Preparation method and application of RNA fluorescent probe based on biological orthogonal reaction

Abstract

The application belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of an RNA fluorescent probe based on biological orthogonal reaction, and the structural formula of the fluorescent probe is: the application provides a preparation method for generating an RNA activated near-infrared fluorescent probe based on biological orthogonal reaction. First, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-en-1-methanal and 6-bromo-1-ethyl-2-methylquinolin-1-ium salt are used as raw materials to synthesize a precursor compound beta-Br, and then the beta-Br is rapidly in-situ generated into a fluorescent probe under physiological conditions. After the fluorescent probe generated based on biological orthogonal reaction is reacted with RNA, the fluorescence intensity at the near-infrared region 670-900 nm is enhanced with the increase of the RNA concentration, and the fluorescence enhancement multiple reaches 400. Secondly, the fluorescent probe generated based on biological orthogonal reaction shows high selectivity to RNA, and is not affected by other various ions, active oxygen, RNA bases, biological thiols and amino acids. Finally, the fluorescent probe can be washed-free and fast imaging in living cells, and can be in-situ fast generated into a fluorescent probe in cells, and can be used for monitoring intracellular ribosomal RNA, distinguishing between living cells and dead cells, and real-time monitoring of intracellular oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a method for preparing RNA fluorescent probes based on biological orthogonal reactions and their applications. Background Technology

[0002] Bioorthogonal chemistry is a class of chemical reactions characterized by high rate, high yield, and excellent selectivity. These reactions can proceed efficiently under physiological conditions without interfering with surrounding biological processes (Scinto SL, Bilodeau DA, Hincapie R., et al. Bioorthogonal chemistry. Nature Reviews Methods Primes. 2021, 1(1): 30). In recent years, bioorthogonal reactions have achieved groundbreaking progress in several research fields, including biomedical imaging, medicinal chemistry, protein engineering, and biomaterials. These reactions are hailed as revolutionary tools in chemical biology, primarily due to their several core characteristics: First, their exceptional selectivity, or "orthogonality." The functional groups involved (such as azides, alkynes, and tetrazides) have almost no natural counterparts in vivo, thus enabling highly specific targeting and preventing cross-reactions with the vast number of endogenous groups within cells. Second, their excellent biocompatibility ensures that the reactions can be completed rapidly under mild conditions, typically without the need for toxic catalysts, and without significant impact on cell viability. Furthermore, bioorthogonal reactions are non-invasive and allow for precise control of labeled events in both temporal and spatial dimensions, significantly expanding their applicability in medical and clinical research (Wang LP, Huang YJ, Wang J., et al. Bioorthogonal reaction of β-chloroacroleins with meta-aminothiophenol to develop near-infrared fluorogenic probes for simultaneous two-color imaging. Journal of the American Chemical Society, 2025, 147(8): 6707–6716), providing an important research tool for elucidating complex molecular mechanisms in living systems. Fluorescence imaging technology itself possesses significant advantages such as high sensitivity, strong specificity, and the ability to achieve real-time dynamic imaging. Therefore, developing high-performance fluorescent probes based on the principle of bioorthogonal reactions has significant scientific value and broad application prospects.

[0003] In the field of biomedical research, the visualization and dynamic tracking of RNA are of great significance for in-depth analysis of core biological issues such as gene expression regulation mechanisms, RNA metabolism processes, and intracellular signal transduction (Yang M., Prestwood P. R., Passalacqua LFM, et al. Structure-informed design of anultrabright RNA-activated fluorophore. Nature Chemistry, 2025, 17(8): 1188–1195). RNA molecules have complex spatial distribution characteristics and dynamic changes. After transcription, they undergo a series of processes such as processing, transport, translation, and degradation, and play diverse functions at different life stages (Zuo FT, Jiang, L., Su, N., et al. Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA. Nature Chemical Biology, 2024, 20(10): 1272–1281). Since RNA itself does not have autofluorescence properties, imaging of it must rely on fluorescently labeled probes to selectively label the target RNA. Currently, RNA labeling strategies are mainly divided into two categories: one is the polymerized RNA motif labeling system based on RNA-binding proteins (such as the MS2-MCP system). Although this method has been widely adopted and is considered one of the gold standards for RNA labeling, it often results in a low signal-to-background ratio during imaging due to the background signal of unbound fluorescent fusion proteins. The other is the labeling system based on fluorescently activated RNA aptamers. This type of strategy can effectively reduce the background signal; however, most systems still rely on exogenous fluorescent dyes, which face difficulties in cell and in vivo delivery. Some dyes may also pose a risk of non-specific activation in complex physiological environments (Zhou WJ, Wu MY, Shao XJ, et al. Fluorogenic interacting protein stabilization for orthogonal RNA imaging. Angewandte Chemie International Edition, 2025, 64(25): e202502350). Therefore, developing novel technologies that can achieve in situ RNA tracing and functional regulation at the living cell level is of great scientific significance and application value for revealing the spatiotemporal dynamic regulatory mechanism of RNA and promoting research on RNA-targeted therapeutic strategies.

[0004] Traditional fluorescent probes are often limited in practical applications by their short excitation wavelengths, resulting in limited tissue penetration depth, and by significant spectral crosstalk between excitation and emission light. Compared to traditional oxanthracene fluorophores, thioxanthracene fluorophores exhibit longer Stokes shifts, thus possessing stronger tissue penetration capabilities during imaging and effectively suppressing interference from background autofluorescence (Yang ZH, Yang ZC, Zhao JT, et al. Triple-synergistic mitochondria-targeted NIR fluorescent probe for mitochondrial hydrogen sulfide: precision monitoring and image-guided resection of metastatic breast cancer. Analytical Chemistry, 2025, 97(40):22318–22329). By introducing a bioorthogonal reaction strategy to synthesize thioxanthracene fluorophores, the imaging signal-to-noise ratio can be significantly improved, providing a powerful tool for precise studies at the cellular level, thereby enabling efficient tracking of RNA dynamics.

[0005] Based on the above advantages, constructing RNA-activated fluorescent probes using bioorthogonal reactions has become an innovative and efficient strategy for monitoring cellular physiological processes, and has important research value for revealing the dynamic regulatory mechanisms of RNA. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes an RNA-activated near-infrared fluorescent probe based on a bioorthogonal reaction, and provides its preparation method and applications. This fluorescent probe can rapidly undergo a bioorthogonal reaction under physiological conditions, generating fluorescent molecules with near-infrared emission characteristics in situ within the physiological environment, thereby effectively reducing background signals and significantly improving the imaging signal-to-noise ratio. Simultaneously, this probe exhibits good specificity for RNA recognition and high detection sensitivity. Therefore, this invention provides a new technical approach to solving the problems of high background interference and insufficient specificity in existing RNA fluorescence imaging techniques.

[0007] The technical solution of this invention is to provide an RNA-activated near-infrared fluorescent probe based on a biological orthogonal reaction, the structural formula of its precursor compound β-Br is as follows:

[0008] .

[0009] This invention provides an RNA-activated near-infrared fluorescent probe based on a biological orthogonal reaction, and the preparation method of its precursor compound β-Br is as follows:

[0010] (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-en-1-carboxaldehyde and 6-bromo-1-ethyl-2-methylquinoline-1-onium salt were added to a round-necked flask, dissolved in anhydrous ethanol, and the mixture was heated and stirred. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain the precursor compound β-Br.

[0011] Preferably, the molar ratio of (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde and 6-bromo-1-ethyl-2-methylquinoline-1-onium salt is 1-1.5 : 1.

[0012] Preferably, the solvent, anhydrous ethanol, contains 10-20 mL.

[0013] Preferably, the reaction temperature is 70-80 °C.

[0014] Preferably, the stirring reaction time is 11-12 hours.

[0015] Preferably, the eluent used for silica gel column chromatography purification is dichloromethane:methanol = 200:1-6.

[0016] The technical solution of this invention is to provide an RNA-activated near-infrared fluorescent probe Q-NH2 based on a biological orthogonal reaction, the structural formula of which is as follows:

[0017] .

[0018] This invention provides a method for preparing an RNA-activated near-infrared fluorescent probe Q-NH2 based on a biological orthogonal reaction, as follows:

[0019] The precursor compound β-Br was dissolved in H2O (containing 20% ​​DMSO) solution, and then m-aminothiophenol was added. The mixture was shaken at room temperature to allow for a rapid reaction, thus obtaining the target fluorescent probe Q-NH2.

[0020] Preferably, the molar ratio of β-Br to m-aminothiophenol is 1:2.5-3.

[0021] This invention provides an RNA-activated near-infrared fluorescent probe that rapidly generates a fluorescent signal under physiological conditions in vivo via a bioorthogonal reaction, significantly reducing background noise and improving the signal-to-noise ratio of imaging. This fluorescent probe exhibits high selectivity for RNA and high sensitivity, facilitating precise monitoring of RNA metabolism and biological functions in vivo. Furthermore, the near-infrared fluorescent probe generated based on the bioorthogonal reaction can specifically locate ribosomal RNA in the cytoplasm, and its unique subcellular localization mechanism enables real-time monitoring of changes in cellular physiological states. Under oxidative stress conditions, the fluorescent probe translocates from ribosomes in the cytoplasm to the nucleolus, thus allowing for monitoring of intracellular oxidative stress processes. In addition, this probe exhibits strong near-infrared fluorescence emission in response to RNA, showing potential for imaging applications in deep tissues and live animals.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) Highly sensitive fluorescence signal generation: The precursor compound undergoes a rapid bioorthogonal reaction with m-aminothiophenol to generate a fluorescent probe in situ. After RNA activation, the fluorescence signal of the probe increases by 400-fold in situ, and with the increase of RNA concentration, the fluorescence intensity of the probe in the range of 700 nm to 900 nm is significantly enhanced, showing high sensitivity.

[0024] (2) Cell viability differentiation: Near-infrared fluorescent probes generated based on bioorthogonal reactions can accurately distinguish between live and dead cells. In live cells, the fluorescent probes specifically bind to ribosomal RNA in the cytoplasm; when cells die, leading to nuclear membrane rupture and increased permeability, the probes enter the nucleus and bind to RNA within the nucleolus. This difference in distribution provides a reliable basis for imaging cell viability.

[0025] (3) Monitoring oxidative stress: Near-infrared fluorescent probes generated based on bioorthogonal reactions can sensitively respond to the oxidative stress state of cells. Under normal physiological conditions, the fluorescent probes are specifically located in the ribosomal RNA of the cytoplasm; while under oxidative stress stimulation such as H2O2, the fluorescent probes undergo significant subcellular translocation and enter the nucleolar region, becoming an effective monitoring tool for the cellular oxidative stress process. Attached Figure Description

[0026] Figure 1 This is a synthetic route diagram for the fluorescent probe Q-NH2.

[0027] Figure 2 For the precursor compound β-Br 1 H NMR spectrum.

[0028] Figure 3 For the fluorescent probe Q-NH21 H NMR spectrum.

[0029] Figure 4 This is a high-resolution mass spectrum of the fluorescent probe Q-NH2.

[0030] Figure 5 The absorption spectrum of the precursor compound β-Br (20 μM) reacting with m-aminothiophenol in H2O (containing 20% ​​DMSO) solution and responding to RNA (900 ug / mL) is shown.

[0031] Figure 6 The fluorescence spectrum of the precursor compound β-Br (20 μM) reacting with m-aminothiophenol in H2O (containing 20% ​​DMSO) solution and responding to RNA (900 ug / mL) is shown.

[0032] Figure 7 The fluorescence spectra of the precursor compound β-Br (20 μM) reacting with m-aminothiophenol in H2O (containing 20% ​​DMSO) solution and then reacting with different concentrations of RNA are shown.

[0033] Figure 8 The linear relationship between the precursor compound β-Br (20 μM) reacting with m-aminothiophenol in H2O (containing 20% ​​DMSO) solution and then with different concentrations of RNA is shown.

[0034] Figure 9 The fluorescence intensity at 745 nm is shown for the precursor compound β-Br (20 μM) reacting with m-aminothiophenol in H2O (containing 20% ​​DMSO) solution, followed by reactions with different analytes (10 mM).

[0035] Figure 10 Confocal fluorescence imaging of live and dead cells using near-infrared fluorescent probes generated based on bioorthogonal reactions (scale bar 10 μm).

[0036] Figure 11 Confocal fluorescence imaging (scale bar 10 μm) of near-infrared fluorescent probes generated based on bioorthogonal reactions to monitor H2O2-stimulated oxidative stress in 4T1 cells. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to the prior art, and reagents or instruments used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0038] Example 1:

[0039] Synthesis of precursor compound β-Br and fluorescent probe Q-NH2

[0040] Synthetic routes such as Figure 1 As shown. (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-en-1-carboxaldehyde (0.1278 g, 0.74 mmol) and 6-bromo-1-ethyl-2-methylquinoline-1-onium salt (0.1548 g, 0.62 mmol) were added to a round-necked flask, dissolved in anhydrous ethanol (10 mL), and heated to 80 °C with stirring for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding a red solid, which was the precursor compound β-Br (0.089 g), in a yield of approximately 35.6%.

[0041] The prepared compounds were characterized using proton nuclear magnetic resonance spectroscopy: 1 ¹H NMR (400 MHz, DMSO) δ 9.01 (d, J = 8.7 Hz, 2H), 8.73 (s, 2H), 8.56 (d, J = 9.4 Hz, 2H), 8.35 (d, J = 9.5 Hz, 2H), 8.17 (d, J = 8.0 Hz, 2H), 5.76 (s, 1H), 4.98 (d, J = 7.6 Hz, 4H), 3.09 (s, 4H), 1.51 (s, 3H), 1.23 (s, 2H). The ¹H NMR spectrum is shown below. Figure 2 As shown, this confirms that the compound is the precursor compound β-Br.

[0042] The precursor compound β-Br was dissolved in H2O (containing 20% ​​DMSO) solution, and then m-aminothiophenol was added. The mixture was shaken at room temperature to allow for a rapid reaction, thus obtaining the target fluorescent probe Q-NH2.

[0043] The prepared compounds were characterized using proton nuclear magnetic resonance spectroscopy: 1H NMR (400 MHz, DMSO) δ8.85 (d, J = 9.1 Hz, 1H), 8.67 (d, J = 2.3 Hz, 1H), 8.56 (d, J = 9.1 Hz, 1H), 8.47 (d, J = 9.5 Hz, 1H), 8.29 (s, 1H), 8.28 – 8.24 (m, 1H), 7.29 (s, 1H),7.08 (d, J = 15.3 Hz, 1H), 5.03 (d, J = 7.6 Hz, 2H), 4.18 – 4.14 (m, 1H),2.70 (t, J = 6.2 Hz, 2H), 1.77 (d, J = 6.7 Hz, 2H), 1.51 (d, J = 7.3 Hz, 2H), 1.26 (d, J = 7.0 Hz, 3H). The proton NMR spectrum is shown below. Figure 3 As shown, this confirms that the compound is the fluorescent probe Q-NH2.

[0044] Furthermore, the prepared compound was further validated by high-resolution mass spectrometry: HR-MS (ESI, m / s): the theoretically calculated molecular mass-to-charge ratio is [M]+ C. 26 H 24 BrN2S + The actual molecular weight obtained was 475.0832, with a value of 475.0838. The high-resolution mass spectrum is shown below. Figure 4 As shown, this further illustrates that the structure of the prepared fluorescent probe is as expected.

[0045] Example 2:

[0046] Fluorescent probes and RNA responses generated based on bioorthogonal reactions

[0047] First, the spectroscopic properties of the fluorescent probe Q-NH2, generated after a bioorthogonal reaction between the precursor compound β-Br and m-aminothiophenol, were examined in response to RNA. For example... Figure 5 As shown, the maximum absorption wavelength of the precursor compound β-Br is 475 nm. Upon addition of m-aminothiophenol, the maximum absorption wavelength red-shifts to 625 nm, indicating the formation of the fluorescent probe Q-NH2. Simultaneously, after the reaction of the precursor compound β-Br with aminothiophenol, the absorption of the probe at 625 nm is enhanced, indicating that the fluorescent probe Q-NH2 responds to RNA. Then, the fluorescence spectra of the precursor compound β-Br, the reaction of the precursor compound β-Br with m-aminothiophenol, and the response of the precursor compound β-Br with m-aminothiophenol followed by RNA were investigated, as shown below. Figure 6As shown, the precursor compound β-Br itself is non-fluorescent, but produces weak fluorescence after reacting with m-aminothiophenol. However, after reacting with RNA, the fluorescence signal of the probe at 745 nm rapidly increases by 400-fold, indicating that the fluorescent probe generated based on the bioorthogonal reaction has high sensitivity for RNA detection. Finally, we further investigated the fluorescence spectra of the fluorescent probe generated based on the bioorthogonal reaction with different concentrations of RNA. Figure 7 As shown and Figure 8 As shown, the characteristic peak of the fluorescent probe at 745 nm gradually increased with increasing RNA concentration, and the fluorescence intensity of Q-NH2 showed a good linear relationship with RNA concentration when the RNA concentration was between 200 ug / mL and 900 ug / mL (R0.05). 2 =0.9913).

[0048] Example 3:

[0049] Selectivity of fluorescent probes generated based on bioorthogonal reactions

[0050] To verify the selectivity of the fluorescent probe for RNA, the selectivity of the fluorescent probe generated based on a bioorthogonal reaction with different interfering substances (such as metal ions Na+) was measured. 2+ K + Mg 2+ Cu 2+ Zn 2+ etc., reactive oxygen species ClO - H2O2, ONOO - Fluorescence spectra after interaction with RNA bases A, U, C, G, biothiols Cys, CSH, and related amino acids L-Ile, L-Phe, L-Lys, L-Leu, etc. For example... Figure 9 As shown, the fluorescence signal at 745 nm only shows a significant enhancement after the probe responds to RNA, indicating that the fluorescent probe exhibits high selectivity for RNA.

[0051] Example 4:

[0052] Intracellular applications of fluorescent probes generated by bioorthogonal reactions

[0053] 10 μM of the precursor compound β-Br was added to normal and dead 4T1 cells, respectively, followed by the addition of m-aminothiophenol. This rapidly generated the fluorescent probe Q-NH2 in situ within the cells, which was excited using a 640 nm laser, with the acquisition wavelength ranging from 650 to 750 nm. Figure 10As shown, in normal cells, the fluorescence signal of the fluorescent probe generated based on the orthogonal reaction is mainly located in the cytoplasmic ribosomes, indicating that the fluorescent probe specifically binds to ribosomal RNA. However, when cell death leads to nuclear membrane rupture and increased permeability, the fluorescence signal of the fluorescent probe generated based on the orthogonal reaction is mainly located in the nucleolus, indicating that the probe can also bind to ribosomal RNA within the nucleolus. This clear difference in distribution provides a reliable imaging basis for distinguishing cell viability.

[0054] 4T1 cells were incubated with H2O2 to induce oxidative stress, and then a near-infrared fluorescent probe was generated in situ using the precursor compound β-Br and m-aminothiophenol. The results are as follows: Figure 11 As shown, after the addition of H2O2, the fluorescence signal of the fluorescent probe generated based on the bioorthogonal reaction enters the nucleolar region from the ribosomes in the cytoplasm. This experimental phenomenon provides a reliable imaging basis for real-time monitoring of intracellular oxidative stress processes and cell viability.

Claims

1. An RNA fluorescent probe based on a biological orthogonal reaction, characterized in that, Its chemical structure is as follows: 。 2. The method for preparing RNA fluorescent probes based on bioorthogonal reactions according to claim 1, characterized in that, Its synthetic route is as follows: 。 3. The method for preparing RNA fluorescent probes based on bioorthogonal reactions according to claim 2, characterized in that, The reaction steps include the following: (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-en-1-carboxaldehyde and 6-bromo-1-ethyl-2-methylquinoline-1-onium salt were added to a round-necked flask, dissolved in anhydrous ethanol, and the mixture was heated and stirred. After the reaction was completed, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain the precursor compound β-Br. The precursor compound β-Br was dissolved in H2O (containing 20% ​​DMSO) solution, and then m-aminothiophenol was added. The mixture was shaken at room temperature to allow for a rapid reaction, thereby obtaining the fluorescent probe Q-NH2.

4. The method for preparing RNA fluorescent probes based on bioorthogonal reactions according to claim 3, characterized in that, The molar ratio of (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde and 6-bromo-1-ethyl-2-methylquinoline-1-onium salt is 1-1.5:1; the molar ratio of β-Br and m-aminothiophenol is 1:2.5-3.

5. The application of the fluorescent probe Q-NH2 based on bioorthogonal reaction according to claim 1, characterized in that, The fluorescent probe Q-NH2, generated by a bioorthogonal reaction, responds to RNA, and its fluorescence intensity at 745 nm in the near-infrared region increases with increasing RNA concentration, exhibiting a good linear relationship.

6. The application of the fluorescent probe Q-NH2 based on bioorthogonal reaction according to claim 1, characterized in that, The fluorescent probe Q-NH2, generated based on a bioorthogonal reaction, reacts with RNA, and its fluorescence rapidly increases by 400 times.

7. The application of the fluorescent probe Q-NH2 based on bioorthogonal reaction according to claim 1, characterized in that, The precursor compound β-Br undergoes a rapid in-situ bioorthogonal reaction within 4T1 cells, generating the fluorescent probe Q-NH2, which can be used to monitor ribosomal RNA within the cells.

8. The application of the fluorescent probe Q-NH2 based on bioorthogonal reaction according to claim 1, characterized in that, The fluorescent probe Q-NH2, generated based on a bioorthogonal reaction in 4T1 cells, can effectively distinguish between live and dead cells.

9. The application of the fluorescent probe Q-NH2 based on bioorthogonal reaction according to claim 1, characterized in that, The fluorescent probe Q-NH2, generated based on a bioorthogonal reaction, can monitor intracellular oxidative stress processes in 4T1 cells.

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