A DNA structure for targeted regulation of EGFR dimerization on cell membrane surface and its preparation method and application

Abstract

This invention discloses a DNA structure for targeting and regulating EGFR dimerization on the cell membrane surface. The DNA structure is a double-stranded DNA bridge constructed using nucleic acid self-assembly. The double-stranded DNA bridge includes aptamer probes A and B, and a complementary strand C, which is complementary to aptamer probes A and B. This invention utilizes the specific recognition and binding of aptamer probes A and B to the EGFR receptor on the cell membrane, each exposing a DNA sequence. When complementary strand C is present, the exposed sequences on aptamer probes A and B can self-assemble with complementary strand C through base pairing principles, forming a double-stranded DNA bridge. By using double-stranded DNA bridges of different lengths, the distance for EGFR dimerization can be controlled. This achieves an effective spatial distance for inhibiting receptor dimerization, ultimately inhibiting cellular behavior. This provides a new approach for effectively regulating the occurrence and development of cancer and has significant implications for targeted cancer therapy.

Description

Technical Field

[0001] The present invention relates to the field of EGFR dimerization regulation, and in particular to a DNA structure for regulating EGFR dimerization on the cell membrane surface, and a preparation method and application thereof. Background Art

[0002] Cancer is a complex, multifactorial disease that has puzzled researchers for many years. Among all the malignant tumors faced by humans, lung cancer is the second most common cancer after breast cancer and the main cause of cancer death, so it has received widespread attention. Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, and non-small cell lung cancer accounts for 80% to 85%. Studies have shown that high expression or abnormal activation of EGFR is associated with the progression and treatment resistance of many cancers, including non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, colorectal cancer and breast cancer. Therefore, EGFR is used as an effective target to block key links in the signal transduction pathway to control the growth, proliferation and migration of tumors. It has the advantages of strong targeting and low incidence of adverse drug reactions, which provides hope for the treatment of cancers that rely on EGFR activation.

[0003] EGFR is a transmembrane glycoprotein widely distributed on the surface of mammalian epithelial cells and a member of the ErbB tyrosine kinase family. As a key regulator of cell behavior, 58 different receptor tyrosine kinases (RTKs) have been identified, belonging to 20 subfamilies. RTKs have similar structural regions, consisting of an extracellular ligand binding region, a transmembrane hydrophobic region, and an intracellular kinase region. Under normal circumstances, EGFR exists as an inactive monomer. When the signaling molecules epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) bind to its extracellular domain, the two monomers form homologous or heterologous dimers, and the tail protein kinase function of the intracellular domain is activated, becoming a binding site (multiple intracellular signaling proteins are activated after binding to it). After that, the signal complex is amplified through different signal transduction pathways, causing a series of cellular biochemical reactions, such as cell proliferation, migration, and differentiation. Normally, after EGFR binds to its cognate ligand EGF, there are three pathways for signal transduction: 1. EGFR-GRB2-SOS-RAS-RAF-Erk1 / 2; 2. EGFR-STAT; 3. EGFR-Gab1-PI3K-Akt. EGFR plays an indispensable role in the growth and differentiation of normal epidermal cells, but it can also activate downstream pathways through its overexpression or abnormal mutation, leading to uncontrolled cell growth, differentiation, proliferation, migration, invasion and anti-apoptosis. Therefore, EGFR has become a popular target for the treatment of malignant tumors.

[0004] At present, the commonly used EGFR-targeted drugs for NSCLC include tyrosine kinase inhibitors such as erlotinib and gefitinib, and monoclonal antibodies (mAbs) such as cetuximab and panitumumab. Tyrosine kinase inhibitors exist in the form of ATP analogs, and inhibit EGFR signal transduction by competitively binding to the ATP binding site on the intracellular catalytic kinase domain of the receptor. The drug needs to enter the cell to work, and the drug resistance caused by EGFR mutations due to various complex reasons makes its efficacy poor. In addition, the drug will produce side effects such as skin toxicity and rash. Another class of drugs, cetuximab and panitumumab, work by blocking the binding of ligands to the extracellular domain of EGFR. Antibodies have inherent disadvantages such as heat denaturation and easy contamination, expensive production and large batch production differences. The drug resistance generated by antibodies is also unclear in research. Although these drugs have achieved encouraging results in the early stage, their clinical response rates are not satisfactory. With the continuous understanding of the regulatory role of RTKs, we are increasingly aware that the spatial regulation of receptor dimerization may be of great significance for both regenerative therapy and cancer targeted therapy. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a DNA structure that targets and regulates the dimerization of EGFR on the cell membrane surface, and targets and regulates the dimerization state of EGFR through a double-stranded DNA bridge constructed by nucleic acid self-assembly, thereby providing a new idea for the related treatment of non-small cell lung cancer.

[0006] The second purpose of the present invention is to provide a method for preparing a DNA structure that targets and regulates EGFR dimerization on the cell membrane surface.

[0007] The third purpose of the present invention is to provide an application of a DNA structure for targeted regulation of EGFR dimerization on the cell membrane surface.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A DNA structure for targeted regulation of EGFR dimerization on the cell membrane surface, wherein the DNA structure is a double-stranded DNA bridge constructed by nucleic acid self-assembly, the double-stranded DNA bridge comprises a nucleic acid aptamer probe A and a nucleic acid aptamer probe B, and a complementary chain C complementary to the aptamer probe A and the aptamer probe B, wherein the aptamer probe A and the aptamer probe B both comprise EGFR aptamers.

[0010] Preferably, the double-stranded DNA bridge is one of double-stranded DNA bridges 1 to DNA bridge 7, and the double-stranded DNA bridges 1 to DNA bridge 7 are respectively composed of nucleic acid aptamer probes A1, B1 and complementary strands C1, A2, B2 and complementary strands C2, A3, B3 and complementary strands C3, A4, B4 and complementary strands C4, A5, B5 and complementary strands C5, A6, B6 and complementary strands C6, A7, B7 and complementary strands C7, and the nucleic acid sequences of A1, B1, C1, A2, B2, C2, A3, B3, C3, A4, B4, C4, A5, B5, C5, A6, B6, C6, A7, B7, C7 are as shown in SEQ ID NO:1-21.

[0011] Preferably, the double-stranded DNA bridge 3 also includes an auxiliary chain D3, and the nucleic acid sequence of the auxiliary chain D3 is shown in SEQ ID NO:22.

[0012] The second object of the present invention is achieved by adopting the following technical solution:

[0013] The preparation method of the above-mentioned DNA structure for targeted regulation of EGFR dimerization on the cell membrane surface comprises the following steps: taking the single nucleic acid chain mother liquid A1, B1, C1, A2, B2, C2, A3, B3, C3, D3, A4, B4, C4, A5, B5, C5, A6, B6, C6, A7, B7, C7 of each double-stranded DNA bridge respectively, heating them in a water bath and then cooling them to room temperature, the single nucleic acid chain mother liquids having the same concentration are mixed in a volume ratio of 1:1:1 to form the corresponding single nucleic acid chain mother liquids of each double-stranded DNA bridge, and the double-stranded DNA bridges 1 to 7 are obtained after reaction at room temperature.

[0014] Preferably, each single nucleic acid strand master solution is heated in a 95° C. water bath for 10 min.

[0015] Preferably, the reaction is carried out at room temperature for 15 minutes.

[0016] The third object of the present invention is achieved by adopting the following technical solution:

[0017] The application of the above-mentioned DNA structure for targeted regulation of EGFR dimerization on the cell membrane surface comprises the following steps:

[0018] (1) using PBS to prepare mixed probe solutions of nucleic acid aptamers A1 and B1, A2 and B2, A3 and B3, A4 and B4, A5 and B5, A6 and B6, A7 and B7, solutions of complementary chains C1, C2, C3, C4, C5, C6, and C7, and a solution of auxiliary chain D3;

[0019] (2) When the fusion rate of the experimental cells to be cultured is greater than 80%, the original culture medium is discarded, the cells are washed with PBS, and a cell culture medium without fetal bovine serum is added to continue culturing for 24 hours to starve the cells. The cells are washed with PBS again and a mixed probe solution composed of corresponding nucleic acid aptamer probes is added respectively. After incubation at 37°C for a period of time, the excess unbound probes are washed away with PBS, the corresponding complementary chains are added, incubated again for a period of time, and the unbound nucleic acid chains are washed away with PBS. The process is completed;

[0020] Preferably, in step (1), the concentration of the single nucleic acid strand in the mixed probe solution is the same as that of the corresponding complementary strand and auxiliary strand.

[0021] Preferably, the cells in step (2) are A549 cells.

[0022] Compared with the prior art, the beneficial effect of the present invention is that the present invention utilizes the EGFR aptamers in the aptamer probes A and B to specifically recognize and bind to the receptor EGFR on the cell membrane, and each exposes a DNA sequence. When a complementary chain C is present, the exposed sequences on the aptamer probes A and B can self-assemble with the complementary chain C through the principle of base complementary pairing to form a double-stranded DNA bridge. The present invention constructs 7 double-stranded DNA bridges of different lengths. As the distance of the double-stranded DNA bridge increases, that is, the spatial distance of EGFR dimerization becomes farther, the degree of EGFR activation gradually decreases, so as to achieve the distance regulation of EGFR dimerization. Thereby, the spatial distance that effectively inhibits receptor dimerization is obtained, and the purpose of inhibiting cell behavior is finally achieved. The spatial distance of the receptor is controlled at the nanoscale to regulate the receptor function, which is more helpful to fully understand the mechanism of action of the receptor, and provides a new idea for effectively regulating the occurrence and development of cancer, which is of great significance for the targeted treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis characterization results (left figure) and the fluorescence spectrum based on FRET (right figure) of the double-stranded DNA bridge 7 of the present invention;

[0024] Figure 2 The effects of seven different lengths of double-stranded DNA bridges 1 to 7 on EGFR and downstream proteins in A549 cells, where the numbers before the proteins represent the molecular weight of each protein, and the unit is kilodaltons (kDa);

[0025] Figure 3 This is a bar chart showing the effects of seven different lengths of double-stranded DNA bridges 1 to 7 on EGFR and downstream proteins in A549 cells. # It means that the difference between the experimental group and the EGF group is statistically significant. # P < 0.05;

[0026] Figure 4 It is a flow cytometric analysis diagram of the incubation of nucleic acid aptamer probe in double-stranded DNA bridge 7 with A549 cells;

[0027] Figure 5 This is a laser confocal image of double-stranded DNA bridge 7 incubated with A549 cells;

[0028] Figure 6 This is the result diagram of the effect of double-stranded DNA bridge 7 on the proliferation ability of A549 cells;

[0029] Figure 7 The figure shows the effect of double-stranded DNA bridge 7 on the migration ability of A549 cells (4×);

[0030] Figure 8 The figure shows the effect of double-stranded DNA bridge 7 on the invasion ability of A549 cells (10×);

[0031] Fig. 9 This is a schematic diagram of the principle of the double-stranded DNA bridge of the present invention for regulating EGFR dimerization;

[0032] Fig.10 This is a schematic diagram of the principle of using double-stranded DNA bridges of different lengths for regulating the dimerization distance of EGFR in the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0034] The experimental materials and instrument information used in each example are listed as follows: Human non-small cell lung cancer cells (A549) were purchased from ATCC, USA.

[0035] The main reagents and materials used in the experiment are shown in Table 1, the main instruments and equipment used in the experiment are shown in Table 2, the nucleic acids used in the experiment were purchased from Shanghai Shenggong Biological Co., Ltd., and the specific sequences are shown in Table 3. Milli-Q water (resistivity greater than 18.2 MΩ·cm) was used in the experiment.

[0036] Table 1

[0037]

[0038]

[0039] Table 2

[0040] Instrument Name Model and manufacturer Vertical ultra-low temperature storage box DW-86L386, Qingdao Haier Group Co., Ltd. Centrifuge TGL-20M, Hunan Xiangyi Company Ultrapure water device Millipore Corporation Steam boiler for autoclaving HEV-50, Japan HIRAYAMA Cell culture incubator SANYO Japan Electric constant temperature incubator DHG-9146A, Shanghai Jinghong Experimental Equipment Co., Ltd. Digital display electronic constant temperature water bath HH-2, Jintan Danshwe Electric Appliance Factory pH Meter pH213, HANNA, Italy Laser confocal fluorescence microscopy TCSSP8, Leica, Germany Flow cytometry AccuriC6, Becton Dickinson, USA Gel Imager Bio-rad, USA Precision electronic balance FA1204, Qingdao Jinke Instrument Co., Ltd. Horizontal Shaker WD-9405B, Beijing Liuyi Instrument Factory Multi-function microplate reader WELLSCANMK3, Thermo Scientific, USA Oven XH-C, Jintan Guowang Experimental Instrument Factory FS-5 Fluorescence Spectrometer LB960, Berthold, Germany Inverted microscope CKX41, Japan OLYMPUS Vortex mixer XH-C, Changzhou Langyue Instrument Manufacturing Co., Ltd. Electrophoresis Apparatus DYCZ25D, Beijing Liuyi Biotechnology Co., Ltd. Electrophoresis power supply Model DYY-7C, Beijing Liuyi Instrument Factory Chemiluminescence imager Amersham Imager 600, GE Healthcare, USA Ice Maker Sanyo Corporation

[0041] Example 1

[0042] Preparation of double-stranded DNA bridges: Take the single nucleic acid chain master solutions A1, B1, C1, A2, B2, C2, A3, B3, C3, D3, A4, B4, C4, A5, B5, C5, A6, B6, C6, A7, B7, C7 (sequences as shown in Table 3) of each double-stranded DNA bridge with TE buffer as solvent and a concentration of 10 μmol / L, put them in a constant temperature water bath at 95°C for 10 min, take them out, wait for them to slowly cool to room temperature, mix the single nucleic acid chain master solutions corresponding to each double-stranded DNA bridge in a volume ratio of 1:1:1, and adjust the concentration of each mixed single nucleic acid chain to 2 μmol / L with PBS solution. React at room temperature for 15 min to obtain double-stranded DNA bridges 1 to double-stranded DNA bridges 7, with lengths of 0 bp, 10 bp, 20 bp, 30 bp, 50 bp, 70 bp and 100 bp, respectively.

[0043] Table 3

[0044]

[0045]

[0046] Example 2

[0047] The application of the DNA structure for targeted regulation of EGFR dimerization on the cell membrane surface comprises the following steps:

[0048] (1) Probe preparation: Prepare 1 mL each of A1 and B1 mixed probes (A1 and B1 in the mixed probes are 50 nmol / L, the same below), A2 and B2 mixed probes, A3 and B3 mixed probes, A4 and B4 mixed probes, A5 and B5 mixed probes, A6 and B6 mixed probes, and A7 and B7 mixed probes with PBS as the solvent, with the concentrations of each being 50 nmol / L; 1 mL each of 50 nmol / L complementary chain C1, C2, C3, C4, C5, C6, and C7 solutions;

[0049] (2) Cell intervention: A549 cells in the logarithmic growth phase were collected after trypsin digestion, centrifuged, resuspended in DMEM high-glucose medium, and then counted. The cell density was adjusted to 5×10 4 / mL was inoculated into a six-well plate, with 2mL inoculated into each well; when the cell fusion rate was greater than 80%, the original culture medium was discarded, the cells were rinsed twice with PBS, and a cell culture medium without fetal bovine serum was added to continue culturing for 24h (starvation treatment); thereafter, the cells were rinsed twice with PBS buffer, and the corresponding A and B mixed probes were added to each well and incubated at 37°C for 6min; then, the cells were rinsed twice with PBS to wash away excess unbound probes, and the corresponding complementary chains were added to each well and incubated again for 12min; the cells were rinsed twice with PBS to wash away unbound nucleic acid chains, and the process was completed.

[0050] Test Example 1

[0051] 15% non-denaturing polyacrylamide gel electrophoresis was used for verification, and the difference in migration speed between the single nucleic acid strand and the double-stranded DNA bridge in electrophoresis was used to determine whether the probe in Example 1 successfully hybridized to form a double-stranded DNA bridge.

[0052] Taking double-stranded DNA bridge 7 as an example, the results are as follows Figure 1 As shown in the left figure: the lanes of DNA single strands A7, B7 and C7 all show a clear band, and the lane where A7 and B7 are mixed shows two bands, and the positions are consistent with the single strands, indicating that A7 and B7 have no hybridization; while the lane where A7 or B7 is mixed with the complementary strand C7 shows a band whose position is lagging behind the single strand and whose brightness is significantly enhanced, indicating that the complementary strand C7 and the probe A7 or B7 undergo self-assembly hybridization. In addition, the lane where A7, B7 and C7 are mixed shows a band with significantly stronger brightness and more lagging position, indicating that the three DNA single strands have successfully hybridized to form double-stranded DNA bridge 7, from which it is inferred that double-stranded DNA bridge 7 is successfully assembled in the solution. The results of the other six double-stranded DNA bridges are the same.

[0053] Test Example 2

[0054] Fluorescence characterization of double-stranded DNA bridge: Taking double-stranded DNA bridge 7 as an example, fluorescent groups (Alexa488 and Cy5) were labeled at both ends of the nucleic acid aptamer probe, namely A7-Cy5 and B7-Alexa488 (sequences are shown in Table 3). Take an appropriate amount of single nucleic acid chain master solution labeled with fluorescent groups and complementary chain, mix the single nucleic acid chain master solution in a volume ratio of 1:1:1, and use binding buffer 1 (20mmol / LTris, 0.1mol / LNaCl, 10mmol / LMgCl 2 6H 2 O) diluting the concentration of each single nucleic acid chain to 100 nmol / L, then reacting at room temperature for 15 min, and performing fluorescence spectroscopy characterization.

[0055] Take 50 μL of A7-Cy5, B7-Alexa488, and A7 / B7 / C7-Cy5Alexa488 in a micro-cuvette, set the excitation wavelength to 488 nm, the slit width to 1.5 nm, and the emission wavelength scanning range to 500-730 nm to scan and measure each group. Figure 1As shown in the right figure, under 488nm excitation, only A7 / B7 / C7 has a clear peak at 688nm (as indicated by the arrow in the figure); the results indicate that under 488nm excitation, the emission spectrum of B7-Alexa488 is absorbed by A7-Cy5, so A7 / B7 / C7 has a clear peak at 688nm, the best emission of Cy5. This FRET result shows that the double-stranded DNA bridge 7 was successfully assembled. The same is true for the verification methods of other double-stranded DNA bridges.

[0056] Test Example 3

[0057] In Example 2, after washing away the unbound nucleic acid chains, 100 ng / mL EGF was added to each well and incubated with the cells at 37° C. for 30 min to study the effects of the seven double-stranded DNA bridges of different lengths on EGFR dimerization in A549 cells.

[0058] In order to verify the regulation of EGFR dimerization and the effect on EGFR function by double-stranded DNA bridges of different lengths, after incubating cells with different double-stranded DNA bridges, EGF was used to stimulate EGFR activation, and then the activation state of the receptor and downstream signaling pathways was detected, thereby judging the effect of seven double-stranded DNA bridges of different lengths on EGFR dimerization on A549 cells. The present invention selected four proteins, EGFR, p-EGFR, p-Akt and p-Erk1 / 2, as research objects for Western Blot detection. The first two can help understand the changes in EGFR expression and its phosphorylation level by double-stranded DNA bridges of different lengths, and the latter two, as key proteins in the signaling pathway, detecting their phosphorylation levels helps to know the effect of double-stranded DNA bridges on the downstream signaling pathways in cells. The specific process is as follows:

[0059] 1. Total protein extraction of each group of cells:

[0060] (1) Place the cells on ice to stop stimulation and rinse twice with PBS.

[0061] (2) Add 150 μL of the prepared cell lysis buffer to each well of cells, gently pipette the cells on ice to fully lyse them, and collect the cell lysate after 5 minutes.

[0062] (3) The collected lysate was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was transferred to a new EP tube for cryogenic storage.

[0063] 2. BCA protein quantification kit was used to detect the total protein concentration of each group

[0064] (1) Preparation of protein standards: Dissolve the protein standard in PBS according to the kit instructions and dilute to 0.5 mg / mL.

[0065] (2) Preparation of working solution: Mix working solution A and working solution B in the kit at a ratio of 1:50, shake gently to mix well and set aside.

[0066] (3) Dilution of protein standard: 0.5 mg / mL protein standard was added into a 96-well plate at the following times: 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL and 20 μL, followed by the addition of 20 μL, 19 μL, 18 μL, 16 μL, 12 μL, 8 μL, 4 μL and 0 μL of PBS, so that the total volume of each well was 20 μL.

[0067] (4) Dilution of protein samples of each group: 4 μL of each group of samples to be tested was added to a 96-well plate in sequence, followed by 16 μL of PBS to make the total volume of each well 20 μL.

[0068] (5) Addition of working solution: Add 200 μL of working solution to the protein standard and protein sample wells, and then shake at 37°C for 30 min.

[0069] (6) Determination of absorbance: Use a multifunctional plate reader to detect the absorbance of the wells of a 96-well plate at 562 nm.

[0070] (7) Drawing a standard curve: Draw a standard curve with the concentration of the protein standard as the horizontal axis and the absorbance value of the protein standard as the vertical axis. Substitute the absorbance value of each group of protein samples into the fitting equation to calculate the protein concentration of each group of samples.

[0071] (8) Protein preservation: Add 5× protein loading buffer to each protein sample, denature at 100°C for 5 min, and place in a -20°C refrigerator for later use.

[0072] 3. Western Blot detection of relative protein content in each group

[0073] (1) SDS-PAGE electrophoresis: Prepare 8 mL of lower gel and 2 mL of upper gel according to the instructions of the SDS-PAGE gel preparation kit; add 20 μg of the above protein sample to each well and 5 μL of protein marker to the wells on both sides; first run the electrophoresis at a constant voltage of 80 V for 30 min, then adjust the voltage to 120 V for 90 min.

[0074] (2) Transfer: After the electrophoresis is completed, the gel containing the target protein is appropriately cut according to the molecular weight indicated by the protein marker, and the length and width are measured. Then, the gel is placed in a tray containing 1× electrotransfer solution for later use. Next, a PVDF membrane of the same length and width is cut, and after being activated by soaking in methanol for 1 min, it is also placed in the tray for later use. Subsequently, two portions of filter paper of the same size (3 sheets each) are cut and also placed in the tray for later use. The "sandwich" transfer method is prepared in the order described. Afterwards, it is fixed with a clamp and placed in the electrotransfer tank, and the membrane is transferred at a constant current of 200 mA for 90 min.

[0075] (3) Blocking: After the transfer is completed, the PVDF membrane is taken out and placed in the prepared 5% skimmed milk powder, and incubated for 2 h at room temperature with shaking.

[0076] (4) Incubation with primary antibody: After blocking, remove the PVDF membrane and place it in TBST buffer and wash it three times, 8 min each time. Then, cut the PVDF membrane according to the target protein and place it in the primary antibody of the corresponding protein and incubate it at 4°C overnight.

[0077] (5) Incubation with secondary antibody: After the primary antibody incubation is completed, the PVDF membrane is removed and placed in TBST buffer and eluted three times, each time for 8 minutes; then the PVDF membrane is placed in the corresponding secondary antibody and shaken at room temperature for 1 hour.

[0078] (6) Development and imaging: After the secondary antibody incubation is completed, the PVDF membrane is removed and placed in TBST buffer and eluted three times, each time for 8 min. Then, the PVDF membrane is removed and placed on a development tray, and the excess buffer is wiped off with filter paper. The prepared developer is then evenly added to the PVDF membrane and placed in a chemiluminescence imager for imaging.

[0079] (7) Data analysis: The developed images were quantified using ImageJ software, and the relative content of the target protein was obtained by dividing the gray value of the target protein by the gray value of the internal reference protein. The data were then analyzed and further graphed using Graphpad Prism 8 software. The results are shown in the figure. Figure 2 and Figure 3 shown.

[0080] Depend on Figure 2 and Figure 3It can be seen that the A549 cells after starvation treatment were used as the blank group; in contrast, when the double-stranded DNA bridge 1 (0bp) was incubated with the cells, the expression levels of p-EGFR and p-Erk1 / 2 increased significantly, indicating that the double-stranded DNA bridge 1 can shorten the distance between EGFR, promote EGFR dimerization and activate downstream signals. As the length of the double-stranded DNA bridge increases, the expression levels of the three proteins p-EGFR, p-Akt and p-Erk1 / 2 decrease, and inhibition occurs at the double-stranded DNA bridge 5 (50bp), that is, the expression levels of the three proteins are lower than those of the control group; it may be due to the further increase in the receptor dimerization distance, and the double-stranded DNA bridge 7 (100bp) has the best effect in inhibiting EGFR function and downstream signaling pathways. In summary, the double-stranded DNA bridge can be used to regulate EGFR dimerization and downstream signaling pathways, and the double-stranded DNA bridge 7 can effectively inhibit EGFR dimerization activation and downstream protein activation. In addition, the activation of downstream signaling pathways is affected by regulating the function of the receptor. It also suggests that EGFR controls cellular responses by transmitting signals through two pathways: EGFR–GRB2–SOS–RAS–RAF–Erk1 / 2 and EGFR–Gab1–PI3K–Akt.

[0081] Test Example 4

[0082] Taking double-stranded DNA bridge 7 as an example, the binding of the probe to the cell is further verified by the change in the fluorescence intensity of the cell. The steps are as follows:

[0083] (1) Cell preparation: A549 cells in the logarithmic growth phase were collected after trypsin digestion, centrifuged and resuspended, and then the cells were counted. The cell density was then adjusted to 2×10 5 / mL, transfer to 4 5mL centrifuge tubes and add 1mL of cell suspension to each tube.

[0084] (2) Probe incubation: Use binding buffer 2 (1g / LBSA, 5mmol / LMgCl 2 6H 2 O, 25mmol / L glucose) was used to prepare 200nmol / L A7-Cy5, B7-Alexa488, and A7-Cy5 and B7-Alexa488 mixed probes. At the same time, the four tubes of cell suspension were centrifuged at 1000rpm at room temperature for 3min, and the supernatant was discarded. Then, they were incubated with 200μL of A7-Cy5, B7-Alexa488, A7-Cy5 and B7-Alexa488 mixed probes and binding buffer 2 (blank group) at room temperature with shaking for 15min.

[0085] (3) Flow cytometry: After incubation, the fluorescence intensity of cells in the four tubes was detected using a flow cytometer. The results were as follows: Figure 4 shown.

[0086] like Figure 4 As shown, the horizontal axis represents the fluorescence intensity of Cy5, and the vertical axis represents the fluorescence intensity of Alexa488; the red scattered dots represent the blank group (only cells); the blue scattered dots represent the control group 1 (cells incubated with A7-Cy5), and the collected Cy5 fluorescence intensity is significantly enhanced compared with the blank group; the orange scattered dots represent the control group 2 (cells incubated with B7-Alexa488), and the collected Alexa488 fluorescence intensity is significantly enhanced compared with the blank group; the green scattered dots represent the experimental group (cells incubated with A7-Cy5 and B7-Alexa488 at the same time), and the collected Cy5 fluorescence intensity and Alexa488 fluorescence intensity are significantly enhanced compared with the blank group, indicating that the EGFR aptamer selected in the experiment can bind to the EGFR on the cell membrane.

[0087] Test Example 5

[0088] Laser confocal characterization of the binding of double-stranded DNA bridges to cells: Taking double-stranded DNA bridge 7 as an example, laser confocal characterization of the binding of double-stranded DNA bridges to cells was performed. The FRET phenomenon was observed under a laser confocal fluorescence microscope to determine whether the probe was bound to the cell and whether a double-stranded DNA bridge was formed on the cell membrane surface. The specific operation steps are as follows:

[0089] (1) Cell preparation: A549 cells in the logarithmic growth phase were collected after trypsin digestion, centrifuged and resuspended, and then the cells were counted. Then, 1×10 5 The cells were seeded in a 30 mm confocal culture dish and cultured in an incubator for 24 h.

[0090] (2) Probe incubation: Use binding buffer 2 (1g / LBSA, 5mmol / LMgCl 2 6H 2 O, 25mmol / L glucose) was used to prepare 200nmol / L of A7-Cy5 and B7-Alexa488 mixed probes and complementary chain C7, and the cells were rinsed twice with PBS, then incubated with 300μL of A7-Cy5 and B7-Alexa488 mixed probes for 6min, then rinsed twice with PBS to wash away excess unbound probes, and then incubated with 300μL of complementary chain C7 for 12min, and then rinsed twice with PBS to wash away excess unbound nucleic acid chains.

[0091] (3) Cell imaging: Live cells were imaged in real time using a laser confocal fluorescence microscope with a 63× oil objective. Figure 5 shown.

[0092] Using sequential scanning, first under 488nm and 552nm excitation, the fluorescence of Alexa488 channel (500-550nm) and Cy5 channel (650-730nm) were collected, and the outline of the cell was clearly seen, indicating that A7-Cy5 and B7-Alexa488 were bound to EGFR on the cell membrane; then under 488nm excitation light, the fluorescence of Cy5 was collected, indicating that the emission spectrum of Alexa488 was absorbed by Cy5. This result confirmed that the complementary chain C7 was successfully assembled with A7 and B7 on the cell membrane surface, and Alexa488 and Cy5 underwent FRET, that is, a double-stranded DNA bridge 7 was successfully constructed on the cell membrane surface.

[0093] Experimental Example 6: Using double-stranded DNA bridge 7 as an example to study its effect on A549 cell behavior

[0094] CCK-8 assay:

[0095] (1) Cell plating: Take A549 cells in the logarithmic growth phase, digest them with trypsin, collect them, centrifuge and resuspend them, and then count the cells. Adjust the cell density to 5×10 4 / mL was inoculated in a 96-well plate, with 200 μL in each well. A blank group was set up at the same time, with only DMEM high-glucose medium added.

[0096] (2) Probe preparation: Prepare 150 μL of 40 nmol / L, 120 nmol / L, 200 nmol / L, 400 nmol / L, 1000 nmol / L, 4000 nmol / L A7 and B7 mixed probes and 150 μL of C7 solution of corresponding concentrations.

[0097] (3) Cell intervention: After the cells adhered to the wall for 24 h, the original culture medium was discarded, the cells were rinsed twice with PBS, and cell culture medium without fetal bovine serum was added to continue culturing for 24 h (starvation treatment); then, the cells were rinsed twice with PBS, 50 μL of mixed probes of different concentrations of A7 and B7 were added (2 replicate wells) and incubated at 37°C for 6 min, 50 μL of C7 solution of corresponding concentration was added and incubated at 37°C for 12 min, and then 100 μL of 200 ng / mL EGF was added and incubated with the cells for 24 h; the final concentrations of double-stranded DNA bridge 7 in each well were 10 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L, 250 nmol / L, and 500 nmol / L, respectively, and the EGF concentration was 100 ng / mL.

[0098] (4) Add CCK-8: Discard the intracellular solution in each well, rinse twice with PBS, add 10 μL CCK-8 solution and 190 μL cell culture medium without fetal bovine serum to each well, and continue incubating in the incubator for 1 hour.

[0099] (5) Detection: When the solution turns yellow, measure the absorbance of the 96-well plate at 450 nm using a multifunctional microplate reader.

[0100] (6) Calculate the cell proliferation inhibition rate.

[0101]

[0102] The results are shown in Table 4 and Figure 6 As shown in the figure, compared with the control group, each experimental group showed an effect on the proliferation ability of A549 cells, that is, double-stranded DNA bridge 7 can inhibit the proliferation ability of A549 cells, and as the concentration of double-stranded DNA bridge 7 increases, the inhibitory effect on cell proliferation is more obvious (P<0.05). When the intervention concentration of double-stranded DNA bridge 7 is lower than 50nmol / L, each experimental group has a weaker inhibitory effect on cell proliferation; when its intervention concentration is higher than 50nmol / L, it shows a stronger ability to inhibit proliferation.

[0103] Table 4

[0104]

[0105] Note: # It means that the difference between the experimental group and the EGF group is statistically significant. # P<0.05.

[0106] Cell scratch assay:

[0107] (1) Cell plating: Take A549 cells in the logarithmic growth phase, digest them with trypsin, collect them, centrifuge and resuspend them, and then count the cells. Adjust the cell density to 1×10 5 / mL was inoculated into a 12-well plate, with 1 mL in each well.

[0108] (2) Probe preparation: Prepare 750 μL of 200 nmol / L and 400 nmol / L mixed probes of A7 and B7, and 750 μL of C7 solution of corresponding concentrations.

[0109] (3) Cell scratching: When the cell confluence reached 80%, the original culture medium was discarded, the cells were rinsed twice with PBS, and cell culture medium without fetal bovine serum was added to continue culturing for 24 h (starvation treatment); then, a scratch wound was scraped in the cell monolayer of each well with a 200 μL pipette tip, and then rinsed twice with PBS to remove free cells; at this time, a microscope was used to take pictures and record the width of the cell scratch at 0 h.

[0110] (4) Cell intervention: Then, 250 μL of mixed probes of A7 and B7 of different concentrations (two replicate wells) were added and incubated at 37°C for 6 min. Then, 250 μL of C7 solution of corresponding concentration was added and incubated at 37°C for 12 min. Then, 500 μL of 200 ng / mL EGF was added and the cells were incubated continuously. Finally, the concentrations of double-stranded DNA bridge 7 in each well were 50 nmol / L and 100 nmol / L, respectively, and the EGF concentration was 100 ng / mL.

[0111] (5) Photo recording: Take out the 12-well plate at 4 h, 8 h, and 24 h, and take photos of the scratches on the cells in each well.

[0112] (6) Data analysis: ImageJ software was used to quantify the scratch width and calculate the migration rate.

[0113]

[0114] The results are as follows Figure 7 As shown: According to the results of the inhibition rate of cell proliferation by double-stranded DNA bridge 7, the two concentrations of 50nmol / L and 100nmol / L were selected for the experiment. The cells were divided into a blank group, a control group (EGF), and an experimental group (EGF+50nmol / LA7 / B7 / C7 and EGF+100nmol / LA7 / B7 / C7) for different treatments. Compared with the EGF group, each experimental group showed an effect on the migration ability of A549 cells in the 4h, 8h and 24h time periods, that is, double-stranded DNA bridge 7 can inhibit the migration ability of A549 cells. Under the intervention of 50nmol / L and 100nmol / L double-stranded DNA bridge 7, the cell scratch migration rate was significantly decreased (P<0.05).

[0115] Transwell invasion assay:

[0116] (1) Laying Matrigel: Melt the Matrigel at 4°C, then dilute it with serum-free medium at a ratio of 1:8. After dilution, take 50 μL and evenly spread it on the upper polycarbonate membrane surface of the Transwell chamber. Then, place the chamber in a cell culture incubator at 37°C for 1 hour to allow it to solidify. Note that the operation should be performed on ice to prevent the Matrigel from solidifying prematurely.

[0117] (2) Cell plating: Take A549 cells in the logarithmic growth phase, digest them with trypsin, collect them, and count the cells after centrifugation and resuspending. Adjust the cell density to 5×10 5 / mL was inoculated, 100 μL was inoculated into each chamber, and 500 μL of cell culture medium was added to the 24-well plate.

[0118] (3) Probe preparation: Prepare 75 μL of 200 nmol / L and 400 nmol / L A7 and B7 mixed probes, and 75 μL of C7 solution of corresponding concentrations.

[0119] (4) Cell intervention: After the cells adhered to the wall and grew, the original culture medium was discarded, and the cells were rinsed twice with PBS. The chamber and the 24-well plate were added with cell culture medium without fetal bovine serum and cultured for 24 hours (starvation treatment); then they were rinsed twice with PBS, and then 25 μL of mixed probes of different concentrations of A7 and B7 (2 replicate wells) were added and incubated at 37°C for 6 minutes, and then 25 μL of C7 solution of corresponding concentration was added and incubated at 37°C for 12 minutes, and then 50 μL of 200 ng / mL EGF was added and the cells were incubated for 24 hours, and 500 μL of cell culture medium was added to the 24-well plate; the final concentrations of double-stranded DNA bridge 7 in each well were 50 nmol / L and 100 nmol / L, respectively, and the EGF concentration was 100 ng / mL.

[0120] (5) Cell fixation and staining: Remove the chambers, discard the solution in each chamber, and gently wipe off the matrix gel and cells in the upper chamber with a cotton swab; take a new 24-well plate and add 600 μL of 4% paraformaldehyde to each well, allowing the chambers to be immersed and fixed for 20 to 30 minutes; gently rinse with PBS three times, and stain with crystal violet solution for 5 to 10 minutes.

[0121] (6) Photographing and counting: Rinse gently with PBS three times, wipe the upper chamber gently with a cotton swab, air-dry appropriately, and take photos under a microscope. Select three fields of view to observe and count the cells.

[0122] (7) Data analysis: ImageJ software was used to quantify the number of cells that passed through the matrix gel and calculate the invasion rate.

[0123]

[0124] The results are as follows Figure 8 As shown: According to the inhibitory effect of double-stranded DNA bridge 7 on cell proliferation and cell migration, the two concentrations of 50nmol / L and 100nmol / L and the 24h observation point were selected for the experiment. The cells were divided into a blank group, a control group (EGF), and an experimental group (EGF+50nmol / LA7 / B7 / C7 and EGF+100nmol / LA7 / B7 / C7) for different treatments. Compared with the EGF group, each experimental group showed an effect on the invasion ability of A549 cells, and the number of cells that passed through the matrix gel and entered the lower chamber was significantly reduced, that is, double-stranded DNA bridge 7 can inhibit the invasion ability of A549 cells. Under the intervention of 50nmol / L and 100nmol / L double-stranded DNA bridge 7, the cell invasion rate was significantly decreased (P<0.05).

[0125] From the above experiments, it can be seen that the nucleic acid self-assembled double-stranded DNA bridge of the present invention can be successfully constructed on A549 cells and regulate the dimerization state of EGFR. During the regulation process, as the distance of the double-stranded DNA bridge increases, that is, the spatial distance of EGFR dimerization becomes farther, the degree of EGFR activation gradually decreases. The 100bp double-stranded DNA bridge 7 can effectively inhibit the dimerization activation of EGFR and successfully inhibit the proliferation, migration and invasion of A549 cells by inhibiting the downstream signaling pathway in the cell.

[0126] The nucleic acid self-assembled double-stranded DNA bridge of the present invention is used to regulate the dimerization of EGFR on the cell membrane surface. The principle diagram is as follows: Fig. 9 As shown: Each double-stranded DNA bridge includes three or four DNA chains, namely aptamer probes A and B, complementary chain C (and auxiliary chain D). After the EGFR aptamers in the aptamer probes A and B specifically recognize and bind to the receptor EGFR on the cell membrane, each exposes a DNA sequence; when complementary chain C is present, the exposed sequences on probes A and B can self-assemble with complementary chain C through the principle of base complementary pairing to form a double-stranded DNA bridge.

[0127] The present invention involves seven double-stranded DNA bridges of different lengths (0 bp, 10 bp, 20 bp, 30 bp, 50 bp, 70 bp and 100 bp), which are respectively defined as double-stranded DNA bridge 1 (A1 / B1 / C1, 0 bp), double-stranded DNA bridge 2 (A2 / B2 / C2, 10 bp), double-stranded DNA bridge 3 (A3 / B3 / C3 / D3, 20 bp), double-stranded DNA bridge 4 (A4 / B4 / C4, 30 bp), double-stranded DNA bridge 5 (A5 / B5 / C5, 50 bp), double-stranded DNA bridge 6 (A6 / B6 / C6, 70 bp) and double-stranded DNA bridge 7 (A7 / B7 / C7, 100 bp). The specific nucleic acid sequences involved are shown in Table 3. The seven double-stranded DNA bridges of different lengths are incubated with cells respectively to induce and regulate the dimerization of EGFR at different distances. The principle is as follows: Fig.10 As shown. By regulating double-stranded DNA bridges of different lengths, the effective distance at which EGFR dimerization can be inhibited is explored, thereby inhibiting the level of EGFR dimerization activation, further preventing intracellular signal transduction, and ultimately achieving the purpose of controlling cell behavior. The present invention not only helps to explore in detail the relationship between receptor dimerization and receptor activation degree and intracellular downstream signal transduction at nanoscale intervals, but also helps to fully understand the receptor mechanism of action, providing a new idea for effectively regulating the occurrence and development of cancer, which is of great significance for the targeted treatment of cancer.

[0128] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A DNA molecule that targets and regulates EGFR dimerization on the cell membrane surface, characterized in that: The DNA molecule is a double-stranded DNA bridge constructed by nucleic acid self-assembly, and the double-stranded DNA bridge includes aptamer probe A and aptamer probe B, and a complementary strand C complementary to aptamer probe A and aptamer probe B; The double-stranded DNA bridge is one of double-stranded DNA bridges 5 to DNA bridge 7, and the double-stranded DNA bridges 5 to DNA bridge 7 are respectively composed of nucleic acid aptamer probes A5, B5 and complementary chains C5, A6, B6 and complementary chains C6, A7, B7 and complementary chains C7, and the nucleic acid sequences of A5, B5, C5, A6, B6, C6, A7, B7, C7 are as shown in SEQ ID NO:13-21.

2. The method for preparing a DNA molecule for targeted regulation of EGFR dimerization on the cell membrane surface as claimed in claim 1, characterized in that: The method comprises the following steps: taking the single nucleic acid chain master solutions A5, B5, C5, A6, B6, C6, A7, B7, C7 of each double-stranded DNA bridge respectively, heating them in a water bath and then cooling them to room temperature; the concentrations of the single nucleic acid chain master solutions are the same, and the single nucleic acid chain master solutions corresponding to each double-stranded DNA bridge are mixed in a volume ratio of 1:1:1; after reaction at room temperature, double-stranded DNA bridges 5 to 7 are obtained.

3. The method for preparing a DNA molecule for targeted regulation of EGFR dimerization on the cell membrane surface according to claim 2, characterized in that: Each single nucleic acid strand master solution was heated in a 95°C water bath for 10 min.

4. The method for preparing a DNA molecule for targeted regulation of EGFR dimerization on the cell membrane surface according to claim 2, characterized in that: React at room temperature for 15 minutes.

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