SELF-ASSEMBLED DNA NANOWIRE SENSOR BASED ON HYBRID CHAIN REACTION FOR IMAGING OF MRNA-125b IN A549 CELLS

Abstract

The invention provides a DNA nanowire sensor based on hybridization chain reaction self-assembly and an application of the DNA nanowire sensor in miRNA-125b imaging in an A549 cell. The sensor is triggered by an aptamer Apt-T, a dendritic nanowire skeleton is formed through cascade self-assembly of H1 and H2 hairpin probes (4: 4), and an antisense chain (anti chain) is anchored to be combined with miRNA-125b. The fluorescent group FAM (the 3'end of an anti chain) and the quenching group BHQ1 (the 5 'end of a H1 / H2 probe) realize dual-mode switching of signal closure-recovery through an FRET effect; miRNA-125b triggers a chain displacement reaction to dissociate the anti chain, recover fluorescence and synchronously inhibit the expression of a target gene. The assembly ratio of the sensor is Apt-T: H1: H2: anti = 1: 4: 4: 8, and the sensor has high sensitivity (the detection limit is 0.176 nM, and the linear range is 0.5-45 nM), strong selectivity, excellent stability and low cytotoxicity. The sensor can realize in-situ dynamic detection and expression regulation of miRNA-125b in living cells, and provides an efficient tool for integration of cancer diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry detection, and specifically relates to a self-assembled DNA nanowire sensor based on hybridization chain reaction - for imaging miRNA-125b in A549 cells. Background Art

[0002] miRNA is a short single-stranded non-coding RNA molecule (about 21-23 nucleotides in length), which can be used to regulate the expression of other genes and is one of the most active research topics in life science. [Li D, Xia L, Zhou Q. Label-Free Detection of miRNA Using Surface-Enhanced Raman Spectroscopy. Anal Chem, 2020, 92(19):12769~12773] Among them, miRNA-125b has been confirmed to be closely related to the progression of various malignant tumors such as lung cancer and breast cancer, and its abnormal expression can be used as an important biomarker for early cancer diagnosis, prognosis evaluation and treatment monitoring. However, due to the lack of appropriate technical means, in-situ real-time imaging of miRNA in living cells cannot be achieved, and there are problems such as complex operation and insufficient sensitivity, which greatly limit its application in clinical diagnosis and basic research.

[0003] In recent years, the molecular probe technology based on nucleic acid self-assembly has provided new ideas for the detection of miRNA in living cells. Hybridization chain reaction (HCR), as an enzyme-free isothermal amplification strategy, can form long-chain DNA nanostructures through the cascade self-assembly of trigger hairpin probes, and has advantages such as strong signal amplification ability and flexible design. Due to the characteristics of isothermal, enzyme-free and high amplification efficiency, HCR is often used as a signal amplification technology in various biosensing and biomedical fields. However, as an enzyme-free self-assembly reaction, traditional HCR probes still face multiple challenges in living cell applications: firstly, the conventional HCR products are linear structures and lack targeted delivery ability, making it difficult to efficiently enter specific cells; secondly, existing probes mostly rely on single fluorescence labeling, are easily interfered by the complex intracellular environment, and have a low signal-to-noise ratio; thirdly, most designs only focus on the static detection of miRNA, and it is difficult to achieve the synergistic effect of dynamic monitoring and its functional regulation. [Zhou R, Zeng Z, Sun R, et al. Traditional and new applications of the HCR in biosensing and biomedicine[J]. Analyst, 2021, 146(23):7087-7103.]

[0004] To address the above technical bottlenecks, the present invention proposes a DNA nanowire sensor based on HCR self-assembly, which breaks through the limitations of existing probes by integrating ligand-targeted recognition, strand displacement-triggered signal conversion, and antisense gene regulation functions. The sensor uses the Apt-T aptamer as the trigger core and constructs a nanowire structure with dendritic side chains by precisely regulating the assembly ratio of H1 / H2 hairpin probes (1:4:4), significantly enhancing cell targeting and probe loading capacity. An antisense strand (anti-strand) is innovatively introduced as a functional inhibitory unit for miRNA-125b, and through the precise spatial arrangement of the FAM-BHQ1 fluorescence resonance energy transfer (FRET) pair, a "signal off - recovery" dual-mode switch is achieved. When the anti-strand is anchored to the nanowire side chain through base complementary pairing, the proximity effect of FAM and BHQ1 leads to fluorescence quenching; while the intervention of miRNA-125b can trigger a strand displacement reaction, causing the anti-strand to dissociate and restore the fluorescence signal. This dynamic response mechanism not only endows the probe with high-sensitivity detection ability but also synchronously realizes the down-regulation intervention of miRNA-125b, providing a new tool for cancer diagnosis and treatment integration. Summary of the Invention

[0005] The object of the present invention is to design a self-assembled DNA nanowire sensor based on hybridization chain reaction for imaging miRNA-125b in A549 cells.

[0006] The object of the present invention is achieved by the following technical solutions.

[0007] A self-assembled DNA nanowire sensor based on hybridization chain reaction for imaging miRNA-125b in A549 cells, characterized by the following steps:

[0008] (1) Preparation of DNA sensor: First, stock solutions of all oligonucleotide strands were prepared using TE buffer (10 mM Tris, 0.1 mM EDTA, pH = 8.0). Then, according to experimental requirements, phosphate-buffered saline (PBS, 0.01 M) containing 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4, and 10 mM Mg(Ac)2 was prepared, and its pH was adjusted to 7.4 with hydrochloric acid and sodium hydroxide. Hairpin probes H1 and H2 were diluted to 20 μM with PBS buffer, annealed at 95 °C for 5 min, and the annealed hairpins were placed in a refrigerator at 4 °C for more than half an hour. The experimental probe was synthesized at a ratio of Apt-T:H1:H2:anti = 1:4:4:8. This probe is an aptamer-triggered nanowire probe. First, the three strands of Apt-T, H1, and H2 were added and incubated at 37 °C for 5 h to form a nanowire. Then, the anti strand was added and incubated at 37 °C for 5 h to form a DNA sensor. Finally, the concentration of the anti strand was used as the final probe concentration, and a 4 μM probe was synthesized. After synthesis, it was stored in a refrigerator at 4 °C.

[0009] (2) Native polyacrylamide gel electrophoresis (PAGE) experiment: 4 mL of 30% acrylamide was mixed with 4 mL of ultrapure water and 2 mL of 5×TBE buffer. Subsequently, 35 μL of ammonium persulfate (APS) and 3.5 μL of tetramethylethylenediamine (TEMED) were added, and the mixture was stirred well and polymerized at 37 °C into a 12% native polyacrylamide gel. A 6×Tris-glycerol gel loading buffer was mixed with the sample at a ratio of 1:5 for loading, and electrophoresis was carried out at a voltage of 120 V for 50 min. The gel was stained with 4S Gel blue nucleic acid dye for 20 min and photographed and recorded using a gel imaging system (JY04S-3E).

[0010] (3) In vitro fluorescence measurement experiment: 2.5 μL of the successfully prepared 4 μM DNA nanowire sensor was added to different concentrations of the target miRNA-125b, and the volume was made up to 200 μL with PBS buffer. The above mixed solution was incubated at 37 °C and then the fluorescence intensity was measured. The excitation wavelength of the fluorescent group FAM was set at 488 nm, and the fluorescence emission was collected on a FL-7000 spectrometer, with the collection range controlled at 508 - 650 nm.

[0011] (4) Stability test: To prove the stability of the sensor, the DNA sensor loaded with nanoparticles was mixed with 2 U / mL endonuclease I (DNase I), 5 mM glutathione (GSH), and 10% human serum sample respectively, and made up to 200 μL with PBS buffer and incubated for different times. The integrity of the DNA sensor structure was explored by phosphorescence test. And the sensor was mixed with 10% (v / v) fetal bovine serum (FBS) and incubated for different times, and the integrity of the sensor structure was explored by PAGE experiment.

[0012] (5) Cell culture and imaging: Non-small cell lung cancer cells (A549) were cultured in complete medium (DMEM) with 10% (v / v) fetal bovine serum in a 5% carbon dioxide, 37 °C saturated humidity incubator. An appropriate amount of A549 cells were seeded in a confocal dish with a diameter of 15 mm and continued to be cultured until fully adherent and reached an appropriate proliferation state. After washing three times with PBS (pH = 7.4), the DNA sensor and DMEM medium were added and co-incubated for a certain time. After washing three times with PBS, confocal imaging was performed.

[0013] (6) Cytotoxicity analysis: A549 cells were seeded in a 96-well plate (200 μL) and continued to be cultured for 24 h until adherent. The medium was removed and the cells were washed twice with PBS. DMEM medium containing DNA sensors with different concentrations was added respectively. At the same time, the cells cultured with only medium were used as the blank control group. After incubation for 24 h, the medium was removed and the cells were washed twice with PBS. Subsequently, 20 μL of MTT solution with a concentration of 5 mg / mL was added to each well and incubated in the incubator for 4 h. Then the MTT solution was removed and 150 μL of DMSO solution was added and shaken well for 15 min to fully dissolve the purple crystals produced in the wells. The absorbance value at 490 nm was measured by a microplate reader to calculate the cell viability.

[0014] (7) Cell co-localization analysis: A549 cells were seeded in a culture dish with a pore size of 15 mm and cultured for 24 h. After washing three times with PBS, the DNA sensor (100 nM) was added and co-incubated with A549 cells for 6 h. After washing three times with PBS, Lyso-Tracker dye (final concentration of 1 μM) and DMEM were added and mixed to a total volume of 1 mL and then co-incubated with the cells for 30 min. After washing three times with PBS, imaging was performed. The excitation wavelength of the DNA sensor in confocal imaging was 488 nm, and the excitation wavelength of the lysosome tracking dye was 561 nm.

[0015] (8) Flow cytometry analysis: An appropriate amount of A549 cells was seeded in a 24-well plate. After incubating in an incubator for 24 h, the cells were washed twice with PBS. According to the experimental requirements, the cells in the 24-well plate were pretreated, and then a DNA sensor was added and co-incubated with the cells for 6 h. The cells were washed three times with PBS buffer, trypsin was added to digest the cells, and then the digestion was terminated with DMEM. The cell suspension was transferred to a 1.5 mL centrifuge tube and centrifuged (1500 rpm, 5 min). After washing, the supernatant was removed and the cells were resuspended in 200 μL of PBS. The samples were analyzed using a flow cytometer (DxP AthenaTM). Description of the Drawings

[0016] Figure 1 Schematic diagram of the construction of the DNA nanowire sensor and its detection of miRNA-125b.

[0017] Figure 2 Gel verification analysis of the DNA nanowire sensor.

[0018] Figure 3 Feasibility analysis of the DNA nanowire sensor for detecting miRNA-125b.

[0019] Figure 4 Time optimization of the DNA nanowire sensor for detecting miRNA-125b. Error bars represent the standard deviation of three parallel experiments.

[0020] Figure 5 Fluorescence spectra of the DNA nanowire sensor for detecting miRNA-125b (A) Concentration linear range and linear equation graph (B). Error bars represent the standard deviation of three parallel experiments.

[0021] Figure 6 Homologous selectivity (A) and specificity experiment (B) of the DNA nanowire sensor for detecting miRNA-125b. Error bars represent the standard deviation of three parallel experiments.

[0022] Figure 7 Enzyme stability experiment (A), GSH stability experiment (B), human serum stability experiment (C), time stability experiment (D) of the DNA nanowire sensor, and non-denaturing polyacrylamide gel experiment to analyze the structural integrity of the DNA nanowire sensor in 10% FBS at different times (E) (F0 is the initial fluorescence intensity of the sensor, F is the fluorescence intensity at the corresponding time when the sensor coexists with biological samples). Error bars represent the standard deviation of three parallel experiments.

[0023] Figure 8 Cytotoxicity analysis of the DNA nanowire sensor on A549 cells.

[0024] Figure 9 ​​​​​​​​​Co-localization analysis of DNA nanowire sensors and lysosome dyes in A549 cells.

[0025] Figure 10 Cell imaging comparison diagrams after treatment with mimics and inhibitors. On the right are flow cytometry analysis diagrams of three groups of samples. The scale bar is 20 μm

[0026] Figure 11 Confocal imaging comparison diagrams of DNA nanowire sensors and mismatched DNA nanowire sensors. On the right are comparative flow cytometry analysis diagrams of the samples. The scale bar is 20 μm.

[0027] Figure 12 Probe sequences required in this experiment. Specific implementation methods

[0028] Here, in combination with the accompanying drawings and embodiments, the specific implementation methods of the present invention are further described in detail. The following embodiments are used to illustrate the present invention, but do not limit the application scope and extension of the present invention.

[0029] Example 1: Construction of a DNA nanowire sensor and its schematic diagram for detecting miRNA-125b.

[0030] As Figure 1 shown, the DNA nanowire sensor is a large structure composed of Apt-T-triggered HCR, which is cascaded in a ratio of Apt-T:H1:H2 = 1:4:4 to form the main configuration nanowire shape of the sensor. Apt-T triggers the opening of hairpins H1 and H2, making the sides of the nanowire dendritic. Then, according to base complementary pairing, the antisense strand of miRNA-125b is connected to the side of the nanowire to downregulate miRNA-125b in A549 cells. The fluorescent reporter group FAM is labeled at the 3' end of the anti-strand, and the quenching group BHQ1 is modified at the 5' end of H1 and H2. When the formed nanowire is connected to the anti-strand, the fluorescent group FAM and the quenching group BHQ1 will be close to each other, resulting in fluorescence quenching. Through reasonable design, 8 bases are left free at the 5' end of the domain of the anti-single strand as the foothold for target miRNA-125b strand displacement triggering. In the presence of miRNA-125b, it will hybridize with the anti-strand and undergo a strand displacement reaction, resulting in the dissociation of the anti-strand with FAM, and the fluorescent group FAM moving away from the quenching group BHQ1, and the fluorescence recovers, and the fluorescence intensity continuously increases. Therefore, this DNA nanowire sensor can be applied to the sensitive detection and imaging analysis of miRNA-125b in living cells.

[0031] Example 2: Gel verification analysis of DNA nanowire sensors.

[0032] ​​​The construction of the DNA nanowire sensor was preliminarily verified by non-denaturing polyacrylamide gel electrophoresis experiment. As Figure 2 shown, lane 1 is the Maker, lanes 2-3 are the single-strand lanes of H1 and H2, lane 4 is the mixed lane of H1 and H2. In the absence of the trigger strand, H1 and H2 are in a metastable state and no cascade occurs. Lane 5 is the mixture of single-stranded Apt-T, H1, and H2 at a concentration ratio of 1:4:4 to undergo a hybridization chain reaction. After incubation at 37 °C for 5 h, a cascaded nanowire is formed, with a relatively large molecular weight and a low mobility. Lane 6 is the addition of a certain concentration of anti-strand to the nanowire and then incubation at 37 °C for 5 h to form a complete DNA nanowire sensor, where the ratio of single-stranded Apt-T, H1, H2, and anti is 1:4:4:8. Lane 7 adds the target miRNA-125b verification strand equivalent to the concentration of the anti-strand of the sensor to verify the strand displacement reaction, and it can be seen that bands with a faster mobility are generated. And lane 8 is the double-stranded structure formed by the binding of the anti-strand and the miRNA-125b strand. According to lanes 7-8, it can be shown that the DNA nanowire sensor is successfully formed and its feasibility for detecting the target miRNA-125b is successfully verified in gel electrophoresis.

[0033] Example 3: Feasibility analysis of the DNA nanowire sensor for detecting miRNA-125b.

[0034] The fluorescence spectrum also successfully proves again that in the presence of the target miRNA-125b, the fluorescence of the DNA nanowire sensor has changed significantly. As Figure 3 shown, the fluorescence background signal of the DNA nanowire sensor is low and there is no leakage of false positive signals. Because after the successful construction of the DNA nanowire sensor, BHQ1 on H1 and H2 will be closely close to the fluorescent group FAM and the fluorescence is quenched. After adding the target miRNA-125b, the anti-strand is dissociated from the foothold and FAM is far away from the quenching group BHQ1, and there will be different degrees of fluorescence recovery according to different concentrations of the target. When the concentration ratio of the DNA nanowire sensor and miRNA-125b is 1:1, a significant increase in fluorescence intensity can be observed. The successful construction of the DNA nanowire sensor and the fluorescence feasibility for detecting the target miRNA-125b are verified again by the fluorescence spectrum.

[0035] Example 4: Time optimization of the DNA nanowire sensor for detecting miRNA-125b.

[0036] As Figure 4As shown in the figure, the reaction time for the DNA nanowire sensor to detect miRNA-125b was optimized. Two different concentrations of miRNA-125b and a DNA nanowire sensor (4 μM) with a final concentration of 50 nM in the system were incubated at 37 °C for different times, and then the corresponding fluorescence intensity was measured. As Figure 4 shown in the figure, when miRNA-125b was 20 nM and 40 nM, the fluorescence intensity of the DNA nanowire sensor increased sharply with the extension of the reaction time and reached the maximum value of F / F0 at 5 min (F0 is the initial fluorescence intensity of the sensor, and F is the fluorescence intensity obtained by the sensor detecting the target). Thus, the designed DNA nanowire sensor has high-efficiency detection of miRNA-125b.

[0037] Example 5: Sensitivity analysis of the DNA nanowire sensor for detecting miRNA-125b.

[0038] To evaluate the sensitivity of the DNA nanowire sensor for detecting miRNA-125b, different concentrations of miRNA-125b were added to the reaction system, and fluorescence measurement was performed after incubation at 37 °C. Only in the presence of miRNA-125b, it will specifically hybridize and complement with the anti-strand, causing the anti-strand to detach from the DNA nanowire sensor. The fluorescent reporter group FAM on the anti-strand is far from the quenching group BHQ1 at the H1 and H2 ends, and the fluorescence of FAM recovers and the fluorescence intensity increases. From Figure 5 (A) and Figure 5 (B), it can be seen that as the concentration of the target miRNA-125b increases continuously, the fluorescence intensity of the DNA nanowire sensor also shows a gradually increasing trend. This result verifies the specific response ability of the DNA nanowire sensor to miRNA-125b. As Figure 5 (B) shows, when the concentration range of miRNA-125b is 0.5 - 45 nM, there is a good linear relationship between the fluorescence intensity of the DNA nanowire sensor and the concentration of miRNA-125b. The obtained linear regression equation is Y = 0.42C + 0.99, R2 = 0.999, where Y represents the F / F0 value and C is the concentration of the target miRNA-125b. Calculated by the 3σ / k theory, the detection limit of this DNA nanowire sensor for detecting miRNA-125b is 0.176 nM. After verification by this experiment, the DNA nanowire sensor constructed in this study shows significant potential in the sensitive detection of miRNA-125b.

[0039] Example 6: Performance analysis of the DNA nanowire sensor.

[0040] To comprehensively evaluate the selectivity of the DNA nanowire sensor for the target miRNA-125b, a homologous selectivity analysis experiment was conducted. In the experiment, other homologous miRNAs with the same concentration as miRNA-125b were selected, including miRNA-122-5p, miRNA-155, miRNA-141, and miRNA-21, as selective samples for comparative analysis. According to Figure 6 (A), it can be seen that only when miRNA-125b is present, the F / F0 value of the DNA nanowire sensor can reach the optimal state, indicating that this strategy has high selectivity for the target miRNA-125b. In contrast, the presence of other homologous miRNAs did not cause significant changes in fluorescence intensity.

[0041] To deeply analyze the selectivity, sequences with single-base mutation (SM), double-base mutation (DM), and triple-base mutation (TM) with the target were selected for detailed selectivity exploration. From Figure 6 (B), it can be seen that when miRNA-125b undergoes base mutation, it cannot cause significant changes in the fluorescence intensity of the DNA nanowire sensor. As the number of base mutations increases, the fluorescence intensity decreases accordingly. Therefore, the DNA nanowire sensor constructed in this study has the ability to specifically recognize and respond to miRNA-125b from the complex miRNAs family, and can effectively exclude the interference of other miRNAs. This characteristic lays a solid foundation for its accurate detection of target miRNAs in complex biological samples.

[0042] Meanwhile, to explore the stability of the DNA nanowire sensor in the presence of DNase Ⅰ. From Figure 7 (A), it can be seen that in the presence of 2 U / mL of DNase Ⅰ, the background signal of the DNA nanowire sensor did not leak, indicating good anti-enzyme stability. Figure 7 (B) verified the anti-biol-thiol performance of the DNA nanowire sensor. In the system with 10 mM of GSH added, the fluorescence intensity of the DNA nanowire sensor could be stably maintained for at least 8 h or more, indicating that the DNA nanowire sensor has a certain anti-biol-thiol stability. Further exploration was carried out on the performance of the DNA nanowire sensor in human serum samples. Figure 7 (C) When the DNA nanowire sensor was added to 10% human serum, its background signal remained stable within 8 h, indicating that the sensor has good anti-interference stability. Figure 7 (D) shows that the DNA nanowire sensor was stored at 4 °C, and the fluorescence signal of the sensor was tested every day thereafter. It could maintain stability for at least 7 days without fluorescence leakage, further verifying its good time stability.

[0043] In the complex intracellular environment, the stability of DNA sensors is crucial. Therefore, the stability of DNA nanowire sensors was comprehensively explored to ensure their accurate detection of targets in complex biological samples. First, to investigate the stability of DNA nanowire sensors in a system containing 10% serum (FBS), this study used polyacrylamide gel electrophoresis (PAGE) experimental methods for analysis. As Figure 7 (E) shows, no diffused bands appeared after the DNA nanowire sensors were co-incubated with 10% serum for 8 h, once again confirming that the DNA nanowire sensors can stably exist in a 10% FBS environment for a long time.

[0044] Example 7: Cytotoxicity analysis of DNA nanowire sensors on A549 cells.

[0045] Cytotoxicity is a key factor in evaluating whether an in vitro constructed DNA sensor is suitable for intracellular research. The cell viability after co-incubating A549 cells with different concentrations of DNA nanowire sensors for 24 h was investigated. As Figure 8 shown, even after 24 h of incubation, the activity of the sensors at different concentrations on A549 cells still remained at a high level of over 80%. Thus, it can be seen that the DNA nanowire sensors have little effect on the viability of A549 cells. In addition, in the cell imaging experiment, the co-incubation time of the DNA nanowire sensors with A549 cells was 6 h, and 50 nM of the sensors were selected to enter the cells. Considering the combined effects of concentration and time on cell activity, it can be reasonably inferred that the DNA nanowire sensors are basically non-toxic to A549 cells, so their impact in cell experiments can be ignored.

[0046] Example 8: Cell imaging analysis.

[0047] To explore the final accumulation location of DNA nanowire sensors in cells after entering via the endocytic pathway, a lysosome co-localization experiment was carried out using Lyso-Tracker dye (1 μM). After co-incubating the DNA nanowire sensors with A549 cells for 6 h, the lysosomes were fluorescently labeled and imaged using Lyso-Tracker dye. As Figure 9 shown, where Figure 9 (A) presents the green fluorescence image emitted by FAM in the DNA nanowire sensors; Figure 9 (B) is the red fluorescence image emitted by the Lyso-Tracker dye; Figure 9 (C) is the superimposed image of FAM green light and Lyso-Tracker red light. It can be seen that the yellow fluorescence produced by their overlap is not significant, preliminarily indicating that there is no obvious co-localization phenomenon in the spatial distribution of the two fluorescence signals. In addition, Figure 9(D) The red-green channel intensity correlation diagram also shows a low correlation. In summary, it can be inferred that after the DNA nanowire sensor enters the cell, its main fluorescence signal accumulates mainly in the cytoplasm.

[0048] To further explore the applicability of the DNA nanowire sensor in distinguishing the expression levels of miRNA-125b in living cells, before the experiment, A549 cells were first incubated with mimics and antisense strands of miRNA-125b for 12 h, and the cells without pretreatment were used as the control group. Subsequently, the same concentration of DNA nanowire sensor and A549 cells were added and incubated for another 6 h for imaging. Figure 10 It was clearly observed that compared with the blank group, when the cells were pretreated with miRNA-125b mimics and antisense strands, an increase and a decrease in fluorescence signals could be observed respectively. The right side of the figure shows the corresponding flow cytometry analysis diagram, which also indicates the change in intracellular fluorescence intensity. From the above comparison, it can be seen that the output of the fluorescence signal is closely related to the expression level of miRNA-125b, indicating that the DNA nanowire sensor can successfully distinguish the expression levels of miRNA-125b in different living cells.

[0049] To further explore the specificity of the DNA nanowire sensor in living cells, mismatched DNA nanowire sensors synthesized with H1, H2, and anti with sequence mismatches were designed and incubated with A549 cells. As Figure 11 shown, after the mismatched DNA nanowire sensor was incubated and entered the cells, the fluorescence signal in A549 cells was significantly lower than that in the A549 cells of the DNA nanowire sensor group. This result indicates that the DNA nanowire sensor also has high specificity in living cells. At the same time, flow cytometry was used to analyze and collect the fluorescence intensities of the DNA nanowire sensor and the mismatched DNA nanowire sensor. As shown in the right figure of 11, this is consistent with the confocal imaging results.

Claims

1. DNA nanowire sensor based on hybridization chain reaction (HCR) self-assembly - for imaging miRNA-125b in A549 cells, characterized in that The sensor includes: (1) Aptamer Apt-T, as the triggering core, for targeting and recognizing A549 cells; (2) H1 and H2 hairpin probes, which self-assemble through HCR to form a nanowire structure with dendritic side chains, where the molar ratio of H1 to H2 is 4:4; (3) Antisense strand (anti-strand), which is anchored to the nanowire side chain through base complementary pairing and is used to undergo a strand displacement reaction with miRNA-125b; (4) Fluorescent group FAM and quenching group BHQ1, which are respectively labeled at the 3' end of the anti-strand and the 5' end of the H1 / H2 probe to form a fluorescence resonance energy transfer (FRET) pair; wherein, the assembly ratio of the sensor is Apt-T:H1:H2:anti = 1:4:4:8, and the sensor triggers the restoration of the fluorescence signal through a strand displacement reaction to achieve the dynamic detection and functional inhibition of miRNA-125b.

2. The DNA nanowire sensor according to claim 1, wherein The assembly of the H1 and H2 hairpin probes is achieved through the following steps: (1) Dilute the H1 and H2 hairpin probes with phosphate buffer solution (PBS) to 20 μM respectively, anneal at 95 °C for 5 minutes and then cool to 4 °C; (2) Mix Apt-T, H1 and H2 in a ratio of 1:4:4, incubate at 37 °C for 5 hours to form a nanowire skeleton; (3) Add the anti-strand and continue to incubate at 37 °C for 5 hours to form a complete DNA nanowire sensor.

3. The DNA nanowire sensor according to claim 1, wherein The 5' end of the antisense strand (anti-strand) is designed with a free sequence of 8 bases as the triggering foothold for the miRNA-125b strand displacement reaction.

4. The fluorescence signal response range of the sensor according to claim 1 is 0.5 - 45 nM for the concentration of miRNA-125b, the detection limit is 0.176 nM, and the fluorescence intensity has a linear relationship with the concentration of the target (linear equation Y = 0.42C + 0.99, R 2 = 0.999).

5. The sensor according to claim 1 maintains structural stability under the following conditions: (1) Co-incubate with 2 U / mL DNase I for at least 8 hours; (2) Co-incubate with 5 mM glutathione (GSH) or 10% human serum for at least 8 hours; (3) Stable storage at least 7 days under the storage condition of 4 °C.

6. A method for preparing the DNA nanowire sensor according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Configure TE buffer solution (10 mM Tris, 0.1 mM EDTA, pH = 8.0) and PBS buffer solution (containing 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4 and 10 mM Mg(Ac)2, pH = 7.4); (2) After annealing the H1 and H2 hairpin probes respectively, mix them with Apt-T in proportion and incubate to form a nanowire skeleton; (3) Add the anti-strand and incubate to form a complete sensor, and the final probe concentration is 4 μM.

7. Use of the DNA nanowire sensor according to any one of claims 1 to 5 in imaging miRNA-125b in A549 cells, characterized in that, It includes the following steps: (1) Co-incubate the sensor with A549 cells for 6 hours and detect the fluorescence signal through a confocal microscope; (2) Combine with a lysosome dye (Lyso-Tracker) for co-localization analysis to confirm that the sensor is mainly distributed in the cytoplasm; (3) Quantitatively analyze the intracellular fluorescence intensity by flow cytometry to distinguish the expression level of miRNA-125b.

8. The sensor according to claim 7 has an impact on the survival rate of A549 cells lower than 20%, and can achieve high signal-to-noise ratio imaging at a concentration of 50 nM.

9. Use of the DNA nanowire sensor according to any one of claims 1 to 5 in integrated cancer diagnosis and treatment, characterized in that, Down-regulate the expression of miRNA-125b through the antisense strand (anti-strand), and simultaneously achieve the synergistic effect of detection and treatment functions.

10. A method for verifying the specificity of the DNA nanowire sensor according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Verify the selectivity through miRNA sequences with single-base mutation (SM), double-base mutation (DM), and triple-base mutation (TM); (2) Construct a control sensor through mismatched H1, H2, and anti-strands, and compare the results of confocal imaging and flow cytometry analysis.

Images