A dual DNA tetrahedron structure, its preparation method and application

Abstract

The present invention provides a dual DNA tetrahedron structure, a preparation method and an application thereof. The dual DNA tetrahedron of the present invention includes a first DNA tetrahedron structure, a second DNA tetrahedron structure and a connecting chain. This structure can specifically recognize two cell surface proteins and be labeled by fluorescence; at the same time, due to the steric hindrance effect of the structure, it is difficult for the two proteins to approach each other, affecting the interaction between the two. This structure can exist on the cell surface for a long time, thus having a good cell regulation effect on these two cell membrane surface proteins, specifically reflected in reducing the phosphorylation level of HER2 protein, promoting the expression of apoptosis-related proteins, and ultimately leading to apoptosis of tumor cells and reducing the cell activity of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical fields of fluorescent structures and tumor detection, and particularly relates to a dual DNA tetrahedron structure, a preparation method of the structure, and an application thereof in tumor cell detection. Background Technique

[0002] The statements in this part only provide background technical information related to the present invention and do not necessarily constitute prior art.

[0003] DNA molecules can be combined with inorganic nanomaterials, such as modified on the surface of gold, quantum dots, or loaded in mesoporous nanoparticles, for intracellular cancer biomarker analysis. In addition, structures assembled from DNA strands provide more opportunities for bioanalysis due to their good biocompatibility, excellent anti-degradation stability, controllable shape, size, and easy endocytosis by cells. Analytical methods based on DNA structures have been widely used for the sensitive detection of intracellular proteins, ions, miRNAs, etc.

[0004] Membrane proteins are located on the cell surface and show complexity in variability and quantity. These changes may all be related to different cell identities, and abnormally expressed proteins are generally present on the membranes of cancer cells. Compared with intracellular genomic or transcriptional marker molecules (such as RNA or DNA), membrane proteins have excellent accessibility and specificity, so they are excellent candidates for the accurate detection and diagnosis of early tumors.

[0005] HER2 is a member of the human epidermal growth factor receptor family, and other members also include ErbB1, HER3, and HER4. These receptors activate a variety of downstream signaling pathways through protein dimerization, thereby regulating cell proliferation and growth. The content of this family of proteins in normal cells is very low, while the overexpression of HER2 protein is very common in many tumor cells, especially in breast cancer cells. This abnormality is found in about 30% of cases, leading to uncontrolled proliferation of tumor cells. Nucleolin is a protein commonly present on the surface of cancer cells and participates in the basic processes of cell proliferation and growth. The level of nucleolin on the surface of the vast majority of cancer cells is increased and participates in cell oncogenic transformation and cancer development. Existing studies have found that the overexpression of cell surface nucleolin enhances the interaction between nucleolin and HER2 protein and can activate the HER2 receptor and its regulated signaling pathways, which is closely related to tumor cell growth. Therefore, targeting these two cell membrane surface proteins may achieve the regulation of cell behavior.

[0006] Currently, methods based on nucleic acid aptamers to recognize cell surface membrane proteins have been widely developed and applied. Many studies have proved that nucleic acid aptamer chains have good cell recognition specificity and certain regulatory capabilities. However, simple DNA chains inevitably have shortcomings such as poor stability and integration, which lead to the existence of false signals and limit the regulatory effect. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a dimer-DNA tetrahedron structure (dimer-TDN), which can specifically bind to the target protein on the surface of tumor cells, can more sensitively detect the target protein on the surface of tumor cells or cell surface protein extracts, and show better recognition specificity for tumor cells; in addition, the dimer-DNA tetrahedron structure (dimer-TDN) can regulate the spatial position of the target protein to a certain extent, thereby regulating the cell life course. The present invention also provides a preparation method of the dimer-DNA tetrahedron structure (dimer-TDN) and its application in cell recognition and regulation of cell life course.

[0008] In the present invention, the inventor provides a dimer-DNA tetrahedron structure (dimer-TDN) integrating two aptamers. The structure is composed of aptamer DNA tetrahedrons connected to HER2 protein and nucleolin protein (NCL) connected by a connecting chain. The connecting chain is modified with a fluorescent group and a quenching group, and the fluorescent group is quenched due to its proximity to the quenching group.

[0009] With the help of cell membrane fluidity, the two aptamers can synergistically and specifically bind to the target protein on the cell surface. This specific binding allows the quenching group labeled on the dimer-TDN to be unblocked, and the fluorescence of the fluorescent group is restored for cell recognition; at the same time, due to the steric hindrance of the dimer-TDN bound to the target protein, it is difficult for the two proteins to approach each other, affecting the interaction between the two. This structure can exist on the cell surface for a long time, and thus has a good cell regulation effect on the proteins on the surface of the two cell membranes, which is specifically reflected in reducing the phosphorylation level of HER2 protein and promoting the expression of proteins related to cell apoptosis, and ultimately leads to tumor cell apoptosis and reduces the cell activity of tumor cells. The dimeric DNA tetrahedron structure (dimer-TDN) in the present invention can not only realize the specific recognition of tumor cells, but also realize the effective regulation of cells by changing the relative position of cell surface proteins, which is helpful to provide new research ideas for the diagnosis and treatment of related diseases.

[0010] Specifically, the technical solution of the present invention is as follows:

[0011] In a first aspect of the present invention, a dimeric DNA tetrahedron structure is provided, which includes a first DNA tetrahedron structure, a second DNA tetrahedron structure, and a linking strand.

[0012] The first DNA tetrahedron structure is composed of four single-stranded DNAs S1, S2, S3, S4 and BN, and the S4 contains an aptamer domain of the target protein; the BN can base-complementary pair with the S4 aptamer domain.

[0013] The second DNA tetrahedron structure is composed of four single-stranded DNAs S1, S2, S3', S4' and BH, and the S4' contains an aptamer domain of the target protein; the BH can base-complementary pair with the S4' aptamer domain.

[0014] The linking strand is composed of three single-stranded DNAs S5, S6 and S7.

[0015] The S6 connects the first DNA tetrahedron structure and the second DNA tetrahedron structure, and the S6 is complementary to the S5 sequence at the same time; the S7 is complementary to the S5 sequence; the S5 and S7 are also complementary to BH and BN respectively; the ends of the S5 and S6 that are base-complementary are respectively modified with a quenching group and a luminescent group.

[0016] In some embodiments of the present invention, the sequences of S1-S4 are as shown in SEQ ID NO.1-4; the sequences of S3' and S4' are as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively; the sequence of BN is as shown in SEQ ID NO.7; the sequence of BH is as shown in SEQ ID NO.8; the sequence of S5 is as shown in SEQ ID NO.9; the sequence of S6 is as shown in SEQ ID NO.10; the sequence of S7 is as shown in SEQ ID NO.11.

[0017] In some embodiments of the present invention, the 5' end of the S5 is modified with a quenching group; the 3' end of the S6 is modified with a luminescent group; further, the luminescent group is Cy3; the quenching group is BHQ.

[0018] In a second aspect of the present invention, a method for preparing the dimeric DNA tetrahedron structure (dimer-TDN) described in the first aspect is provided, which includes mixing the first DNA tetrahedron structure, the second DNA tetrahedron structure and the linking strand in equal proportions, and co-incubating to assemble the dimeric DNA tetrahedron structure.

[0019] The incubation conditions are: incubating at 35°C - 38°C for 3 - 6 h; specifically, the incubation conditions are: incubating at 37°C for 4 h.

[0020] In some embodiments of the present invention, the method for preparing the first DNA tetrahedron structure is as follows: single-stranded DNAs S1, S2, S3, S4, and BN that constitute the tetrahedron are mixed in a TAE-Mg 2+ buffer solution at a molar ratio of 1:1:1:1:1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product. Preferably, the mixed solution is heated at 95 °C for 5 min and then slowly cooled to room temperature;

[0021] Alternatively, the method for preparing the second DNA tetrahedron structure is as follows: single-stranded DNAs S1, S2, S3’, S4’, and BH that constitute the tetrahedron are mixed in a TAE-Mg 2+ buffer solution at a molar ratio of 1:1:1:1:1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product. Preferably, the mixed solution is heated at 95 °C for 5 min and then slowly cooled to room temperature.

[0022] In some embodiments of the present invention, the method for preparing the linking chain is as follows: single-stranded DNAs S5, S6, and S7 are mixed and annealed to obtain a linking chain structure. Specifically, the annealing conditions are as follows: single-stranded DNAs S5, S6, and S7 are mixed in a TAE-Mg 2+ buffer solution at a molar ratio of 1:1:1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product.

[0023] In a third aspect of the present invention, the present invention provides a method for detecting tumor cells, which is realized by using the dual DNA tetrahedron structure described in the first aspect above; the detection is not for the purpose of disease diagnosis and treatment.

[0024] In some embodiments of the present invention, it includes co-incubating the cells to be detected or a sample containing the cells to be detected with the dual DNA tetrahedron structure described in the first aspect above at 35-38 °C for 0.5-5 h, and then performing fluorescence imaging; the detection is not for the purpose of disease diagnosis and treatment.

[0025] In a fourth aspect of the present invention, the present invention provides the application of the dual DNA tetrahedron structure described in the first aspect above in the preparation of tumor cell detection products, and the products include experimental reagents, detection chips, and detection systems; the tumor cells include breast cancer cells.

[0026] In a fifth aspect of the present invention, the present invention provides the application of the dual DNA tetrahedron structure described in the first aspect above in the preparation of products for promoting tumor cell apoptosis, and the products include detection chips and detection systems; the tumor cells include adenocarcinoma cells.

[0027] The above one or more technical solutions have the following beneficial effects:

[0028] In the present invention, the dimer DNA tetrahedron structure (dimer-TDN) can specifically recognize target proteins / tumor cells to be detected and regulate tumor cells. The dimer-TDN can specifically target two membrane proteins through aptamers, can perform good logical responses through intermolecular binding, and can report fluorescence signals through strand displacement reactions within the structure for detection, enabling highly sensitive and strongly specific recognition of tumor cells to be detected. At the same time, the steric hindrance effect of the dimer-TDN hinders the interaction between the two proteins; the dimer-TDN is more likely to target the cell membrane surface than the single DNA tetrahedron structure, and this structure can exist on the cell surface for a longer time. The dimer-TDN regulates cell signal transduction, thereby changing the life course of tumor cells and promoting tumor cell apoptosis, providing a new idea for the accurate recognition of tumor cells and cancer diagnosis.

[0029] The advantages of the additional aspects of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0031] Figure 1 : Schematic diagram of the principle of dimer DNA tetrahedron for the analysis and regulation of HER2 and nucleolin on the cell surface.

[0032] Figure 2 : Enlarged schematic diagram of the dimer-TDN structure.

[0033] Figure 3 : Gel electrophoresis diagram of the strand substitution reaction performed by the linker sequence

[0034] Figure 4 : DN NCL-apt 、TDN HER2-apt and the assembly and formation process of dimer-TDN; (a) Native polyacrylamide gel electrophoresis characterization. Lane M: Maker; Lanes 1 and 6: S1; Lanes 2 and 7: S1+S2; Lane 3: S1+S2+S3; Lane 4: S1+S2+S3+S4; Lane 5: S1+S2+S3+S4+BN; Lane 8: S1+S2+S3; Lane 9: S1+S2+S3’+S4’ Lane 10: S1+S2+S3’+S4’+BH; (b) Agarose gel characterization diagram; Lane M: Maker; Lane 1: TDN NCL-apt; Lane 2: TDN HER2-apt ; Lane 3: dimer-TDN

[0035] Figure 5 : Atomic force microscope (AFM) image characterization of (a) TDN NCL-apt (b) TDN HER2-apt and (c) dimer-TDN structure; Sample tests were all imaged in liquid phase mode, scale bar: 100 nm

[0036] Figure 6 : TDN NCL-apt 、TDN HER2-apt and the dynamic light scattering characterization diagrams of dimer-TDN structure

[0037] Figure 7 : Fluorescence spectrum feasibility verification of dimer-TDN, excitation wavelength: 538 nm

[0038] Figure 8 : (a) Confocal laser imaging of cell-specific recognition, scale bar: 25 μm; (b) Flow cytometry analysis diagram

[0039] Figure 9 : (a) Fluorescence imaging diagrams of dimer-TDN structure and (b) TDNNCL-apt structure incubated with cells over time, scale bar: 25 μm

[0040] Figure 10 : (a) Fluorescence imaging diagrams of dimer-TDN structure and (b) TDNNCL-apt structure stained with cell membrane, scale bar: 10 μm

[0041] Figure 11 : TIRFM co-localization imaging diagrams of NCL and HER2 proteins on cell membrane; Scale bar: 2 μm

[0042] Figure 12 : (a) Immunoblotting diagrams of the expression levels of p-HER2 and Bax proteins in SK-BR-3 cells after treatment with dimer-TDN structure for different times; (b) Flow cytometry analysis diagrams for cell apoptosis detection

[0043] Figure 13 : (a) Effects of dimer-TDN structure on different cell viabilities; (b) Effects of different concentrations of dimer-TDN structure on the viability of SK-BR-3 cells Detailed implementation manners

[0044] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0045] It should be noted that the terms used herein are merely for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention.

[0046] In the case of no conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0047] Glossary:

[0048] TDN NCL-apt Refers to a DNA tetrahedron structure capable of targeting NCL, which is composed of four single-stranded DNAs S1, S2, S3, S4 and BN.

[0049] TDN HER2-apt Refers to a DNA tetrahedron structure capable of targeting HER2, which is composed of four single-stranded DNAs S1, S2, S3', S4' and BH.

[0050] Detection principle of the present invention:

[0051] The present invention first prepared a dyad DNA tetrahedron structure and then applied it to cell recognition and tumor cell apoptosis. The present invention designed a suitable aptamer according to the target protein to be detected and set it in S4 or S4' of the DNA tetrahedron structure. The aptamer regions of S4 and S4' were both designed with BN and BH that could play a blocking role; S3 or S3' of each DNA tetrahedron structure could be complementary paired with different regions of S6 in the linker chain respectively, so that the two tetrahedron structures could be connected. The linker chain also included S5 and S7. The 5'-end region of S5 could be complementary paired with a part of the 3'-end of S6, the 3'-end region of S5 could be complementary paired with a part of S7, and the 5'-end of S5 was modified with a quenching group, and the 3'-end of S6 was modified with a fluorescent group, so that the linker chains approaching each other would not show high-intensity fluorescence. In addition, it should be noted that BN could bind to a part of the sequence of S7 to release S7 from S5; BH could bind to a part of the sequence of S5 to release S5 from S6.

[0052] After the structure binds to the cell surface protein to be detected, BN and BH that block the aptamer region can be released. BN and BH can bind to S7 and S5 respectively and displace them, which will cause S5 to separate from S6, releasing the fluorophore, so that detection can be carried out under a suitable spectrum for cell recognition. At the same time, the dyad DNA tetrahedron structure has a certain steric hindrance, which can adjust the relative positions of cell membrane surface proteins, thus changing the cell signal transduction pathway and then enabling the regulation of the cell life process. The specific principle is shown in Figure 1 , S1-S7, BN, BH, S3', and S4' are all single-stranded DNAs.

[0053] Example

[0054] 1. Reagents and Raw Materials

[0055] The DNA sequences (Table 1) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Diethyl pyrocarbonate (DEPC)-treated water, 40% acrylamide / bisacrylamide (19:1) solution, ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED), Tris, and ethylenediaminetetraacetic acid tetrasodium (EDTA) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). SYBR Gold nucleic acid gel stain was purchased from Thermo Fisher Scientific. Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co., LLC (Missouri, USA). Annexin V-FITC apoptosis detection kit was purchased from Beyotime (Shanghai, China). Alexa Fluor488-labeled wheat germ agglutinin for cell membrane staining was purchased from Thermo Fisher Scientific (Waltham, USA). Protein primary antibodies β-actin, phospho-HER2, Bax, and HRP-labeled anti-rabbit IgG secondary antibody were all purchased from Cell Signaling Technology (the Chinese subsidiary of CST-US in Shanghai). Fetal bovine serum (FBS), DMEM cell culture medium, and phosphate buffered saline (PBS) buffer were purchased from Biological Industries (Israel). Throughout the experiment, ultrapure water (18.25 MΩ·cm) obtained from a UP water purification system was used.

[0056] The TU-1901 spectrometer (Purkinje General Instrument Co., China) was used to record the ultraviolet-visible absorption spectra. The F-320 fluorescence spectrophotometer (Hongdong Co., Tianjin) was used to record the fluorescence emission spectra. The polyacrylamide gel was imaged on a Gel DocTM XR+ imaging system (Bio-RAD, USA). The nanoparticle size and zeta potential analyzer (Malvern, UK) was used for dynamic light scattering measurements. The atomic force microscope (Bruker, USA) was used to characterize the samples. The immunoblot imaging was performed using an Amersham Imager 600 imaging system (GE Healthcare, USA). The confocal fluorescence images were recorded with an oil immersion objective lens (63×) on a laser confocal microscope (Leica, Germany). Flow cytometry analysis was performed using a NovoCyte 3130 flow cytometer (Agilent, USA). Single molecule fluorescence imaging analysis was performed using a multi-color total internal reflection microscope (Olympus, Japan). The absorbance measurements in the MTT assay were carried out on a multimode microplate reader (Tecan, Switzerland).

[0057] Table 1 Oligonucleotide sequences used in the present invention

[0058]

[0059]

[0060] Note: The DNA sequences at the tetrahedron junctions are marked in bold. The aptamer sequences for HER2 protein are marked in italic and gray, and the aptamer sequences for nucleolin protein are marked The remaining identical sequences with single underlines indicate complementary pairing sequences.

[0061] 2. Experimental procedures

[0062] (1) Preparation of the DNA tetrahedron dimer

[0063] Annealing treatments were performed on TDN NCL-apt and TDN HER2-apt respectively. The strands S1, S2, S3, S4, BN that constitute the tetrahedron, or S1, S2, S3’, S4’, BH were mixed in a TAE-Mg 2+ (40 mM Tris, 1.5 mM Mg(CH3COO)2, 0.01 mM EDTA, pH 8.0) buffer solution at a molar ratio of 1:1:1:1:1. The mixed solution was heated at 95 °C for 5 min and then slowly cooled to room temperature. The equimolar amounts of S5, S6, S7 strands were mixed in the TAE-Mg 2+ buffer solution in the same way, and the mixture was heated at 95 °C for 5 min and slowly cooled to room temperature to obtain the linker chain structure. TDN NCL-apt and TDN HER2-aptMix with the connecting chain in equal proportion and incubate at 37 °C for 4 h to obtain the dimer-TDN structure. The synthesized dimer-TDN is stored in a refrigerator at 4 °C for later use.

[0064] (2) Polyacrylamide gel electrophoresis

[0065] Mix the DNA sample with the loading buffer and load it onto a 6% polyacrylamide gel. The 6% polyacrylamide gel is prepared by mixing 16.25 mL of ultrapure water, 3.75 mL of 40% acrylamide / bisacrylamide solution (19:1), 5 mL of 5×TAE-Mg 2+ buffer (200 mM Tris-Ac, 7.5 mM Mg(CH3COO)2, 0.05 mM EDTA, pH 8.0), 180 μL of 0.1 g / mL APS, and 18 μL of TEMED. The gel is run in 1×TAE buffer at 4 °C and 100 V for 3.5 h, stained with 1×SYBR Gold for 30 min, and then imaged using a system.

[0066] (3) Agarose gel electrophoresis characterization

[0067] Prepare a 2% agarose gel. Add 0.6 g of agarose powder to 30 mL of TAE-Mg 2+ buffer for dissolution, heat it in a microwave oven until it becomes clear and transparent, cool it to about 60 °C, pour it into a gel maker, and let it solidify for half an hour before loading the sample. The sample concentration is 300 nM. After running at 100 V for 2 h, stain and image it.

[0068] (4) Atomic force microscopy characterization

[0069] Tear off a fresh mica sheet with tape and place it in a petri dish containing 20 μL of APTES to let it volatilize. After incubating for 5 min, rinse it three times with ultrapure water. Respectively, drop 20 μL of 100 nM TDN NCL-apt , TDN HER2-apt and the dimer-TDN structure solution onto the mica and deposit for 10 min, then rinse the mica with ultrapure water, add 20 μL of ultrapure water to the mica sheet, and then perform the test. Characterize the sample using an atomic force microscope.

[0070] (5) Dynamic light scattering characterization

[0071] Perform structural dynamic light scattering characterization on a nanoparticle size potentiometer. Respectively, add 50 μL of 100 nM TDN NCL-apt , TDN HER2-apt and the dimer-TDN structure into a 50-μL disposable cuvette (Sarstedt, Germany) for dynamic light scattering measurement.

[0072] (6) Fluorescence spectrum measurement

[0073] The concentration of the sample dimer-TDN structure was 50 nM. The fluorescence emission spectrum was recorded in the range of 550 - 700 nm at an excitation wavelength of 538 nm.

[0074] (7) Cell culture

[0075] SK-BR-3, HeLa, and HL-7702 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin-streptomycin. The cells were all cultured at 37 °C in a humidified environment containing 5% CO2.

[0076] (8) Flow cytometry

[0077] Three different types of cells were seeded into 24-well plates at a density of 2×10 5 cells / well and incubated overnight. After the cells adhered, the original medium was removed, and 500 μL of medium containing 200 nM structure was added. The cells were incubated at 37 °C for 1 h. After discarding the medium, the cells were washed with PBS, then digested with trypsin and collected into centrifuge tubes. The cells were collected by centrifugation at 1000 rpm for 3 min and resuspended in 100 μL of PBS containing 2% fetal bovine serum for flow cytometry analysis.

[0078] (9) Laser confocal fluorescence imaging

[0079] To test the response of the dimer-TDN structure to different types of cells, SK-BR-3 cells (or HeLa, HL-7702 cells) were seeded in confocal dishes overnight. After adhesion, the old medium was removed, the cells were washed with PBS buffer, and incubated with fresh medium containing 200 nM structure at 37 °C for 2.5 h for confocal imaging. To investigate the entry of single DNA tetrahedron and dimer DNA tetrahedron structures into cells, SK-BR-3 cells were seeded in confocal dishes overnight, and then dimer-TDN and TDN were separately NCL-aptFluorescence imaging was performed after incubating with cells for a series of times. In the experiment of co-localization of cell membrane and structure, the cell membrane stain WGA-AF488 was added 30 minutes before imaging. After washing with PBS before imaging, fresh medium was replaced for imaging. The experimental conditions in the reference of this experiment (Zheng J, Wang Q, Shi L, et al. Calcium-differentiated cellular internalization of allosteric framework nucleic acids for targeted payload delivery[J]. Anal. Chem., 2022, 94(25): 9097-9105.) added 20 mM of Ca 2+ and 50 mM of K + .

[0080] (10) Single-molecule fluorescence imaging

[0081] SK-BR3 cells were incubated with a mixture of S3-AF488 labeled with NCL aptamer and S3'-Cy3 labeled with HER2 aptamer for 1 h, or incubated with dimer-TDN structures labeled with two fluorescent groups synthesized from S3-AF488 and S3'-Cy3 for 2 h at a structure concentration of 200 nM. After washing three times with PBS, fresh medium was replaced for live cell single-molecule fluorescence imaging analysis. Single-molecule fluorescence images were taken on an Olympus microscope, and the labeled samples were excited with 488 nm and 561 nm lasers respectively.

[0082] (11) Immunoblot analysis

[0083] SK-BR-3 cells were seeded onto 6-well plates and the experiment was performed when the cells reached 90% confluence. The cells were treated with 400 nM of the structure for 12 h or 24 h respectively. Then the cells were lysed with RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. Bax and p-HER2 were separated using 12% and 6% SDS-PAGE gels respectively, and transferred to PVDF membranes with 0.22 μm and 0.45 μm after activation with methanol. After cutting the membranes according to the molecular weight, they were incubated with the corresponding primary antibodies overnight at 4 °C. The next day, they were removed, washed, incubated with secondary antibodies and washed again. Finally, the enhanced developing solution from Beyotime was used to image the target proteins.

[0084] (12) Cell apoptosis analysis

[0085] SK-BR-3 cells were seeded at a density of 2×10 5The cells were seeded at a density of [number of cells] per well in a 24-well plate and incubated overnight. Subsequently, the cells were incubated with 200 nM TDN NCL-apt , TDN HER2-apt , and the dimer-TDN construct for 12 h, and then washed with PBS. After transferring the cells to a flow cytometry tube, 195 μL of Annexin V binding buffer, 5 μL of Annexin V-TITC binding solution, and 5 μL of PI binding solution were added successively. After incubating in the dark at room temperature for 20 min, the cells were characterized using a flow cytometer.

[0086] (13) Cell viability assay

[0087] SK-BR-3 cells were seeded at a density of 4000 cells per well in a 96-well cell culture plate and cultured under conditions of 37 °C and 5% CO2. After 24 h, the original culture medium was removed. The cells were treated with fresh medium containing 200 nM dimer-TDN construct for 24 h, and then 20 μL of 5 mg / mL MTT was added. After incubating in the dark at 37 °C for 4 h, the MTT solution was carefully discarded, and 100 μL of DMSO was added to dissolve the formazan. The absorbance at 490 nm of each well was measured using a microplate reader, and the cell viability was calculated. When investigating the effect of different concentrations of the construct on cell viability, after adding a series of concentration gradients of the construct, the detection was carried out using the same method.

[0088] 3. The following results were obtained for the above experiments (1)-(13)

[0089] Since dimer-TDN is composed of three parts: TDN NCL-apt and TDN HER2-apt connected by a linking strand formed by annealing three strands (S5, S6, S7), where the linking strand not only connects the two tetrahedrons but also performs an "AND" logic response to the complementary strands (BH, BN) released after the binding of the two aptamers to the protein, thereby releasing a fluorescence signal ( Figure 2 ), the feasibility of its sequence was first verified. The release process after the specific binding of the aptamer to the target protein was simulated by adding two aptamer blocking strands, BN and BH. As shown in Figure 3 (a), lane 4 represents the linking strand structure formed by annealing S5-1, S6-1, and S7-1. When the BH strand was added to the system containing the linking strand to simulate the strand displacement reaction when binding only to the HER2 protein, as shown in Figure 3 (a), lane 6, double-stranded bands of S5-1 and BH and the S6-1 band appeared, indicating that when only the HER2 protein was present in the system, it could directly bind to the fluorescent blocking strand (S5-1), proving that this sequence could not fully achieve the logic response effect. Therefore, the number of base complementary pairs of the linking strand was increased and the verification was carried out again, as shown in Figure 3As shown in (b), when only one of the complementary strands BN or BH is present, the S6 strand does not appear in lanes 5 and 6. However, when both complementary strands are present, in lane 7, double-stranded structures formed with the two complementary strands, namely S7+BN and S5+BH, and the single S6 strand can be seen, proving that this set of sequences can exhibit good logical responses.

[0090] After determining the sequence of the linker strand, the dimeric DNA tetrahedron structure was synthesized and assembled. As Figure 4 shown in (a), the assembly processes of two types of monomeric DNA tetrahedrons containing different aptamer sequences were respectively demonstrated by native polyacrylamide gel electrophoresis (PAGE). Among them, lanes 1 to 5 show the formation process of the TDN NCL-apt structure, and lanes 6 to 10 show the formation process of the TDN HER2-apt structure. The main structure of the tetrahedron is composed of four strands (S1, S2, S3, S4 or S1, S2, S3’, S4’). Among them, both the S3 strand and the S3’ strand contain a single-stranded sequence of 15 bases for subsequent formation of the dimer-TDN structure with the linker strand structure. Additionally, the S4 strand and the S4’ strand respectively carry the aptamer sequence of nucleolin and the aptamer sequence of HER2, and are blocked by two single strands BN and BH respectively during the synthesis process. As each component strand is added, it can be clearly seen in the figure that the migration rate of the bands gradually slows down and the bands in each lane are single, proving the gradual formation of the basic structure of the tetrahedron. Among them, two bands can be seen in lane 4, and the band returns to being single after adding the blocking strand BN. It is considered that when the blocking strand BN is not added, the interaction caused by the nucleolin aptamer sequence on the S4 strand due to its rich G bases leads to the cross-linking of the tetrahedron, but this phenomenon disappears after the addition of the blocking strand BN. The single bands in lanes 6 to 10 prove that the monomeric tetrahedron containing the HER2 aptamer has good synthesis effect and high assembly efficiency. Anneal S5, S6, and S7 to form the linker structure of the tetrahedron monomer and combine it with the two assembled TDN NCL-apt and TDN HER2-apt in a 1:1:1 ratio in a TAE-Mg 2+ buffer solution. To verify the successful assembly of the dimer-TDN structure, 2% agarose gel electrophoresis was used for analysis. The results are as Figure 4 shown in (b). Lanes 1 and 2 are TDN NCL-apt and TDN HER2-apt respectively. The migration rate of the band in lane 3 significantly slows down and the band is clear and single, indicating the successful preparation of the dimer-TDN structure. As Figure 5 shown, to further prove the successful synthesis of the structure, TDN NCL-apt , TDN HER2-aptCharacterizations by atomic force microscopy and dynamic light scattering were carried out on dimer-TDN respectively. It can be clearly seen from Figure 5 (c) that there are two interconnected tetrahedral structures, demonstrating the successful assembly of the designed structure. The results of dynamic light scattering characterization (DLS) are as shown in Figure 6 . In the DLS data, the hydrodynamic diameters of TDN NCL-apt and TDN HER2-apt are approximately 6.5 nm and 7.5 nm respectively, while the hydrodynamic diameter of the product formed after connection reaches 18.2 nm, which is approximately the sum of the hydrodynamic diameters of the two tetrahedrons. Therefore, the DLS data indicates the formation of the dimer-TDN structure.

[0091] The feasibility of fluorescence recovery for the recognition of the dimer-TDN structure was verified in vitro by measuring the fluorescence emission spectrum. The fluorescent group Cy3 was labeled on the connecting strand S6 of the two tetrahedrons and was close to the S5 strand labeled with the quenching group BHQ2 after the synthesis of dimer-TDN. Therefore, at the excitation wavelength of 538 nm, Cy3 exhibited a very low fluorescence value. The complementary strands of the NCL aptamer and the HER2 aptamer complementary strand were respectively used to simulate the intrastructural strand displacement process that occurs after the binding of the aptamer to the protein. As shown in Figure 7 , when the complementary sequence of the NCL aptamer was added to the system to simulate the situation where only the NCL protein exists on the cell membrane, the fluorescence intensity of Cy3 in the system still showed a very low fluorescence value; similarly, when the complementary sequence of the HER2 protein aptamer was introduced into the system to simulate the situation where only the HER2 protein exists on the cell membrane, the measured fluorescence intensity of Cy3 in the system hardly increased. Only when the complementary strands of the two aptamers were present simultaneously, the BHQ2 quenching group labeled on the structure was successfully replaced through the strand displacement reaction in the structure, and at this time, a significantly enhanced signal of the Cy3 fluorescent group was obtained by scanning. It shows that when the dimer-TDN structure recognizes two membrane proteins on the cell surface simultaneously, it will show enhanced Cy3 fluorescence, and the intensity is sufficient for subsequent cell imaging and detection.

[0092] To investigate the specific recognition effect of the dimer-TDN structure, the dimer-TDN structure was incubated with three kinds of cells, namely HL-7702, HeLa, and SK-BR-3, for 2 h respectively, and then confocal imaging images were collected for the three kinds of cells. As shown in Figure 8As shown in (a), the dimer-TDN structure showed yellow fluorescence of the Cy3 group on the cell membrane of SK-BR-3 cells where both NCL and HER2 membrane proteins were expressed, while no yellow fluorescence was observed on HL-7702 cells with low expression of both membrane proteins and HeLa cells with only high expression of NCL. This indicates that the aptamer sequence on the dimer-TDN structure only responds to target cells, and through strand displacement inside the structure, the DNA strand labeled with a quenching group is replaced from the structure, causing the restoration of the yellow fluorescence of Cy3 labeled on the dimer-TDN.

[0093] To further prove the specificity of the dimer-TDN structure for target cell recognition, we also analyzed the responses of a large number of cells by flow cytometry. As Figure 8 shown in (b), SK-BR-3 cells showed high fluorescence intensity. Quantitative data were directly given by the software, and the value of Mean X in the figure represents the relative average fluorescence intensity of the cells. The data showed that the fluorescence in the Cy3 detection channel on the target SK-BR-3 cells was enhanced by an order of magnitude compared to the fluorescence intensities of the two control cells, indicating that the dimer-TDN structure can specifically target two proteins on the cell surface and has good discrimination ability for normal cells and different types of tumor cells.

[0094] The dimer-TDN structure and SK-BR-3 cells were incubated for 1 h, 2 h, 3 h, and 4 h respectively, and laser confocal fluorescence images were recorded. As Figure 9 shown in (a), when the dimer-TDN structure was incubated with the cells for 1 h, weak yellow fluorescence of Cy3 was observed on the cell membrane, indicating that at this time, a small amount of the dimer-TDN structure had specifically bound to the two proteins on the cell surface, causing the restoration of the fluorescence of Cy3. As the incubation time continued to extend, the fluorescence intensity on the cell membrane gradually increased, indicating an increase in the number of structures bound to the cell surface. After the cells were incubated with the structure for 4 h, very obvious yellow fluorescence of Cy3 could be observed on the cell membrane, and co-localization of the structure with the stained cell membrane showed that the position of the structure was still distributed on the cell membrane ( Figure 10 (a)). After the TDN NCL-apt structure was incubated with the cells for the same time, confocal imaging maps of the cells were collected. From Figure 9 the results shown in (b), it can be seen that when the TDN NCL-apt structure was incubated with the cells for only 1 h, very few fluorescent dots were seen inside the cells, indicating that at 1 h, the TDN NCL-apt structure began to be recognized on the cell membrane and was internalized into the cells. As time extended, the number of structures engulfed also gradually increased. Compared with the imaging map of incubation with dimer-TDN, the TDN NCL-aptThe structure was hardly observed to stay on the cell membrane, but was quickly internalized by the cell, which may be related to the endocytosis mediated by a single membrane protein. At the same time, the co-localization imaging of the structure and the cell membrane also proved that the structure was internalized into the cell ( Figure 10 (b)). The results of confocal imaging show that the dimer-TDN structure can stay on the cell membrane longer than the TDN structure, which provides an opportunity for the structure to regulate proteins on the cell membrane surface. It is worth noting that in addition to showing the difference in the location of the structure on the cell, the fluorescence intensity of cells treated with the dimer-TDN structure is brighter after being treated with the same concentration of the structure, indicating that there are more structures bound to the cells. This may be related to the fact that the dimer-TDN structure is labeled with two aptamer sequences, which increases the chance of cell recognition. Recent studies have also shown that the affinity with target cells can be enhanced by controlling the number of ligands.

[0095] Two-color single-molecule total internal reflection fluorescence microscopy (TIRFM) was used to investigate the co-localization of NCL protein and HER2 protein on the cell surface. Figure 11 ). SK-BR-3 cells were treated with aptamer chains mixed with S4-AF488 and S4'-Cy3 (represented as Free-Apt in the figure) and then TIRFM imaging was performed. The two chains were assembled into a tetrahedral structure, and the dimer-TDN structure labeled with two fluorescent groups was prepared and incubated with cells in the same way and TIRFM imaging was also performed. The TIRFM imaging results showed that after treatment with the fluorescently labeled aptamer chain, NCL and HER2 were specifically recognized by the aptamer, and the overlapping fluorescent spots on the cell membrane were displayed in yellow after the two fluorescent signals were superimposed. After treatment with the dimer-TDN structure, the results of the superposition of the fluorescent signals showed that the fluorescent spots were obviously dispersed, showing green and red separate fluorescent spots, indicating that the distance between the two proteins in the cells treated with dimer-TDN was controlled to a certain extent. Therefore, the co-localization results of the two proteins show that the dimer-TDN structure has a regulatory effect on the relative position of NCL and HER2 proteins on the cell membrane surface.

[0096] To investigate the regulatory effect of dimer-TDN structure on cells, we treated SK-BR-3 cells with the structure for 12h or 24h, and then performed immunoblotting analysis on the levels of phosphorylated HER2 protein (p-HER2) and proteins related to cell apoptosis. Figure 12As shown in (b), after structural treatment, the level of p-HER2 in cells decreased, and the expression of the pro-apoptotic protein Bax was promoted. The apoptosis of cells was analyzed by flow cytometry. After treating SK-BR-3 cells with the dimer-TDN structure, the proportions of cells in the early and late apoptosis stages increased significantly ( Figure 11 (b)), which was consistent with the protein expression results in the immunoblotting. The reason for achieving this regulatory effect may be that the simultaneous binding of the structure to two target membrane proteins on the cell surface affects the interaction between the two proteins, inhibits the activation of the HER2 protein, and thus affects the pathways it regulates, promoting the occurrence of apoptosis.

[0097] Next, the MTT method was used to further analyze the cell viability ( Figure 13 (a)). After incubating the structure with HL-7702 and HeLa cells for 24 h, the effects on the viabilities of the two types of cells were within 10%. However, the viability of SK-BR-3 cells treated with the structure under the same conditions decreased significantly. This indicates that the regulatory effect of the dimer-TDN structure is specific, that is, it can precisely induce apoptosis in tumor cells that simultaneously express the two target membrane proteins. To further investigate the effect of the dimer-TDN structure on the viability of SK-BR-3 cells, SK-BR-3 cells were incubated with a series of concentrations of the dimer-TDN structure at 0 nM, 50 nM, 100 nM, 200 nM, and 400 nM for 24 h, and then the MTT method was used to measure the cell survival rate. The results are shown in Figure 13 (b). As the structure concentration increased, the viability of SK-BR-3 cells decreased significantly. When the structure concentration increased to 400 nM, the cell viability was only 53% of that of the blank control group, indicating that this structure can effectively inhibit cell viability, which is consistent with the results of flow cytometry and immunoblotting of cell apoptosis.

[0098] In summary, the present invention demonstrates that the designed dimer DNA tetrahedron (dimer-TDN) can be used for tumor cell recognition and promotion of tumor cell apoptosis. The dimer DNA tetrahedron structure responds only to tumor cells that co-express two membrane proteins, HER2 and NCL. The Cy3 fluorescence signal is reported through the strand displacement reaction within the structure for cell recognition; at the same time, due to the steric hindrance effect of the combined structure, it is difficult for the two proteins to approach each other, affecting their interaction. This structure is more easily targeted to the cell membrane surface than the single DNA tetrahedron structure, and it can exist on the cell surface for a longer time. Therefore, it has a better cell regulation effect on these two cell membrane surface proteins, specifically reflected in reducing the phosphorylation level of the HER2 protein, promoting the expression of proteins related to apoptosis, and ultimately leading to tumor cell apoptosis and reducing the cell viability of tumor cells. This dimer DNA tetrahedron structure helps to provide new research ideas for the diagnosis and treatment of related diseases by effectively regulating cells by changing the relative positions of cell surface proteins.

[0099] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A dual DNA tetrahedron structure, characterized in that, The structure includes a first DNA tetrahedron structure, a second DNA tetrahedron structure, and a linking strand; The first DNA tetrahedron structure is composed of four single-stranded DNAs S1, S2, S3, S4 and BN, and S4 contains an aptamer domain of the target protein; BN can base-complementarily pair with the aptamer domain of S4; The second DNA tetrahedron structure is composed of four single-stranded DNAs S1, S2, S3', S4' and BH, and S4' contains an aptamer domain of the target protein; BH can base-complementarily pair with the aptamer domain of S4'; The linking strand is composed of three single-stranded DNAs S5, S6 and S7; S6 connects the first DNA tetrahedron structure and the second DNA tetrahedron structure, and S6 is complementary to the sequence of S5 at the same time; S7 is complementary to the sequence of S5; S5 and S7 are also complementary to BH and BN respectively; The sequences of S1-S4 are as shown in SEQ ID NO.1-4; the sequences of S3' and S4' are as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively; the sequence of BN is as shown in SEQ ID NO.7; the sequence of BH is as shown in SEQ ID NO.8; the sequence of S5 is as shown in SEQ ID NO.9; the sequence of S6 is as shown in SEQ ID NO.10; the sequence of S7 is as shown in SEQ ID NO.11; The 5' end of S5 is modified with a quenching group; the 3' end of S6 is modified with a luminescent group.

2. The dimeric DNA tetrahedron structure according to claim 1, wherein The luminescent group is Cy3; the quenching group is BHQ.

3. The preparation method of the binary DNA tetrahedron structure according to any one of claims 1-2, characterized in that, It includes mixing the first DNA tetrahedron structure, the second DNA tetrahedron structure and the linking strand in equal proportions, incubating them together, and assembling to obtain a dimeric DNA tetrahedron structure; The incubation conditions are: incubating at 35°C - 38°C for 3 - 6 h; The preparation method of the first DNA tetrahedron structure is as follows: single-stranded DNAs S1, S2, S3, S4, and BN that constitute the tetrahedron are mixed in a TAE-Mg 2+ buffer solution in a molar ratio of 1:1:1:1:

1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product; The preparation method of the second DNA tetrahedron structure is as follows: single-stranded DNAs S1, S2, S3’, S4’ and BH that form the tetrahedron are mixed in a TAE-Mg 2+ buffer solution according to a molar ratio of 1:1:1:1:

1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product; The preparation method of the linking strand is: annealing the DNA single strands S5, S6 and S7 to obtain a linking strand structure.

4. The preparation method of the dimeric DNA tetrahedron structure according to claim 3, characterized in that, It includes that the incubation conditions are: incubating at 37°C for 4 h.

5. The preparation method according to claim 3, characterized in that, The preparation method of the first DNA tetrahedron structure is that the mixed solution is heated at 95°C for 5 min and then slowly cooled to room temperature.

6. The preparation method according to claim 3, wherein The preparation method of the second DNA tetrahedron structure is that the mixed solution is heated at 95°C for 5 min and then slowly cooled to room temperature.

7. The preparation method according to claim 3, characterized in that, During the preparation of the connecting chain, the annealing conditions are as follows: The DNA single strands S5, S6, and S7 are mixed in a TAE-Mg 2+ buffer solution in a molar ratio of 1:1:

1. The mixed solution is heated at 90-98 °C for 3-10 min and then cooled to room temperature to obtain the product.

8. The application of the dimeric DNA tetrahedron structure according to claims 1 - 2 in the preparation of tumor cell detection products, and the products are selected from experimental reagents and detection chips; the tumor cells are selected from breast cancer cells.

9. The application of the dimeric DNA tetrahedron structure according to claims 1 - 2 in the preparation of products for promoting tumor cell apoptosis, and the products are selected from experimental reagents; the tumor cells are selected from breast cancer cells.

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