Multivariable gating signal amplification system and application thereof in miRNA-21 imaging

Abstract

The invention discloses a multivariable gating signal amplification system and an application thereof in miRNA-21 imaging, and relates to the technical field of probe design of in-situ imaging of miRNA in cells. The multivariable gating signal amplification system provided by the invention comprises molecular beacon probes H1 and H2; the nucleotide sequence of the molecular beacon probe H1 is as shown in SEQ ID NO.1, a disulfide bond is embedded between the eighth basic group and the ninth basic group from the 3'end of the nucleotide sequence, and a fluorophore Cy3 is modified on the fifteenth basic group; the nucleotide sequence of the molecular beacon probe H2 is shown as SEQ ID NO.2, a light splitting linking bond (PC-linker) is embedded between the eighth basic group and the ninth basic group from the 3'end of the nucleotide sequence, and a fluorophore Cy5 is modified at the 5 'end. Besides, the molecular beacon probes H1 and H2 and the water-phase up-conversion nanoparticles coated with the polylysine are prepared into a compound, so that the miRNA-21 detection in a non-cell environment and a cell environment is realized, and the miRNA-21 detection probe has the advantages of high specificity, high detection sensitivity and high stability.

Description

Technical Field

[0001] The invention relates to the technical field of probe design for intracellular miRNA in situ imaging, and in particular to a multivariate gated signal amplification system and application thereof in miRNA-21 imaging. Background Art

[0002] Cancer is one of the leading causes of death in humans, posing a great threat to human health and placing a significant burden on the global public health system. MicroRNA (miRNA) is an important endogenous gene regulator that participates in many biological processes in humans / animals, including development, differentiation, apoptosis, and proliferation. Its abnormal expression has been shown to be closely related to the occurrence of various cancers. Studies have shown that some upregulated miRNAs can prevent the expression of downstream proteins and affect many cancer-related pathways, playing a role in all stages of cancer initiation, development, and metastasis. Therefore, accurate imaging of overexpressed target miRNAs in cancer cells is crucial for researchers to understand the occurrence and development of cancer. However, the abundance of miRNAs in cancer cells is low, and their accurate imaging is severely hampered by insufficient sensitivity.

[0003] At present, the industry has developed many miRNA signal amplification strategies to improve the sensitivity of miRNA detection. Among them, enzyme-assisted signal amplification systems, such as rolling circle amplification and loop-mediated isothermal amplification, are severely limited in in vivo applications. The probes used in traditional enzyme-free signal amplification systems, such as hybridization chain reaction (HCR) and catalytic hairpin assembly system (CHA), are usually always in an activated state. However, the target miRNA is usually present outside the target area at a non-negligible level, which makes the amplification reaction of the traditional probe passively triggered by the target before reaching the target area, resulting in serious background signal interference. Therefore, the development of a stable and reliable method to achieve high-sensitivity and specific imaging of miRNA in vivo has become an urgent need in the industry. Summary of the invention

[0004] The present invention provides a multivariate gated signal amplification system and its application in miRNA-21 imaging, which has the advantages of strong specificity, high detection sensitivity and stability for miRNA-21 detection, and realizes spatially and temporally controllable amplification imaging of miRNA-21. It is specifically achieved through the following technologies.

[0005] A method for constructing a multivariate gated signal amplification system comprises the following steps:

[0006] Design of molecular beacon probe H 1 and H 2 ; The molecular beacon probe H 1The nucleotide sequence is shown in SEQ ID NO.1, and the molecular beacon probe H 1 A disulfide bond is embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the 15th base is modified with a fluorescent group Cy3; the molecular beacon probe H 2 The nucleotide sequence is shown in SEQ ID NO.2, and the molecular beacon probe H 2 A photocleavable linker (PC-linker) was embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the fluorescent group Cy5 was modified at the 5' end;

[0007] The multivariate gated signal amplification system is used to detect miRNA-21.

[0008] Furthermore, the method for constructing a multivariate gated signal amplification system further includes the following steps:

[0009] Prepare water-phase upconversion nanoparticles, coat polylysine on the water-phase upconversion nanoparticles, and coat the water-phase upconversion nanoparticles coated with polylysine and the molecular beacon probe H 1 and H 2 Mix and incubate to obtain a multivariate gated signal amplification system.

[0010] In the method for constructing the multivariate gated signal amplification system provided by the present invention, oil-phase upconversion nanoparticles are prepared by pyrolysis, and then the oil-phase upconversion nanoparticles are centrifuged and washed by acid ethanol and ultrapure water to prepare uniform water-phase upconversion nanoparticles UCNPs capable of converting long-wavelength near-infrared light into short-wavelength ultraviolet light; polylysine and the water-phase upconversion nanoparticles are stirred at room temperature to obtain polylysine-coated water-phase upconversion nanoparticles; finally, the polylysine-coated water-phase upconversion nanoparticles are mixed with a molecular beacon probe H 1 and H 2 (The combination of the two is M-CHA) mixed and incubated to obtain the M-CHA / U complex, that is, the multivariate gated signal amplification system.

[0011] The multivariate gated signal amplification system provided by the present invention may include only the molecular beacon probe H 1 and H 2 , the molecular beacon probe H 1 and H 2 Combined with poly-lysine-coated aqueous upconversion nanoparticles to form M-CHA / U complex. According to the different environmental conditions for detecting miRNA-21, select the appropriate multivariate gated signal amplification system. When detecting miRNA-21 in non-cellular environments such as test tubes, directly inject the molecular beacon probe H 1 and H 2Add to the sample to be tested, add GSH (glutathione), incubate and detect.

[0012] Furthermore, poly-lysine-coated aqueous upconversion nanoparticles and molecular beacon probes H 1 and H 2 The molar ratio is 1:(30-50):(30-50).

[0013] Furthermore, poly-lysine-coated aqueous upconversion nanoparticles and molecular beacon probes H 1 and H 2 The molar ratio is 1:50:50.

[0014] The present invention also provides a molecular beacon probe combination, wherein the molecular beacon probe H 1 The nucleotide sequence is shown in SEQ ID NO.1, and the molecular beacon probe H 1 A disulfide bond is embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the 15th base is modified with a fluorescent group Cy3; the molecular beacon probe H 2 The nucleotide sequence is shown in SEQ ID NO.2, and the molecular beacon probe H 2 A photocleavable linker (PC-linker) is embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and a fluorescent group Cy5 is modified at the 5' end; the molecular beacon probe combination is used to detect miRNA-21.

[0015] The present invention also provides a multivariable gated signal amplification system, which is constructed using any one of the construction methods described above.

[0016] The multivariate gated signal amplification system provided by the present invention has the key technical feature of using a molecular beacon probe targeting miRNA-21. 1 The disulfide bond is inserted between the 8th and 9th bases from the 3' end of the sequence, which is used to utilize its ability to break in response to GSH stimulation to achieve H 1 It is activated under the stimulation of GSH; the fluorescent group Cy3 is modified on the 15th base, which acts as a donor for fluorescence resonance energy transfer (FRET). When FRET occurs, Cy3 can transfer its own emitted energy to the receptor; molecular beacon probe H 2 A photocleavable linker (PC-linker) is embedded between the 8th and 9th bases from the 3' end of the sequence. Its function is to utilize its ability to break in response to ultraviolet (UV) stimulation, so that under NIR irradiation, the upconversion nanoparticles convert NIR into UV to H 2 Activation. Molecular beacon probe H 2The fluorescent group Cy5 is modified at the 5' end of the sequence, which acts as a FRET acceptor and emits corresponding fluorescence by receiving energy from the donor.

[0017] Molecular beacon probe H 1 and H 2 , and the nucleotide sequence of miRNA-21 is shown in Table 1 below.

[0018] Table 1

[0019]

[0020] The expression of GSH in tumor tissues was significantly higher than that in normal tissues. 1 It is activated only in tumor tissues, so the multivariable gated signal amplification system provided by the present invention has different activation conditions in tumor tissues and normal tissues, and has spatially controllable imaging capabilities. 2 The PClinker on the miRNA-21 is broken, and can be activated at a specific time point according to the needs of the operator, so that the present invention has a time-controlled imaging capability, that is, it realizes the time-space controllable amplification imaging of miRNA-21.

[0021] The present invention also provides an application of the molecular beacon probe combination, which is specifically used for detecting miRNA-21 in a non-cellular environment for purposes other than disease diagnosis and treatment.

[0022] Furthermore, in the above application, when the detection environment is a non-cellular environment, the sample to be tested is placed in a detection container, and the molecular beacon probe combination and GSH are added; the reaction system is irradiated with ultraviolet light and then incubated at room temperature; finally, the fluorescence intensity of the reaction system is detected to complete the detection.

[0023] The present invention also provides application purposes of the multivariate gated signal amplification system, which is specifically used for detecting miRNA-21 in a cell environment for purposes other than disease diagnosis and treatment.

[0024] The "not for the purpose of disease diagnosis and treatment" claimed in the present invention refers to the detection of miRNA-21 in a cellular environment or a non-cellular environment under non-clinical conditions (for example, in laboratory scientific research or teaching). That is, as long as the application scenario is not directly used for the diagnosis or treatment of diseases / symptoms related to vesicular viruses in the human or animal body, the above-mentioned technical means provided by the present invention can be used. It is easy to think that in clinical practice, the nano-hairpin probe combination provided by the present invention can also be considered for miRNA-21 detection.

[0025] Compared with the prior art, the present invention is beneficial in that:

[0026] 1. The multivariate gated signal amplification system constructed by the present invention uses disulfide bonds and photocleavable linkers as gating units for amplification, and has excellent sensitivity and specificity when performing miRNA-21 detection and imaging under clinical or non-clinical conditions.

[0027] 2. The multivariable gated signal amplification system constructed by the present invention utilizes polylysine-coated aqueous upconversion nanoparticles as a converter for converting near-infrared light into ultraviolet light, thereby avoiding the direct use of exogenous ultraviolet light to activate the system, greatly improving the light penetration depth and biocompatibility; polylysine-coated aqueous upconversion nanoparticles are used as probe carriers, and also have good intracellular probe delivery efficiency and spatiotemporal resolution during cell imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The molecular beacon probe H prepared in Example 1 1 and H 2 (M-CHA) Schematic diagram of the principle of miR-21 specific amplification imaging;

[0029] Figure 2 The feasibility analysis results of programmed activation of the molecular beacon probe M-CHA prepared in Example 1

[0030] Figure 3 Graphs showing (a) sensitivity and (b) specificity of the molecular beacon probe M-CHA prepared in Example 1 for miR-21.

[0031] Figure 4 (a) Preparation schematic diagram, (b) TEM image and (c) fluorescence emission spectrum of the multivariate gated signal amplification system M-CHA / U prepared in Example 2 under 980 nm near-infrared light irradiation.

[0032] Figure 5 This is a cytotoxicity test of the multivariate gated signal amplification system M-CHA / U prepared in Example 2.

[0033] Figure 6 This is a selective imaging image of SW480 cancer cells of the multivariate gated signal amplification system M-CHA / U prepared in Example 2. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0035] The multivariate gated signal amplification system provided by the present invention is specially constructed for miRNA-21 detection. The multivariate gated signal amplification system at least includes a molecular beacon probe H 1 and H 2 This multivariate gated signal amplification system can be used to detect miRNA-21 in a non-cellular environment.

[0036] In addition, aqueous upconversion nanoparticles coated with poly-lysine can be prepared to interact with the molecular beacon probe H 1 and H 2 The M-CHA / U complex is formed. This M-CHA / U complex is used as a multivariate gated signal amplification system to detect miRNA-21 in the cellular environment.

[0037] Regardless of which multivariate gated signal amplification system is used, the principle of detecting miRNA-21 is the same as Figure 1 shown.

[0038] Example 1: Design and preparation of molecular beacon probe H 1 and H 2

[0039] In this example, a molecular beacon probe H was designed based on the nucleotide sequence of miRNA-21 (DNA mimic) (as shown in Table 1 and SEQ ID NO.3). 1 and H 2 .

[0040] As shown in Table 1, molecular beacon probe H 1 The nucleotide sequence is shown in SEQ ID NO. 1, and a disulfide bond is embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the 15th base is modified with a fluorescent group Cy3.

[0041] Molecular beacon probe H 2 The nucleotide sequence is shown in SEQ ID NO.2, and the molecular beacon probe H 2 A photocleavable linker (PC-linker) was embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the fluorescent group Cy5 was modified at the 5' end.

[0042] The molecular beacon probe H designed by the above design method 1 and H 2 , can be activated under the combined stimulation of endogenous GSH and exogenous light. These two stimuli are indispensable, thus realizing the design of multiple gates. This multiple gate design combining endogenous and exogenous gating can better improve the spatiotemporal resolution of the imaging of the present invention, that is, improve the accuracy of imaging in time and space.

[0043] Example 2: Molecular Beacon Probe H 1 and H 2 Feasibility analysis of programmed activation

[0044] The molecular beacon probe H 1 With H 2 After annealing at 95°C, the hairpins were dispersed in HEPES buffer solution and prepared into a 100 nM system. Depending on the corresponding experimental conditions, GSH (10 mM) and miR-21 (10 nM) were added or not. The test tubes were then placed under UV (5 mW / cm 2 After irradiation, the cells were incubated at 37 °C for 180 min. After incubation, the fluorescence intensity of the system was measured using a fluorescence spectrophotometer. The fluorescence excitation wavelength was 490 nm, and the FRET ratio was calculated using the ratio of the fluorescence intensity at the emission wavelengths of 675 nm (Cy5) and 575 nm (Cy3).

[0045] The results are as follows Figure 2 As shown, Figure 2 The "+" on the horizontal axis means that the corresponding substance is added or UV irradiation is performed, and the "-" means that the corresponding substance is not added or UV irradiation is not performed. 1 With H 2 Only after GSH and UV co-activation can it react with miR-21 and produce a strong FRET signal. This indicates that the molecular beacon probe H 1 With H 2 Programmatic activation is possible.

[0046] Example 3: Molecular Beacon Probe H 1 and H 2 Sensitivity and specificity analysis

[0047] The molecular beacon probe H 1 With H 2 The hairpins were annealed at 95°C and dispersed in HEPES buffer solution to prepare a 100 nM system. GSH (10 mM) and different concentrations of miR-21 (10, 5, 2, 1, 0.2, 0.1, 0.05, 0.01, 0.005, 0 nM) were added to the reaction system. Figure 3 a) or different types of miRNAs ( Figure 3 b). The tube was then placed under UV light (5 mW / cm 2After irradiation, the cells were incubated at 37°C for 180 min. After incubation, the fluorescence intensity of the system was measured using a fluorescence spectrophotometer. The fluorescence excitation wavelength was 490 nm, and the FRET ratio was calculated using the ratio of the fluorescence intensity at the emission wavelengths of 675 nm (Cy5) and 575 nm (Cy3).

[0048] Detection results of different concentrations of miR-21 Figure 3 a The results show that the molecular beacon probe H provided by the present invention can be seen 1 With H 2 It has excellent detection sensitivity, and the detection limit is 10 pM calculated according to the 3σ rule.

[0049] Detection results of different types of miRNA Figure 3 b The results show that the molecular beacon probe H provided by the present invention 1 With H 2 FRET signal was generated only when the target miR-21 was added, indicating that M-CHA has good specificity for miR-21 detection.

[0050] Example 4: Preparation of a multivariate gated signal amplification system for miRNA-21 detection in a cellular environment

[0051] 1. Preparation of NaGdF 4 : Yb / Tm@NaGdF 4 Upconversion nanoparticles (oil phase upconversion nanoparticles).

[0052] The nanodevice was prepared by the thermal decomposition method provided in the literature “Upconversion Luminescence-Activated DNA Nanodevice for ATP Sensing in Living Cells” (Jian Zhao, Jinhong Gao, Wenting Xue, Zhenghan Di, Hang Xing, Yi Lu, Lele Li, DOI: 10.1021 / jacs.7b11161).

[0053] 2. Preparation of poly-lysine-coated aqueous upconversion nanoparticles

[0054] NaGdF 4 : Yb / Tm@NaGdF 4The upconversion nanoparticles (oil-phase upconversion nanoparticles) were vigorously stirred with dilute hydrochloric acid at room temperature for 3 h. After the reaction, they were washed three times by centrifugation with 0.01M HCl-ethanol solution and ultrapure water (MQ water) respectively to obtain aqueous upconversion nanoparticles. The aqueous upconversion nanoparticles (UCNPs) were dispersed in 1 mL MQ water and stored at 4°C.

[0055] 1 mL of the above-mentioned aqueous upconversion nanoparticles was stirred with 5 mg of polylysine (PLL) at room temperature for 24 h. After stirring, ultrapure water was used for centrifugal washing three times to obtain polylysine-coated aqueous upconversion nanoparticles.

[0056] Then, the aqueous upconversion nanoparticles coated with poly-lysine were combined with the molecular beacon probe H 1 and H 2 The mixture was mixed at a molar ratio of 1:50 and incubated at 37 °C for 2 h to form the M-CHA / U complex, which is a multivariate gated signal amplification system for detecting miRNA-21 in a cellular environment.

[0057] Preparation process Figure 4 As shown in a, the TEM image of the M-CHA / U composite is Figure 4 The fluorescence emission spectrum under 980 nm near-infrared light irradiation is shown in b. Figure 4 As shown in c.

[0058] Example 5: Cytotoxicity test of M-CHA / U complex

[0059] SW480 cells were cultured in 96-well plates overnight; then the cells were treated with poly-lysine-coated aqueous upconversion nanoparticles, molecular beacon probe M-CHA, and M-CHA / U complex for 4 h, with or without 980 nm NIR (near infrared) irradiation for 10 min (1.2 W / cm 2 After treatment, fresh culture medium was replaced and cultured for 24 h. After culture, cells were treated with the commercial CCK-8 kit.

[0060] like Figure 5 As shown, from the test results, it can be seen that the toxicity of NIR, UCNPs, M-CHA and M-CHA / U to cells is negligible, which indicates that the multivariate gated signal amplification system has good biocompatibility.

[0061] Example 6: Experiment on cancer cell selective imaging of M-CHA / U complex

[0062] SW480 cancer cells and L02 normal cells were evenly seeded into glass-bottom confocal culture dishes and cultured overnight. After overnight, the cells were gently washed with PBS for 3 times, and the culture medium was replaced with Opti-MEM containing M-CHA / U complex (each probe was equal to 100 nM) and cultured for another 4 h. After the culture, the cells were washed 3 times to remove the M-CHA / U complex that did not enter the cells, and then fresh Opti-MEM was replaced and irradiated with 980 nm NIR for 10 min (1.2 W / cm 2 , irradiation for 1 min, rest for 4 min), and then continue to culture for 1 h. Finally, the cells were imaged in a laser confocal system, and fluorescence photos of the corresponding channels of Cy3 and Cy5 were collected at an excitation wavelength of 520 nm to analyze the cell FRET signal.

[0063] like Figure 6 As shown, obvious FRET signals were observed in SW480 cells with high expression of GSH and miR-21, while no obvious FRET signals were observed in L02 cells with low expression of GSH and miR-21; this indicates the feasibility of the M-CHA / U complex for selective imaging of target cancer cells through specific activation.

[0064] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for constructing a multivariate gated signal amplification system, characterized in that: The following steps are involved: Molecular beacon probes H1 and H2 are designed; the nucleotide sequence of the molecular beacon probe H1 is shown in SEQ ID NO.1, and the molecular beacon probe H1 has a disulfide bond embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the 15th base is modified with a fluorescent group Cy3; the nucleotide sequence of the molecular beacon probe H2 is shown in SEQ ID NO.2, and the molecular beacon probe H2 has a photocleavable linker (PC-linker) embedded between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the fluorescent group Cy5 is modified at the 5' end; The multivariate gated signal amplification system is used to detect miRNA-21.

2. The method for constructing a multivariate gated signal amplification system according to claim 1, characterized in that: The following steps are also included: Aqueous upconversion nanoparticles are prepared, polylysine is coated on the aqueous upconversion nanoparticles, and the polylysine-coated aqueous upconversion nanoparticles and the molecular beacon probes H1 and H2 are mixed and incubated to obtain a multivariate gated signal amplification system.

3. The method for constructing a multivariate gated signal amplification system according to claim 1, characterized in that: The molar ratio of the poly-lysine-coated aqueous upconversion nanoparticles and the molecular beacon probes H1 and H2 is 1:(30-50):(30-50).

4. The method for constructing a multivariate gated signal amplification system according to claim 3, characterized in that: The molar ratio of poly-lysine-coated aqueous upconversion nanoparticles and molecular beacon probes H1 and H2 is 1:50:

50.

5. A molecular beacon probe combination, characterized in that: The nucleotide sequence of the molecular beacon probe H1 is shown in SEQ ID NO.1, and the molecular beacon probe H1 embeds a disulfide bond between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the 15th base is modified with a fluorescent group Cy3; the nucleotide sequence of the molecular beacon probe H2 is shown in SEQ ID NO.2, and the molecular beacon probe H2 embeds a photocleavable linker (PC-linker) between the 8th and 9th bases from the 3' end of the nucleotide sequence, and the fluorescent group Cy5 is modified at the 5' end; the molecular beacon probe combination is used to detect miRNA-21.

6. A multivariate gated signal amplification system, characterized in that: It is constructed by the construction method described in any one of claims 1 to 4.

7. A use of the molecular beacon probe combination according to claim 5, characterized in that: Used to detect miRNA-21 in non-cellular environments without the purpose of disease diagnosis and treatment.

8. The use according to claim 7, characterized in that: The sample to be tested is placed in a detection container, and the molecular beacon probe combination and GSH are added; the reaction system is irradiated with ultraviolet light and then incubated at room temperature; finally, the fluorescence intensity of the reaction system is detected to complete the detection.

9. An application of the multivariate gated signal amplification system according to claim 6, characterized in that: Used to detect miRNA-21 in the cellular environment without the purpose of disease diagnosis and treatment.

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