum is located at 486 nm. Therefore, QDCNPy can be calculated at 1 × 10⁻⁶ nm. -5 The Stokes shift of M in aqueous solution was 41 nm, and the fluorescence quantum yield in this aqueous solution was measured to be 0.12. To test the binding ability of this ionic salt to RNA, different equivalents (20 eq, 40 eq, 70 eq, 140 eq) of RNA were added to QDCNPy aqueous solution to establish a concentration gradient, and the emission spectrum and fluorescence quantum yield were measured respectively. Figure 1bAs RNA concentration increased, the fluorescence signal detected by the emission spectrum significantly increased, and the emission peak showed a redshift. The emission wavelength redshifted from 486 nm with zero RNA to 496 nm with 140 eq of RNA. The fluorescence quantum yields of QDCNPy aqueous solutions containing the above gradient equivalents of RNA were 0.22 (20 eq), 0.51 (40 eq), 0.87 (70 eq), and 1.39 (140 eq; the fluorescence quantum yield of this group of RNA solutions without QDCNPy was negligible). These results indicate that QDCNPy has a strong binding ability to RNA, and this binding helps to avoid biological autofluorescence and enhance the brightness of the labeled area, resulting in high-quality fluorescence imaging images.

[0030] Experiment 2: Cytotoxicity assay, cell viability = (Experiment - Blank) / (Control - Blank) x 100%, results are shown in [link to results]. Figure 2 .

[0031] Figure 2 The results showed that healthy cells were digested, centrifuged, and seeded into 96-well plates with 100 μL of cell suspension (approximately 3000-5000 cells / well) added to each well. Different concentration gradients of QDCNPy solutions (20 μM, 40 μM, 60 μM, 80 μM, and 100 μM) were prepared for cell treatment, with 4-6 replicates. Cell controls and culture medium controls were also included. Cells were cultured overnight at 37°C, 5% CO2, and 90% humidity. After treatment, cells were washed 1-2 times with PBS, and 100 μL of culture medium containing 10% CCK-8 solution was added to each well. After incubation in the dark for 2 h, the absorbance at 450 nm was measured using a microplate reader. Cell viability in the 20 μM, 40 μM, and 60 μM groups was above 80%, indicating that QDCNPy at concentrations not exceeding 60 μM exhibits low cytotoxicity and is suitable for live cell labeling.

[0032] Application Example 1: The compound QDCNPy of this invention is used for cell staining imaging. 1. Cell Culture The HeLa cells used in the experiment were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture, under the conditions of 37°C and 5% carbon dioxide.

[0033] 2. Cell imaging Before the confocal imaging experiment, all cells were seeded in a confocal culture dish and cultured for 48 hours.

[0034] Confocal imaging parameters: QDCNPy concentration 5 μM, excitation wavelength (Ex) 415 nm, emission wavelength (Em) 460-560 nm.

[0035] Probe and live cell experiments: Cells were incubated with QDCNPy at 37°C and 5% CO2 for 50 minutes, and then imaged using an FV1200 confocal microscope.

[0036] Probe and fixed cell experiments: Live cells were fixed with 4% paraformaldehyde at room temperature for 2 hours, washed twice with 1 mL PBS, and then co-incubated with QDCNPy for 40 minutes. Finally, the cells were imaged using an FV1200 confocal microscope.

[0037] 3. Results, such as Figure 3 As shown. Figure 3 This indicates that imaging of HeLa live cells 50 minutes after staining with the probe showed no obvious targeting of cell structures, and the fluorescence signal was diffusely distributed, suggesting that it failed to enter the live cells. However, imaging of fixed HeLa cells 40 minutes after staining clearly showed an abnormally increased number of nucleoli and cytoplasm (the bright areas marked by staining within the cell) and an abnormally enlarged nucleus (the dark areas not marked by staining within the cell). The scale bar for this set of images is 10 μm. In application, the concentration of the RNA fluorescent probe can be 1 μM, 10 μM, or 20 μM, and the staining incubation time can be 60 minutes, 30 minutes, or 20 minutes, achieving the same effect.

[0038] Based on the above experimental results, QDCNPy exhibits low cytotoxicity to live cells when used at a concentration not exceeding 60 μM. Cell imaging experiments show that although it cannot effectively stain live cells, it can specifically label the nucleolus and cytoplasm (RNA-rich areas) of fixed cells at a low concentration of 5 μM. However, the nucleus, which is rich in DNA, remains unlabeled, and laser confocal imaging is clear. This indicates that this small organic molecule ionic salt is an effective RNA fluorescent probe, which is expected to overcome technical barriers and has broad application prospects in basic research related to nucleoli and clinical applications closely related to tumor diagnosis.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An RNA fluorescent probe, characterized in that... The fluorescent probe is a compound having the following chemical structural formula: ； Its chemical name is 1-(2-(2-(2,2-dicyanovinyl))-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as QDCNPy, and its chemical formula is C 19 H 19 BrN4O.

2. The method for preparing an RNA fluorescent probe as described in claim 1, characterized in that... Includes the following steps: 1) Mix 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde and malononitrile in toluene solvent, add alumina, and react at room temperature for 2-4 hours. After post-treatment, the intermediate 2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethylene)malononitrile, abbreviated as QDCN, has the chemical formula C 14 H 14 BrN3O; 2) The obtained intermediate was reacted in pyridine solvent at 70-90℃ for 16-20 hours. After cooling, precipitation, filtration and washing, the target product RNA fluorescent probe was obtained.

3. The method for preparing an RNA fluorescent probe as described in claim 2, characterized in that... The synthesis path is as follows: 。 4. The method for preparing an RNA fluorescent probe as described in claim 2, characterized in that... In step 1), the molar ratio of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde to malononitrile is 1:1.5-2.5, and the amount of alumina used is 8-12 equivalents of the molar amount of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde.

5. The method for preparing an RNA fluorescent probe as described in claim 2, characterized in that... In step 1): the post-processing includes filtration and washing the filter cake with dichloromethane. After the solvent is removed by vacuum rotary evaporation, the filtrate is recrystallized from dichloromethane / n-hexane and then filtered to obtain a solid intermediate.

6. The method for preparing an RNA fluorescent probe as described in claim 2, characterized in that... In step 2), the reaction temperature is 80℃ and the reaction time is 18 hours.

7. The application of the RNA fluorescent probe as described in claim 1 in cell nucleolar staining.

8. The application as described in claim 7, characterized in that, The application target is fixed cells, the concentration of the RNA fluorescent probe is 1-20 μM, preferably 5-10 μM, and the staining incubation time is 30-60 minutes.

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