A DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease and its preparation method and application

Abstract

The present invention discloses a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease, a preparation method and an application thereof, wherein the DNA nanomachine is composed of a DNA tetrahedron, an enzyme-activated DNA hairpin E-H1 and a DNA hairpin H2 labeled with Cy5 and BHQ2. When APE1 and target miR-224 coexist, the AP site in E-H1 is specifically recognized and cleaved, triggering a strand displacement reaction, resulting in the separation of Cy5 and BHQ2, and generating an enhanced fluorescence signal. The DNA nanomachine of the present invention has the advantages of high specificity, simple operation, cost-effectiveness and biocompatibility, and provides a new tool for early diagnosis and treatment monitoring of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease, and a preparation method and application thereof. Background Art

[0002] With the development of molecular biology and nanotechnology, the detection and imaging of specific molecules in tumor cells has become an important means for early diagnosis and treatment of cancer. As a class of endogenous small non-coding RNA molecules, miRNA (microRNA) plays a key role in a variety of biological processes, including the occurrence and development of tumors. Therefore, the development of methods that can specifically identify and image miRNA in tumor cells is of great significance for the diagnosis and treatment of cancer.

[0003] Existing miRNA imaging technologies mainly include luciferase reporter gene systems, quantum dot labels, and nucleic acid-based nanostructures. However, these methods have some limitations, such as high background signals, poor specificity, and complex or high costs. Especially in the presence of endogenous enzymes in tumor cells, how to achieve specific response and imaging of miRNA remains a challenge.

[0004] Human apurinic / apyrimidinic endonuclease (APE1) is an enzyme that is widely present in cells and plays a key role in DNA damage repair and gene expression regulation. APE1 can recognize and cut DNA containing AP sites (apurinic / apyrimidinic site), which makes it possible to design DNA nanomachines based on APE1 response.

[0005] Currently, there is a lack of an endogenous human apurinic / apyrimidinic endonuclease-responsive DNA nanomachine for tumor cell-specific miRNA imaging, as well as a preparation method and application thereof. Summary of the invention

[0006] The object of the present invention is to provide a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease for tumor cell-specific miRNA imaging, and a preparation method and application thereof.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for preparing a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease.

[0009] In a second aspect, the present application provides a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease prepared by a preparation method.

[0010] In a third aspect, the present application provides an application of an endogenous human apurinic / apyrimidinic endonuclease-responsive DNA nanomachine in tumor cell-specific miRNA imaging.

[0011] In a fourth aspect, the present application provides a method for tumor cell-specific miRNA imaging using a DNA nanomachine.

[0012] In a fifth aspect, the present application provides a method for detecting the expression of miR-224 in tumor cells.

[0013] In a sixth aspect, the present application provides a method for evaluating APE1 activity in tumor cells.

[0014] In a seventh aspect, the present application provides a kit for imaging miR-224 in tumor cells.

[0015] The first aspect of the present application provides a method for preparing a DNA nanomachine EGDN responsive to endogenous human apurinic / apyrimidinic nuclease APE1, comprising the following steps:

[0016] (1) preparing four single-stranded DNAs S1, DNA S2, DNA S3, and DNA S4, wherein each single-stranded DNA has a specific sequence and can be assembled into a DNA tetrahedron structure through complementary base pairing;

[0017] (2) mixing equal amounts of single-stranded DNA of DNA S1, DNA S2, DNA S3 and DNA S4 in an assembly buffer comprising 20 mM Tris, 10 mM magnesium chloride, 150 mM sodium chloride, and a pH value of 8.0;

[0018] (3) heating the mixed solution to 95°C for 5 minutes, and then immediately cooling to 4°C and maintaining for 30 minutes to assemble into DNA tetrahedrons;

[0019] (4) preparing enzyme-activated DNA hairpin E-H1, the 9th nucleotide of the 3' tail of which is modified with an AP site;

[0020] (5) preparing DNA hairpin H2 labeled with Cy5 and BHQ2;

[0021] (6) The DNA tetrahedron obtained in step (3) was incubated with E-H1 and H2 in an equimolar ratio and maintained at 37°C for 30 minutes to produce a DNA nanomachine EGDN responsive to endogenous human apurinic / apyrimidinic nuclease.

[0022] Further, in step (1), the nucleotide sequence of DNA S1 is shown as SEQ ID NO.1, the nucleotide sequence of DNA S2 is shown as SEQ ID NO.2, the nucleotide sequence of DNA S3 is shown as SEQ ID NO.3, and the nucleotide sequence of DNA S4 is shown as SEQ ID NO.4; in step (4), the DNA tetrahedron serves as the structural backbone, and hairpins E-H1 and hairpin H2 are loaded through base complementary pairing; the nucleotide sequence of hairpin E-H1 is shown as SEQ ID NO.5, the nucleotide sequence of hairpin H2 is shown as SEQ ID NO.6, and the nucleotide sequence of miR-224 is shown as SEQ ID NO.7.

[0023] Furthermore, in step (4), the toehold domain in E-H1 is tightly locked, reducing the non-site signaling of CHA production directly triggered by the target miR-224.

[0024] The second aspect of the present application provides a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease prepared by a preparation method.

[0025] The third aspect of the present application provides an application of an endogenous human apurinic / apyrimidinic nuclease-responsive DNA nanomachine in tumor cell-specific miRNA imaging.

[0026] The fourth aspect of the present application provides a method for tumor cell-specific miRNA imaging using a DNA nanomachine, comprising the following steps:

[0027] a. Incubating the DNA nanomachine with APE1 and miR-224 at 37°C for 30 minutes;

[0028] b. Observe that APE1 specifically recognizes and cleaves the AP site in E-H1, resulting in the separation of the terminal 8-base DNA fragment from E-H1, generating activated hairpin H1;

[0029] c. Target miR-224 hybridizes with the exposed toehold domain in H1, opening the hairpin structure and triggering the strand displacement reaction (SDR) between H1 and H2, resulting in the separation of Cy5 and BHQ2 and generating a significantly enhanced fluorescence signal.

[0030] The fifth aspect of the present application provides a method for detecting the expression of miR-224 in tumor cells, comprising the following steps:

[0031] a. Using DNA nanomachine EGDN;

[0032] b. EGDN was incubated with APE1 and different concentrations of miR-224 at 37°C;

[0033] c. The fluorescence intensity change of Cy5 was detected by fluorescence spectroscopy to determine the concentration of miR-224.

[0034] The sixth aspect of the present application provides a method for evaluating the activity of APE1 in tumor cells, comprising the following steps:

[0035] a. Using DNA nanomachine EGDN;

[0036] b. Incubate EGDN with APE1 and miR-224 at 37°C;

[0037] c. The activity of APE1 was evaluated by observing the changes in the fluorescence signal generated by the separation of Cy5 and BHQ2.

[0038] In a seventh aspect, the present application provides a kit for imaging miR-224 in tumor cells, the kit comprising a DNA nanomachine EGDN; a buffer for incubation; and an indicator for detecting a fluorescent signal.

[0039] Beneficial effects: The DNA nanomachine of the present invention has the advantages of high specificity, easy operation, cost-effectiveness and biocompatibility, and provides a new tool for early diagnosis and treatment monitoring of tumors. The DNA nanomachine utilizes the enzyme activity of APE1 and achieves high-specific recognition and imaging of miRNA through specific DNA structure design, while avoiding interference from nonspecific signals and improving the sensitivity and accuracy of imaging.

[0040] Compared with the prior art, the present invention has the following advantages: (1) The present invention aims to develop a novel endogenous APE1-responsive DNA nanomachine (EGDN) for tumor cell-specific miRNA imaging. Through this design, the present invention not only improves the efficiency of miRNA imaging in tumor cells, but also provides a new tool for early diagnosis and treatment of cancer.

[0041] (2) The DNA nanomachine (EGDN) provided by the present invention can specifically respond to the endogenous APE1 enzyme, thereby achieving highly specific imaging of specific miRNAs in tumor cells and reducing the interference of nonspecific signals. The preparation method of EGDN is simple and can be achieved through standard DNA assembly and incubation steps, which is convenient for laboratory operation and large-scale production.

[0042] (3) Highly specific imaging: Signal amplification: The EGDN design includes a strand displacement reaction (SDR). When the target miR-224 hybridizes with the exposed toehold domain in E-H1, it can trigger the SDR between H1 and H2, resulting in the separation of the fluorescent markers Cy5 and BHQ2, generating a significantly enhanced fluorescence signal, thereby achieving signal amplification. Cost-effectiveness: Compared with traditional imaging techniques, such as quantum dot labeling, the DNA nanomachine used in the present invention is less expensive and more suitable for a wide range of applications.

[0043] (4) Real-time monitoring: EGDN can monitor the expression changes of miRNA under real-time conditions, providing a tool for studying the dynamic changes of miRNA in tumor cells. Low background noise: Due to the high specificity of EGDN, the background noise generated during the imaging process is low, which improves the signal-to-noise ratio of imaging. Biocompatibility: EGDN is composed of DNA and has good biocompatibility. It is suitable for use in vivo and reduces toxicity and immune response to cells. Versatility: The design principle of EGDN can be extended to other types of miRNA and different enzyme response mechanisms, and has broad application prospects.

[0044] (5) Early diagnosis potential: By specifically imaging miRNA, EGDN can help in the early diagnosis of cancer, especially when the miRNA expression pattern is closely related to tumor development. Treatment monitoring: EGDN can be used to monitor the effect of tumor treatment and evaluate the treatment effect by tracking the changes in miRNA, providing support for personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0046] Figure 1 This is an imaging diagram of the endogenous APE1-responsive DNA nanomachine (EGDN) of the present invention for tumor-specific miRNA;

[0047] Figure 2 This is a feasibility analysis diagram of using EGDN of the present invention for miR-224 detection. Figure 2 A is the CHA reaction diagram of the present invention. Figure 2 B is a diagram showing the construction of EGDN of the present invention. Figure 2 C is the fluorescence spectrum of EGDN of the present invention under different conditions. Figure 2 D is a diagram showing the real-time fluorescence changes of EGDN under different conditions of the present invention.

[0048] Figure 3 This is a performance analysis diagram of EGDN of the present invention for miR-224 detection. Figure 3 A is the fluorescence spectrum of EGDN of the present invention at different concentrations of miR-224. Figure 3 B is a graph showing the relationship between fluorescence intensity and miR-224 concentration in FIG. A of the present invention (the inset is a standard curve of low-concentration miR-224). Figure 3 C is the fluorescence spectrum of EGDN of the present invention in response to different miRNAs. Figure 3 D is a graph showing the fluorescence intensity of mEGDN, nEGDN and EGDN of the present invention under different conditions.

[0049] Figure 4 This is an imaging analysis diagram of EGDN used for miR-224 in living cells. Figure 4 A, Figure 4 B is the co-localization analysis of EGDN in living cells of the present invention. Figure 4 C is a diagram showing the working principles of EGDN, mEGDN and nEGDN of the present invention. Figure 4 D is the confocal imaging of EGDN, mEGDN and nEGDN of the present invention in MCF-7 cells. Figure 4 E is a flow cytometry analysis result diagram of EGDN, mEGDN and nEGDN of the present invention in MCF-7 cells. Figure 4 F is the fluorescence quantitative result diagram corresponding to Figure B of the present invention. DETAILED DESCRIPTION

[0050] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0051] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0052] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0053] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0054] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0055] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0056] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0057] The first aspect of the present application provides a method for preparing a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease, comprising the following steps:

[0058] (1) preparing four single-stranded DNAs S1, DNA S2, DNA S3, and DNA S4, wherein each single-stranded DNA has a specific sequence and can be assembled into a DNA tetrahedron structure through complementary base pairing;

[0059] (2) mixing equal amounts of single-stranded DNA of DNA S1, DNA S2, DNA S3 and DNA S4 in an assembly buffer comprising 20 mM Tris, 10 mM magnesium chloride, 150 mM sodium chloride, and a pH value of 8.0;

[0060] (3) heating the mixed solution to 95°C for 5 minutes, and then immediately cooling to 4°C and maintaining for 30 minutes to assemble into DNA tetrahedrons;

[0061] (4) preparing enzyme-activated DNA hairpin E-H1, the 9th nucleotide of the 3' tail of which is modified with an AP site;

[0062] (5) preparing DNA hairpin H2 labeled with Cy5 and BHQ2;

[0063] (6) The DNA tetrahedron obtained in step (3) was incubated with E-H1 and H2 in an equimolar ratio and maintained at 37°C for 30 minutes to prepare a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease.

[0064] In some embodiments, in step (1), the nucleotide sequence of DNA S1 is shown as SEQ ID NO.1, the nucleotide sequence of DNA S2 is shown as SEQ ID NO.2, the nucleotide sequence of DNA S3 is shown as SEQ ID NO.3, and the nucleotide sequence of DNA S4 is shown as SEQ ID NO.4; in step (4), the DNA tetrahedron serves as the structural backbone, and hairpins E-H1 and hairpin H2 are loaded through complementary base pairing; the nucleotide sequence of hairpin E-H1 is shown as SEQ ID NO.5, the nucleotide sequence of hairpin H2 is shown as SEQ ID NO.6, and the nucleotide sequence of miR-224 is shown as SEQ ID NO.7.

[0065] In some embodiments, in step (4), the toehold domain in E-H1 is tightly locked, reducing the non-site signaling of CHA production directly triggered by the target miR-224.

[0066] The second aspect of the embodiments of the present application provides a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease prepared by a preparation method.

[0067] A third aspect of the embodiments of the present application provides an application of an endogenous human apurinic / apyrimidinic endonuclease-responsive DNA nanomachine in tumor cell-specific miRNA imaging.

[0068] A fourth aspect of the present application provides a method for tumor cell-specific miRNA imaging using a DNA nanomachine, comprising the following steps:

[0069] a. Incubating the DNA nanomachine with APE1 and miR-224 at 37°C for 30 minutes;

[0070] b. Observe that APE1 specifically recognizes and cleaves the AP site in E-H1, resulting in the separation of the terminal 8-base DNA fragment from E-H1, generating activated hairpin H1;

[0071] c. Target miR-224 hybridizes with the exposed toehold domain in H1, opening the hairpin structure and triggering the strand displacement reaction (SDR) between H1 and H2, resulting in the separation of Cy5 and BHQ2 and generating a significantly enhanced fluorescence signal.

[0072] The fifth aspect of the present application embodiment provides a method for detecting the expression of miR-224 in tumor cells, comprising the following steps:

[0073] a. Using DNA nanomachine EGDN;

[0074] b. EGDN was incubated with APE1 and different concentrations of miR-224 at 37°C;

[0075] c. The fluorescence intensity change of Cy5 was detected by fluorescence spectroscopy to determine the concentration of miR-224.

[0076] A sixth aspect of the present application provides a method for evaluating the activity of APE1 in tumor cells, comprising the following steps:

[0077] a. Using DNA nanomachine EGDN;

[0078] b. Incubate EGDN with APE1 and miR-224 at 37°C;

[0079] c. The activity of APE1 was evaluated by observing the changes in the fluorescence signal generated by the separation of Cy5 and BHQ2.

[0080] A seventh aspect of an embodiment of the present application provides a kit for imaging miR-224 in tumor cells, the kit comprising a DNA nanomachine EGDN; a buffer for incubation; and an indicator for detecting a fluorescent signal.

[0081] Example 1

[0082] The method for preparing a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease of the present invention comprises the following steps:

[0083] (1) preparing four single-stranded DNAs S1, DNA S2, DNA S3 and DNA S4, wherein each single-stranded DNA has a specific sequence and can be assembled into a DNA tetrahedron structure through complementary base pairing; the nucleotide sequence of DNA S1 is shown in SEQ ID NO.1, the nucleotide sequence of DNA S2 is shown in SEQ ID NO.2, the nucleotide sequence of DNA S3 is shown in SEQ ID NO.3, and the nucleotide sequence of DNA S4 is shown in SEQ ID NO.4,

[0084] (2) mixing equal amounts of single-stranded DNA of DNA S1, DNA S2, DNA S3 and DNA S4 in an assembly buffer comprising 20 mM Tris, 10 mM magnesium chloride, 150 mM sodium chloride, and a pH value of 8.0;

[0085] (3) heating the mixed solution to 95°C for 5 minutes, and then immediately cooling to 4°C and maintaining for 30 minutes to assemble into DNA tetrahedrons;

[0086] (4) Prepare enzyme-activated DNA hairpin E-H1, which has an AP site modified on the 9th nucleotide of the 3' tail; DNA tetrahedron as the structural backbone, loads hairpin E-H1 and hairpin H2 through base complementary pairing. The toe-point domain in E-H1 is tightly locked, reducing the non-site signal generated by CHA directly triggered by the target miR-224. DNA tetrahedron as the structural backbone, loads hairpin E-H1 and hairpin H2 through base complementary pairing; the nucleotide sequence of hairpin E-H1 is shown in SEQ ID NO.5, the nucleotide sequence of hairpin H2 is shown in SEQ ID NO.6, and the nucleotide sequence of miR-224 is shown in SEQ ID NO.7.

[0087] (5) preparing DNA hairpin H2 labeled with Cy5 and BHQ2;

[0088] (6) The DNA tetrahedron obtained in step (3) was incubated with E-H1 and H2 in an equimolar ratio and maintained at 37°C for 30 minutes to prepare a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease.

[0089] The invention discloses a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease prepared by a preparation method.

[0090] The invention discloses an application of an endogenous human apurinic / apyrimidinic nuclease-responsive DNA nanomachine in tumor cell-specific miRNA imaging.

[0091] Example 2

[0092] A method for tumor cell-specific miRNA imaging using a DNA nanomachine of the present invention comprises the following steps:

[0093] a. Incubating the DNA nanomachine with APE1 and miR-224 at 37°C for 30 minutes;

[0094] b. Observe that APE1 specifically recognizes and cleaves the AP site in E-H1, resulting in the separation of the terminal 8-base DNA fragment from E-H1, generating activated hairpin H1;

[0095] c. Target miR-224 hybridizes with the exposed toehold domain in H1, opening the hairpin structure and triggering the strand displacement reaction (SDR) between H1 and H2, resulting in the separation of Cy5 and BHQ2 and generating a significantly enhanced fluorescence signal.

[0096] Example 3

[0097] A method for detecting the expression of miR-224 in tumor cells of the present invention comprises the following steps:

[0098] a. Using DNA nanomachine EGDN;

[0099] b. EGDN was incubated with APE1 and different concentrations of miR-224 at 37°C;

[0100] c. The fluorescence intensity change of Cy5 was detected by fluorescence spectroscopy to determine the concentration of miR-224.

[0101] Example 4

[0102] A method for evaluating APE1 activity in tumor cells of the present invention comprises the following steps:

[0103] a. Using DNA nanomachine EGDN;

[0104] b. Incubate EGDN with APE1 and miR-224 at 37°C;

[0105] c. The activity of APE1 was evaluated by observing the changes in the fluorescence signal generated by the separation of Cy5 and BHQ2.

[0106] A seventh aspect of an embodiment of the present application provides a kit for imaging miR-224 in tumor cells, the kit comprising a DNA nanomachine EGDN; a buffer for incubation; and an indicator for detecting a fluorescent signal.

[0107] Test Example 1

[0108] 1. Experimental part

[0109] 1.1 Materials and Instruments

[0110] All oligonucleotides used in this work were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). APE1, Nt.Bbvc I, and Age I were purchased from New England Biotechnology Co., Ltd. (Beijing, China). Exo I, Kpn I, and EcoRI were purchased from Yisheng Biotechnology Co., Ltd. (Shanghai, China). DNA loading buffer was purchased from Bao Biotechnology Co., Ltd. (Dalian, China). Phosphate-buffered saline (PBS), cell culture medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin, and trypsin cell digestion solution were all from Wuhan Saiweier Biotechnology Co., Ltd. (Wuhan, China). SYBR Gold was purchased from Thermo Fisher Scientific Inc. (Massachusetts, USA). All solutions were prepared by ultrapure water (≥18.2 MΩ cm resistivity). All chemicals were used directly without further purification.

[0111] Gel imaging was performed on a ChemiScope 6200 (Shanghai, China), and fluorescence spectroscopy was performed using a microplate reader (TECAN SPARK, Switzerland). Confocal laser scanning microscopy images were acquired on a Nikon A1 confocal laser scanning microscope (Tokyo, Japan). Flow cytometric analysis was performed on an Agilent flow cytometer (California, USA).

[0112] 1.2 Preparation of EGDN

[0113] Equal amounts of four single-stranded DNAs (S1, S2, S3, and S4) were mixed in an assembly buffer (20 mM Tris, 10 mM magnesium chloride, 150 mM sodium chloride, pH 8.0). The solution was heated to 95°C for 5 min, immediately cooled to 4°C and stored for 30 min to assemble into DNA tetrahedrons. Then, the DNA tetrahedrons were incubated with H1 and H2 in an equimolar ratio and kept at 37°C for 30 min to construct a DNA nanomachine (EGDN).

[0114] 1.3 Gel electrophoresis

[0115] After EGDN was incubated with APE1 and miR-224 at 37°C for 30 min, 2 μL of 6× DNA loading buffer and 1.5 μL of 100× SYBR Gold were added, and the samples were transferred to 2% agarose gel after mixing. The electrophoresis was run in 1× TBE buffer, at 120 V for 60 min, and the ChemiScope6200 system was used for photography.

[0116] 1.4 Fluorescence measurement

[0117] In the sensitivity test, 100 nM EGDN was incubated with different concentrations of miR-224 (0, 0.1, 0.2, 0.5, 1, 2, 4, 6, 8, 10, 20, and 40 nM) in the presence of APE1 (5 U / mL). For specificity analysis, 100 nM EGDN was incubated with miR-224 (10 mM) and other parallel miRNAs (Let-7a, miR-21, miR-155, miR-221, and miR-222). All samples were reacted at 37 °C for 60 min. The excitation wavelength was 625 nm, and the emission spectra were collected in the range of 650-750 nm. All experiments were repeated three times independently.

[0118] 1.5 Laser confocal fluorescence imaging analysis

[0119] MCF-7 cells were seeded in 35 mm glass bottom culture dishes and cultured for 24 h. Then, the cells were incubated with DMEM containing 250 nM EGDN. After 4 h, the cells were washed 3 times with PBS and stained with 0.05 mg / mL Hoechst 33342 for 10 min. The excitation and emission wavelengths were as follows: Hoechst 33342: Ex = 405 nm, Em = 425-470 nm; Cy5: Ex = 640 nm, Em = 663-738 nm.

[0120] 1.6 Flow cytometry analysis

[0121] MCF-7 cells were seeded in 12-well plates for 24 h and incubated with DMEM containing different DNA probes for 4 h. The cells were digested with cell digestion solution, washed three times with PBS, resuspended in 200 μL PBS, and the fluorescence signals were collected by flow cytometry.

[0122] 2. Results

[0123] 2.1 Working Mechanism

[0124] The present invention develops an endogenous human apurinic / apyrimidinic endonuclease (APE1)-responsive DNA nanomachine (EGDN) for tumor cell-specific miRNA imaging, such as Figure 1As shown in Figure 2. EGDN consists of three parts: a DNA tetrahedron assembled from four single-stranded DNAs (S1, S2, S3, and S4), an enzyme-activated DNA hairpin (E-H1) modified with an AP site at the 9th nucleotide of the 3' tail, and a DNA hairpin (H2) labeled with Cy5 and BHQ2. The DNA tetrahedron serves as the structural backbone, loading the hairpins E-H1 and H2 through complementary base pairing. In this case, the toe-point domain in E-H1 is tightly locked, reducing the non-site signal generated by CHA directly triggered by the target miR-224. After entering the tumor cells, the AP site in E-H1 will be specifically recognized and cleaved by endogenous APE1, resulting in the separation of the terminal 8-base DNA fragment from E-H1, and the generation of activated hairpin H1 due to reduced binding. Then, the target miR-224 hybridizes with the exposed toehold domain in H1, opening the hairpin structure, thereby triggering the strand displacement reaction (SDR) between H1 and H2, resulting in the separation of Cy5 and BHQ2, thereby generating a significantly enhanced fluorescence signal. At the same time, the target miR-224 is released from the system, continuing to initiate a new cycle to achieve signal amplification.

[0125] Figure 1 This is an imaging diagram of the endogenous APE1-responsive DNA nanomachine (EGDN) of the present invention for tumor-specific miRNA;

[0126] 2.2 Feasibility Verification

[0127] The CHA reaction and programmable synthesis of EGDN were first explored by 2% agarose gel electrophoresis. Figure 2 As shown in A, E-H1 and H2 can only open the hairpin structure to carry out base complementary pairing and form a DNA double strand in the presence of APE1 and miR-224. Figure 2 As shown in B, with the gradual addition of various DNA chains, a band with significantly reduced mobility was observed in lane 9, demonstrating the successful construction of EGDN. Then, the feasibility of EGDN in the detection of miR-224 in response to APE1 was evaluated in solution. Figure 2 As shown in Figure C, in the absence of APE1 or miR-224, EGDN produced an insufficient fluorescence change. In contrast, when APE1 and miR-224 were present at the same time, the fluorescence signal observed by the present invention was significantly enhanced, which proved that the miRNA sensing mode activated by APE1 was reasonable. The present invention investigated the reaction kinetics of EGDN under different conditions. Figure 2 As shown in D, only in the presence of APE1 and miR-224, the fluorescence intensity of EGDN gradually increased with the extension of incubation time and reached saturation at around 30 min.

[0128] Figure 2 Figure 2 A is the CHA reaction diagram of the present invention. Figure 2 B is a diagram showing the construction of EGDN of the present invention. Figure 2 C is the fluorescence spectrum of EGDN of the present invention under different conditions. Figure 2 D is a diagram showing the real-time fluorescence changes of EGDN under different conditions of the present invention.

[0129] 2.3 Detection Performance Evaluation

[0130] like Figure 3 As shown in A, when APE1 is present, the fluorescence intensity of EGDN increases in response to miR-224 in a concentration-dependent manner, and the limit of detection (LOD) is calculated to be 25.2 pM ( Figure 3 B). In order to study the specificity of EGDN in detecting miR-224, a series of control miRNAs (miR-7a, miR-21, miR-155, miR-221 and miRNA-222) were used as control factors under the same conditions. Figure 3 As shown in Figure C, the target miR-224 produced a significant fluorescence enhancement, while the fluorescence output of these control miRNAs was weak, indicating that EGDN has a high specificity for the target miRNA. In order to further explore the indispensable role of the AP site in this mechanism, the present invention designed a control probe (mEGDN) with the same sequence replaced by a hairpin (mH1) but without an AP site. Figure 3 As shown in D, there was no obvious fluorescence enhancement of mEGDN after the addition of APE1 and miR-224, indicating that the AP site plays a key role in the design of APE1 activation. As another control, the stem sequence of nEGDN, H1, was mutated to inhibit its hybridization with miR-224, and the fluorescence change was negligible, confirming that the fluorescence response was based on CHA triggered by the target miR-224. These results indicate that APE1-responsive EGDN has great promise in the precise imaging of miRNAs in tumor cells.

[0131] Figure 3 . Figure 3 A is the fluorescence spectrum of EGDN of the present invention at different concentrations of miR-224. Figure 3 B is a graph showing the relationship between the fluorescence intensity and the miR-224 concentration in FIG. A of the present invention (the inset is a standard curve of low-concentration miR-224). Figure 3 C is the fluorescence spectrum of EGDN of the present invention in response to different miRNAs. Figure 3 D is a graph showing the fluorescence intensity of mEGDN, nEGDN and EGDN of the present invention under different conditions.

[0132] 2.4 Imaging analysis of miR-224 in living cells

[0133] The present invention selected liver cancer MCF-7 cells as the cell model for miR-224 imaging. First, a fluorescence co-localization experiment was used to evaluate the distribution of EGDN in cells. The results showed that EGDN can enter the cytoplasm well ( Figure 4 A and Figure 4 B). In addition, the present invention introduces two control probes, such as Figure 4 C, mEGDN and nEGDN were used to evaluate the imaging of miR-224 activated by APE1. Figure 4 As shown in D, MCF-7 cells incubated with EGDN showed obvious fluorescence signals, while cells incubated with mEGDN without AP sites or nEGDN containing mutant bases showed weak fluorescence signals, indicating that EGDN has the ability to image miR-224 in response to APE1. Flow cytometry further quantified that the fluorescence intensity of cells treated with EGDN was enhanced by 5.20 times and 4.62 times compared with mEGDN and nEGDN, respectively ( Figure 4 E and Figure 4 F) These results indicate that EGDN enables imaging of APE1-activated miR-224 within tumor cells.

[0134] Figure 4 .( Figure 4 A, Figure 4 B) is a diagram showing the co-localization analysis of EGDN of the present invention in living cells. Figure 4 C is a diagram showing the working principles of EGDN, mEGDN and nEGDN of the present invention. Figure 4 D is the confocal imaging of EGDN, mEGDN and nEGDN of the present invention in MCF-7 cells. Figure 4 E is a flow cytometry analysis result diagram of EGDN, mEGDN and nEGDN of the present invention in MCF-7 cells. Figure 4 F is the fluorescence quantitative result diagram corresponding to Figure B of the present invention.

[0135] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A method for preparing a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease, characterized in that The steps include: (1) preparing four single-stranded DNAs S1, DNA S2, DNA S3, and DNA S4, wherein each single-stranded DNA has a specific sequence and can be assembled into a DNA tetrahedron structure through complementary base pairing; (2) mixing equal amounts of single-stranded DNA of DNA S1, DNA S2, DNA S3 and DNA S4 in an assembly buffer comprising 20 mM Tris, 10 mM magnesium chloride, 150 mM sodium chloride, and a pH value of 8.0; (3) heating the mixed solution to 95°C for 5-10 minutes, and then immediately cooling to 4°C for 30-60 minutes to assemble into DNA tetrahedrons; (4) preparing enzyme-activated DNA hairpin E-H1, the 9th nucleotide of the 3' tail of which is modified with an AP site; (5) preparing DNA hairpin H2 labeled with Cy5 and BHQ2; (6) The DNA tetrahedron obtained in step (3) is incubated with E-H1 and H2 in an equimolar ratio and maintained at 37° C. for 30-60 minutes to prepare a DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease.

2. The preparation method according to claim 1, characterized in that: In step (1), the nucleotide sequence of DNA S1 is shown as SEQ ID NO.1, the nucleotide sequence of DNA S2 is shown as SEQ ID NO.2, the nucleotide sequence of DNA S3 is shown as SEQ ID NO.3, and the nucleotide sequence of DNA S4 is shown as SEQ ID NO.

4. In step (4), the DNA tetrahedron serves as the structural backbone and loads hairpins E-H1 and hairpin H2 through complementary base pairing; the nucleotide sequence of hairpin E-H1 is shown as SEQ ID NO.5, and the nucleotide sequence of hairpin H2 is shown as SEQ ID NO.

6.

3. The preparation method according to claim 1, characterized in that: In step (4), the toehold domain in the E-H1 is tightly locked, reducing the non-site signaling of CHA produced by direct triggering of the target miR-224.

4. A DNA nanomachine responsive to endogenous human apurinic / apyrimidinic nuclease prepared by the preparation method of claim 1.

5. Use of the endogenous human apurinic / apyrimidinic endonuclease-responsive DNA nanomachine according to claim 4 in tumor cell-specific miRNA imaging.

6. A method for tumor cell-specific miRNA imaging using the DNA nanomachine according to claim 1, characterized in that The steps include: a. incubating the DNA nanomachine according to claim 1 with APE1 and miR-224 at 37° C. for 30 minutes; b. Observe that APE1 specifically recognizes and cleaves the AP site in E-H1, resulting in the separation of the terminal 8-base DNA fragment from E-H1, generating activated hairpin H1; c. Target miR-224 hybridizes with the exposed toehold domain in H1, opening the hairpin structure and triggering the strand displacement reaction (SDR) between H1 and H2, resulting in the separation of Cy5 and BHQ2 and generating a significantly enhanced fluorescence signal.

7. A method for detecting miR-224 expression in tumor cells, characterized in that The steps include: a. Using the DNA nanomachine EGDN according to any one of claims 1 to 3; b. EGDN was incubated with APE1 and different concentrations of miR-224 at 37°C; c. The fluorescence intensity change of Cy5 was detected by fluorescence spectroscopy to determine the concentration of miR-224.

8. A method for evaluating APE1 activity in tumor cells, characterized in that The steps include: a. Using the DNA nanomachine EGDN according to claim 1; b. Incubate EGDN with APE1 and miR-224 at 37°C; c. The activity of APE1 was evaluated by observing the changes in the fluorescence signal generated by the separation of Cy5 and BHQ2.

9. A kit for imaging miR-224 in tumor cells, characterized in that: The kit comprises the DNA nanomachine EGDN as claimed in claim 1; a buffer for incubation; and an indicator for detecting a fluorescent signal.

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