Near-infrared RNA (Ribonucleic Acid) selective fluorescent probe, preparation method and application of near-infrared RNA selective fluorescent probe in tracking stress particle dynamics in living cells

Abstract

The invention discloses a near-infrared RNA (Ribonucleic Acid) selective fluorescent probe, a preparation method and application of the near-infrared RNA selective fluorescent probe in tracking stress particle dynamics in living cells, and belongs to the technical field of fluorescent probes. The probe is connected with benzothiazole and quinoline groups through-C = C-bonds, has good biocompatibility and RNA selectivity, and can rapidly and selectively lighten RNA related areas through confocal imaging without water washing. Compared with an existing product, the probe shows higher light stability and selectivity, and stress particles in living cells can be effectively imaged. Experiments find that in addition to the protein-RNA interaction, the protein-protein and RNA-RNA interaction also participates in SG assembly, and the heterostructure of SGs is disclosed. In addition, the probe is utilized to track the formation and depolymerization process of stress particles during oxidative stress and heat stress in real time. The probe has important significance for deeply knowing the dynamic behaviors of RNA and stress particles in living cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular, relates to a near-infrared RNA selective fluorescent probe, a preparation method and an application in tracking stress granule dynamics in living cells. Background Art

[0002] Stress Granules (SGs) are mRNA-protein complexes formed by cells in response to environmental stresses such as heat shock, oxidative stress, nutrient deficiency, viral infection, and tumor formation. The formation of SGs is a protective mechanism of cells against adversity, protecting cells from damage by stagnating protein synthesis. Abnormal accumulation of SGs is associated with the development of a variety of diseases, including certain neurodegenerative diseases and tumors. Therefore, real-time monitoring of the dynamic changes of SGs in living cells is crucial for understanding the pathogenesis of these diseases and developing new treatments.

[0003] Considering that SGs are cohesive combinations of mRNA and proteins, screening RNA-targeting dyes is a logical approach to discover fluorescent probes that selectively aggregate and label SGs. Various tools have been developed to image RNA, including fluorescently labeled beacons, gold nanoparticle-labeled oligonucleotide probes, coordination compounds, carbon dots, and organic small molecules. Among them, small molecule fluorescent probes have higher spatial resolution, membrane permeability, and versatile photophysical properties, making them simpler and more practical for RNA imaging in living cells. However, the development of small molecule probes suitable for RNA analysis has been slow because small molecule fluorescent probes bind more strongly to DNA than to RNA. SYTO RNA Select is the only commercially available probe with RNA selectivity, but its green luminescence and low photostability limit its application. Although these probes have made some progress in studying cellular RNA dynamics, they still face challenges such as poor selectivity, weak long-term imaging ability, susceptibility to photobleaching, and high cytotoxicity, which are necessary to study RNA dynamics in cellular processes. In addition, there are few reports on the use of small molecule probes to label RNA to study the structure and dynamics of SGs, making it challenging to develop small molecule fluorescent probes for SG labeling and structural exploration in the absence of clues. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a near-infrared RNA selective fluorescent probe, a preparation method and an application for tracking stress granule dynamics in living cells. The fluorescent probe has the advantages of simple structure, easy synthesis, good optical stability and low cytotoxicity. Due to TICT, there is almost no fluorescence in aqueous solution, but when it binds to RNA, its fluorescence intensity is enhanced by 33 times. In addition, the probe can quickly enter the cell, bind to the RNA in the cell, emit bright red fluorescence, and has excellent photostability, so that the dynamics of RNA and SGs in living cells can be tracked in real time by confocal imaging.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A near-infrared RNA selective fluorescent probe, referred to as HQBT, the structural formula of the RNA fluorescent probe is as follows:

[0007]

[0008] The second purpose of the present invention is to provide a method for preparing the RNA fluorescent probe, firstly synthesizing an intermediate by reacting 2-methylbenzothiazole and iodomethane, and then synthesizing the RNA fluorescent probe by reacting the intermediate with 8-hydroxyquinoline-5-carboxaldehyde.

[0009] The reaction equation is as follows:

[0010]

[0011] Furthermore, in the above reaction process, the molar ratio of 2-methylbenzothiazole to methyl iodide is: 1:(1.3-1.5).

[0012] Furthermore, in the above reaction process, the 2-methylbenzothiazole reacts with methyl iodide at 83°C.

[0013] Furthermore, in the above reaction process, the molar ratio of the intermediate to 8-hydroxyquinoline-5-carboxaldehyde is 1:(1.1-1.2).

[0014] Furthermore, in the above reaction process, the intermediate reacts with 8-hydroxyquinoline-5-carboxaldehyde under ethanol reflux conditions, and piperidine and glacial acetic acid are added as catalysts.

[0015] The third object of the present invention is to provide the application of the RNA fluorescent probe in imaging RNA in living cells and real-time tracking of the dynamics of stress granules (SGs) in living cells.

[0016] The RNA fluorescent probe of the present invention has an excitation wavelength of 561 nm and a maximum emission wavelength of 620 nm. The molecule has a rotatable single bond, which makes its fluorescence intensity weak in aqueous solution. When bound to RNA, it emits bright red fluorescence, and the fluorescence intensity increases by about 33 times. The fluorescence intensity of the probe does not change in the range of pH = 3-10. The probe does not respond to other analytes and exhibits excellent RNA selectivity. The probe has little effect on the activity of HepG2 cells and shows good biocompatibility. The probe can quickly enter living cells and stain RNA within 10 seconds, showing excellent anti-photobleaching ability. The fluorescence intensity decreases slowly under continuous laser irradiation, which is suitable for long-term tracking and dynamic imaging. The probe successfully locates SGs produced by living cells under stress conditions, and tracks the SGs dynamics of living cells under oxidative stress and heat stress in real time.

[0017] Beneficial effects of the present invention:

[0018] (1) The present invention selects benzothiazole and quinoline derivatives as the parent body, so that the probe has excellent biocompatibility, RNA selectivity and photostability, so that it can quickly enter living cells to stain RNA and emit bright red fluorescence.

[0019] (2) The present invention solves the problems of the prior art methods, which are time-consuming, labor-intensive, expensive, and have invasive cell damage and cannot monitor the dynamics of SGs in real time. The present invention provides a near-infrared RNA selective small molecule fluorescent probe that can monitor the dynamics of SGs in living cells in real time by fluorescence imaging. The synthetic method is simple, the cytotoxicity is low, and long-term real-time imaging can be performed. At the same time, the influence of the molecular concentration itself and the biologically active molecules in the cell can be avoided. The dynamic changes of SGs in living cells can be accurately and real-time observed by fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings.

[0021] Figure 1 is the 1H NMR spectrum of the fluorescent probe HQBT of the present invention;

[0022] Figure 2 is the 13H NMR spectrum of the fluorescent probe HQBT of the present invention;

[0023] Figure 3 is the HRMS spectrum of the fluorescent probe HQBT of the present invention;

[0024] Figure 4 It is the absorption spectrum of the fluorescent probe HQBT of the present invention before and after binding with RNA;

[0025] Figure 5It is the emission spectrum of the fluorescent probe HQBT of the present invention before and after binding with RNA;

[0026] Figure 6 is the absorption spectrum of the fluorescent probe HQBT of the present invention in different solvents;

[0027] Figure 7 It is the emission spectrum of the fluorescent probe HQBT of the present invention in different solvents;

[0028] Figure 8 It is the fluorescence emission spectrum of the fluorescent probe HQBT of the present invention in solutions with different pH values;

[0029] Fig. 9 is the fluorescence emission spectrum of the fluorescent probe HQBT of the present invention in the presence of different analytes;

[0030] Fig.10 is the fluorescence spectrum of the fluorescent probe HQBT of the present invention titrated with yeast RNA;

[0031] Fig.11 is the MTT graph of the fluorescent probe HQBT of the present invention;

[0032] Fig.12 It is a co-localization image of the fluorescent probe HQBT of the present invention and the commercially stained SYTO RNA Select;

[0033] Fig.13 is a time diagram of the fluorescent probe HQBT of the present invention entering cells;

[0034] Fig.14 This is a diagram of the mechanism of the fluorescent probe HQBT of the present invention entering cells;

[0035] Fig.15 This is a comparison chart of the photostability of the fluorescent probe HQBT and SYTO RNA Select of the present invention;

[0036] Fig.16 It is a picture of SGs imaged by the fluorescent probe HQBT of the present invention in cells;

[0037] Fig.17 The fluorescent probe HQBT of the present invention is used to image the dynamic images of SGs in living cells under oxidative stress in real time;

[0038] Fig.18 The fluorescent probe HQBT of the present invention is used to image the dynamic images of SGs in living cells under heat stress in real time;

[0039] Fig.19 It is a schematic diagram of the preparation process of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] See also Figure 1-Figure 18 The embodiments of the present invention provide three technical solutions: near-infrared RNA selective fluorescent probe, preparation method and dynamic application of monitoring SGs in living cells, specifically including the following embodiments:

[0042] Embodiment 1

[0043] The present invention is a novel near-infrared RNA selective fluorescent probe, which contains a cationic group that can selectively bind to RNA. By adjusting the position of the nitrogen atom in the molecule, the binding ability to RNA is significantly enhanced. In addition, the probe introduces an additional hydroxyl functional group to achieve near-infrared emission and stronger RNA binding ability. The probe has an absorption wavelength of 561nm and an emission wavelength of 620nm, and can track the dynamics of RNA and SGs in living cells in real time through fluorescence imaging;

[0044] The structural formula of the RNA fluorescent probe is as follows:

[0045]

[0046] Embodiment 2

[0047] This example describes in detail the synthesis steps of the near-infrared RNA selective fluorescent probe HQBT. Fig.19 shown.

[0048] Step 1: Add iodomethane (0.50 g, 3.52 mmol) and 2-methylbenzothiazole (0.52 g, 3.52 mmol) to 50 mL of acetonitrile and reflux for 12 h, filter the precipitate, and wash with ethanol to obtain the intermediate (0.83 g, yield 82%). The intermediate is directly used in the next reaction without purification.

[0049] Step 2: Add the intermediate (0.20 g, 0.68 mmol), 8-hydroxyquinoline-5-acetaldehyde (0.11 g, 0.68 mmol), piperidine (2 drops), and glacial acetic acid (2 drops) to 50 mL of ethanol and reflux for 6 h. Remove the solvent by rotary evaporation and purify by silica gel chromatography with CH2Cl2:CH3OH=10:1 (V / V) as the eluent to obtain a reddish-brown powder HQBT (0.17 g, yield 57%), i.e., the RNA fluorescent probe.

[0050] 1H NMR(400MHz,d6-DMSO,δ):9.03-9.01(d,J=13.6Hz,1H),8.95-8.94(d,J=7.2Hz,1H),8 .77-8.73(d,J=7.6Hz,1H),8.56-8.54(d,J=7.2Hz,1H),8.40-8.38(d,J=7.2Hz,1H),8. 21-8.19(d,J=8.0Hz,1H),8.02-7.98(d,J=7.2Hz,1H),7.85-7.82(t,1H),7.77-7.76(d ,J=7.2Hz,1H),7.75-7.73(d,J=7.2Hz,1H),7.29-7.26(d,J=7.2Hz,1H),4.34(s,3H). Such as Figure 1 shown.

[0051] 13 C NMR (100 MHz, d6-DMSO, δ) 172.11, 159.21, 149.37, 144.02, 142.52, 138.68, 133.01, 130.94, 129.85, 128.76, 128.52, 128.25, 124.62, 123.87, 121.49, 117.23, 112.49, 112.76, 36.80. Figure 2 shown.

[0052] In positive ion mode, the peak of HQBT [M+H] + found, m / z = 319.0903 and the actual value m / z = 319.0900 also match. Figure 3 shown.

[0053] Embodiment 3

[0054] With respect to the near-infrared RNA fluorescent probe prepared in Example 2, its properties are specifically characterized in this example.

[0055] 1. Please refer to Figure 4-Figure 10 The absorption wavelength of the financial network RNA probe is 561nm ( Figure 4 ), the emission wavelength is 620nm( Figure 5 ). It exhibits solvatochromic effect in different solvents ( Figure 6 ), but its fluorescence intensity in different solvents is much lower than that when bound to RNA ( Figure 7 ), so it can be ignored. The fluorescence intensity of the probe does not change significantly under the condition of pH range 3-10, and is negligible compared with the fluorescence intensity in the presence of RNA ( Figure 8The presence of other analytes did not change the fluorescence intensity of the probe ( Fig. 9 ). As the RNA concentration in the solution increases, the fluorescence intensity of the probe gradually increases ( Fig.10 ). In the presence of 5mM HQBT, the viability of HepG2 cells was above 98% ( Fig.11 The above results indicate that the fluorescent probe HQBT can selectively respond to RNA and emit bright red light, has low cytotoxicity, and is expected to image RNA in living cells.

[0056] 2. Please refer to Fig.12 , Fig.13 , Fig.14 Confocal imaging in HepG2 cells showed that HQBT fluorescence was mainly concentrated in the cytoplasm and nucleolar regions, and the Pearson correlation coefficient after co-staining with SYTO RNA Select was 0.87, indicating a high degree of co-localization between HQBT and RNA. HQBT can quickly (within 10 seconds) enter living cells and selectively label RNA. Multiple inhibitor experiments showed that HQBT enters cells by free diffusion.

[0057] 3. Please refer to Fig.15 Experimental data showed that under continuous illumination, the fluorescence intensity of HQBT only decreased by about 10% within 20 minutes. In contrast, the fluorescence of SYTO RNA Select decreased by more than 80% during the same period, indicating that HQBT has excellent photostability compared to SYTO RNA Select and is expected to be used for long-term tracking of RNA and SGs.

[0058] 4. Please refer to Fig.16 , cells produce SGs under the induction of NaASO2. By co-localizing with the SGs core protein G3BP1 green fluorescent protein dye, it was verified that HQBT can stain SGs produced in living cells.

[0059] 5. Please refer to Fig.17 , HQBT was used to track the dynamic changes of SGs in HepG2 cells induced by NaASO2 within 45 minutes.

[0060] VI. Reference Fig.18 , HQBT was used to track the production of SGs under heat stress conditions and the disaggregation of SGs after the removal of heat stress.

[0061] In summary, the near-infrared RNA selective fluorescent probe HQBT of the present invention has an absorption peak of 561nm and an emission peak of 620nm, has excellent biocompatibility, and has little effect on the luminescence properties of the probe by various bioactive molecules and pH environmental changes in cells, and shows excellent photostability in living cells. Under normal conditions, HQBT is evenly distributed in the cytoplasm and nucleoli, binds to RNA in cells, and has stable fluorescence intensity. Under stress conditions, HQBT can quickly label and image SGs in real time.

[0062] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A near-infrared RNA selective fluorescent probe, characterized in that: The structural formula of the RNA selective fluorescent probe is as follows:

2. The method for preparing a near-infrared RNA selective fluorescent probe according to claim 1, characterized in that: First, 2-methylbenzothiazole and iodomethane react to synthesize an intermediate, and then the intermediate reacts with 8-hydroxyquinoline-5-carboxaldehyde to synthesize the RNA fluorescent probe; The reaction equation is as follows:

3. The method for preparing a near-infrared RNA selective fluorescent probe according to claim 2, characterized in that: The molar ratio of the 2-methylbenzothiazole to methyl iodide is: 1:(1.3-1.5).

4. The method for preparing a near-infrared RNA selective fluorescent probe according to claim 2, characterized in that: The 2-methylbenzothiazole reacts with methyl iodide at 83°C.

5. The method for preparing a near-infrared RNA selective fluorescent probe according to claim 2, characterized in that: The molar ratio of the intermediate to 8-hydroxyquinoline-5-carboxaldehyde is 1:(1.1-1.2).

6. The method for preparing a near-infrared RNA selective fluorescent probe according to claim 2, characterized in that: The intermediate reacts with 8-hydroxyquinoline-5-carboxaldehyde under ethanol reflux conditions, and piperidine and glacial acetic acid are added as catalysts.

7. Use of the RNA selective fluorescent probe according to claim 1 in imaging RNA in living cells and real-time tracking of stress granule dynamics in living cells.

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