Near-infrared rna-selective fluorescent probes, methods of making, and applications in tracking stress granule dynamics in live cells

Abstract

The application discloses a near-infrared RNA selective fluorescent probe, a preparation method and application of tracking stress granule dynamics in living cells, and belongs to the technical field of fluorescent probes. The probe is connected with a benzothiazole and a quinoline group through a -C=C- bond, has good biocompatibility and RNA selectivity, and can quickly, water-washing-free and selectively light up RNA related areas through confocal imaging. Compared with existing products, the probe has higher light stability and selectivity, and can effectively image stress granules in living cells. Through experiments, it is found that in addition to protein-RNA interaction, protein-protein and RNA-RNA interaction also participates in the assembly of SG, and the heterogeneous structure of SGs is revealed. In addition, the probe is used to track the formation and depolymerization process of stress granules in oxidative stress and thermal stress in real time. The probe has important significance for in-depth understanding of the dynamic behavior of RNA and stress granules in living cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and specifically 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 deprivation, viral infection, and tumor formation. SG formation is a cellular protective mechanism against adverse conditions, protecting cells from damage by halting protein synthesis. Abnormal accumulation of SGs has been linked to the development of a variety of diseases, including certain neurodegenerative disorders 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] Given that SGs are cohesive assemblages of mRNA and proteins, screening for RNA-targeting dyes is a logical approach to discover fluorescent probes that selectively aggregate and label SGs. A variety of 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 these, small molecule fluorescent probes offer higher spatial resolution, membrane permeability, and versatile photophysical properties, making RNA imaging in living cells simpler and more practical. However, the development of small molecule probes suitable for RNA analysis has been slow due to their stronger binding to DNA than RNA. SYTO RNA Select is the only commercially available RNA-selective probe, but its green emission 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, limited long-term imaging capability, susceptibility to photobleaching, and high cytotoxicity, all of which are essential for studying RNA dynamics in cellular processes. Furthermore, reports on the use of small molecule probes to label RNA for studying the structure and dynamics of SGs are limited, making the development of small molecule fluorescent probes for SG labeling and structural exploration challenging in the absence of available clues. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art by providing a near-infrared RNA-selective fluorescent probe, a preparation method, and its application in tracking stress granule dynamics in living cells. This fluorescent probe has the advantages of simple structure, ease of synthesis, good optical stability, and low cytotoxicity. Due to TICT, it exhibits almost no fluorescence in aqueous solution, but upon binding to RNA, its fluorescence intensity increases 33-fold. Furthermore, the probe can rapidly enter cells, bind to intracellular RNA, and emit bright red fluorescence. Its excellent photostability enables real-time tracking of RNA and SG dynamics in living cells via 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, has the following structural formula:

[0007]

[0008] The second object 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 reacting the intermediate with 8-hydroxyquinoline-5-carboxaldehyde to synthesize the RNA fluorescent probe.

[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, with piperidine and glacial acetic acid added as catalysts.

[0015] A 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 described in 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, exhibiting excellent anti-photobleaching ability. The fluorescence intensity decreases slowly under continuous laser irradiation, making it suitable for long-term tracking and dynamic imaging. The probe successfully locates SGs produced by living cells under stress conditions, and tracks the dynamics of SGs in 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, 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 existing technologies, such as being time-consuming and labor-intensive, expensive, causing invasive cell damage, and being unable to monitor SGs dynamics 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 through fluorescence imaging. The probe has a simple synthesis method, low cytotoxicity, and is capable of long-term real-time imaging. It can also avoid the influence of the molecule's own concentration and intracellular bioactive molecules. Fluorescence imaging can accurately and real-timely observe the dynamic changes of SGs in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 1 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 is the absorption spectrum of the fluorescent probe HQBT of the present invention before and after binding to RNA;

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

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

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

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

[0029] Figure 9 is the fluorescence emission spectrum of the fluorescent probe HQBT of the present application in the presence of different analytes;

[0030] Figure 10 is the fluorescence spectrum of the fluorescent probe HQBT of the present application in the presence of yeast RNA;

[0031] Figure 11 is the MTT diagram of the fluorescent probe HQBT of the present application;

[0032] Figure 12 is the co-localization diagram of the fluorescent probe HQBT of the present application and commercial dye SYTO RNA Select;

[0033] Figure 13 is the time diagram of the fluorescent probe HQBT of the present application entering cells;

[0034] Figure 14 is the mechanism diagram of the fluorescent probe HQBT of the present application entering cells;

[0035] Figure 15 is the light stability comparison diagram of the fluorescent probe HQBT of the present application and SYTO RNA Select;

[0036] Figure 16 is the SGs imaging diagram of the fluorescent probe HQBT of the present application in cells;

[0037] Figure 17 is the dynamic diagram of the fluorescent probe HQBT of the present application in real-time imaging of SGs in living cells under oxidative stress;

[0038] Figure 18 is the dynamic diagram of the fluorescent probe HQBT of the present application in real-time imaging of SGs in living cells under heat stress;

[0039] Figure 19 is a schematic diagram of the preparation process of the present application. 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 embodiments described 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0041] See also Figures 1-18 The present invention provides 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] Example 1

[0043] This invention is a novel near-infrared RNA-selective fluorescent probe. The probe contains a cationic group that selectively binds to RNA. By adjusting the position of the nitrogen atom in the molecule, its binding to RNA is significantly enhanced. Furthermore, an additional hydroxyl functional group is introduced into the probe to achieve near-infrared emission and stronger RNA binding. With an absorption wavelength of 561 nm and an emission wavelength of 620 nm, the probe can be used to 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] Example 2

[0047] This example describes in detail the synthesis steps of the near-infrared RNA selective fluorescent probe HQBT. Figure 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, 82% yield). The intermediate was used directly in the next reaction without purification.

[0049] Step 2: 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) were added to 50 mL of ethanol and refluxed for 6 h. The solvent was removed by rotary evaporation and purified 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%), which is 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 agree. Figure 3 shown.

[0053] Example 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 4-10 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 conditions 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 ( Figure 9 As the RNA concentration in the solution increases, the fluorescence intensity of the probe gradually increases ( Figure 10 In the presence of 5mM HQBT, the viability of HepG2 cells was above 98% ( Figure 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 be used for RNA imaging in living cells.

[0056] 2. Please refer to Figure 12 、 Figure 13 、 Figure 14 Confocal imaging of HepG2 cells revealed that HQBT fluorescence was primarily concentrated in the cytoplasm and nucleolar regions. A Pearson correlation coefficient of 0.87 was observed after co-staining with SYTO RNA Select, demonstrating a high degree of colocalization between HQBT and RNA. HQBT can rapidly enter living cells (within 10 seconds) and selectively label RNA. Experiments with various inhibitors demonstrated that HQBT enters cells by free diffusion.

[0057] 3. Please refer to Figure 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 Figure 16 Under the induction of NaASO2, cells generate SGs. By co-localizing with the SGs core protein G3BP1 green fluorescent protein dye, it was verified that HQBT can stain SGs generated in living cells.

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

[0060] 6. Reference Figure 18 , used HQBT to track the generation 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 application has an absorption peak of 561 nm and an emission peak of 620 nm, has excellent biocompatibility, and has no influence on the luminescent properties of the probe caused by various biological active molecules and pH environment changes in cells, and shows excellent light stability in living cells. Under normal conditions, HQBT is uniformly distributed in the cytoplasm and nucleolus of cells, is combined with RNA in the cells, and has stable fluorescence intensity. Under stress conditions, HQBT can rapidly label and real-time image SGs.

[0062] The above detailed description part specifically introduces the analysis method involved in the present application. It should be noted that the above introduction is only to help those skilled in the art better understand the method and idea of the present application, and is not a limitation on the related content. Those skilled in the art can also make appropriate adjustments or modifications to the present application without departing from the principles of the present application, and the above adjustments and modifications should also belong to the protection scope of the present application.

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, wherein First, 2-methylbenzothiazole and iodomethane react to synthesize an intermediate, and then the intermediate reacts with 8-hydroxyquinoline-5-carboxaldehyde to synthesize the RNA selective fluorescent probe; The reaction equation is as follows:

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

7. Use of the near-infrared RNA selective fluorescent probe according to claim 1 in preparing an RNA imaging reagent in living cells or a reagent for real-time dynamic tracking of stress granules in living cells.

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