Layered programmable DNA nano device and preparation method and application thereof

Abstract

The invention discloses a layered programmable DNA nano device as well as a preparation method and application thereof. The layered programmable DNA nano device HPDN disclosed by the invention is specially used for space-time selective imaging of mitochondrial miRNA in tumor cells. The layered programmable DNA nano device HPDN is composed of a mitochondrial specific DNA tetrahedron and two cage-shaped HCR hairpin probes, and mitochondrial targeting is achieved through triphenylphosphine (TPP) modification. The HPDN realizes miRNA detection with high sensitivity and specificity by utilizing signal amplification of APE1 activation, ultraviolet light (UV) activation and miR-21 triggering in cells. The technology has the advantages of simplicity and convenience in operation, good biocompatibility, real-time dynamic monitoring and the like, and a new strategy is provided for early diagnosis and treatment monitoring of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a hierarchical programmable DNA nano device and a preparation method and application thereof. Background Art

[0002] In biomedical research and clinical diagnosis, the precise detection and imaging of mitochondrial miRNA in tumor cells is of great significance. MiRNA (microRNA) is a class of endogenous small non-coding RNA molecules that play the role of gene expression regulators in cells and participate in a variety of biological processes, including cell differentiation, proliferation and apoptosis. During tumor occurrence and development, specific miRNA expression patterns will change, so they are considered to be important biomarkers for cancer diagnosis and treatment.

[0003] Traditional miRNA detection methods, such as Northern blot, RT-qPCR and microarray technology, have achieved certain results in terms of sensitivity and specificity, but these methods usually require complex operation procedures, professional experimental equipment and a large number of samples. In addition, these technologies often cannot achieve real-time dynamic imaging of miRNA at specific locations within cells, limiting their application in biomedical research and clinical diagnosis.

[0004] In recent years, with the development of nanotechnology and biosensing technology, biosensors based on nanomaterials have attracted extensive attention due to their high sensitivity, high specificity and designability. Especially in the field of DNA nanotechnology, the programmable properties of DNA molecules can be used to construct nanodevices with specific structures and functions for the detection and imaging of biomolecules. However, existing DNA nanodevices still face challenges in achieving spatiotemporal selective imaging of mitochondrial miRNA in tumor cells. The main problems include:

[0005] Targeting: How to effectively deliver nanodevices to the mitochondria of tumor cells and reduce nonspecific binding to other organelles. Activation mechanism: How to activate nanodevices in a specific microenvironment within the cell to achieve a specific response to miRNA. Signal amplification: How to amplify the signal after detecting the target miRNA to improve the sensitivity of detection. Spatiotemporal selectivity: How to achieve real-time dynamic imaging of miRNA at a specific location within the cell to better understand its biological function.

[0006] Currently, there is a lack of a hierarchical programmable DNA nanodevice for spatiotemporal controllable imaging of mitochondrial miRNA, as well as its preparation method and application. Summary of the invention

[0007] The purpose of the present invention is to provide a hierarchical programmable DNA nanodevice for spatiotemporal controllable imaging of mitochondrial miRNA and a preparation method and application thereof.

[0008] In order to solve the problems of the prior art, the present invention provides the following technical solutions:

[0009] In a first aspect, the present application provides a hierarchical programmable DNA nanodevice.

[0010] In a second aspect, the present application provides a method for preparing a hierarchical programmable DNA nanodevice.

[0011] In a third aspect, the present application provides an application of a hierarchical programmable DNA nanodevice HPDN for detecting mitochondrial miRNA in tumor cells.

[0012] In a fourth aspect, the present application provides a method for imaging mitochondrial miRNA in tumor cells using a hierarchical programmable DNA nanodevice.

[0013] In a fifth aspect, the present application provides a kit for imaging mitochondrial miRNA in tumor cells.

[0014] In a first aspect, the present application provides a hierarchical programmable DNA nanodevice, which is composed of the following modules:

[0015] Mitochondrial-specific DNA tetrahedron DT;

[0016] two caged HCR hairpin probes, cH1 and cH2;

[0017] The DNA tetrahedron serves as a structural backbone and nanocarrier, anchoring the hairpin probes cH1 and cH2, and modifying TPP for targeted delivery into mitochondria;

[0018] Hairpin cH1 was designed by introducing an AP site, which was located at the eighth nucleotide from the 5 end;

[0019] Hairpin cH2 has a short 7-base tail added to the 3' end via a PC linker and is labeled with fluorescein Cy5 and quencher BHQ2 on the opposite side of its stem;

[0020] Signal amplification transduction recognition triggered by target miR-21; the nucleotide sequence of the hairpin probe cH1 is shown in SEQ ID NO.1; the nucleotide sequence of the hairpin probe cH2 is shown in SEQ ID NO.2; the nucleotide sequence of the target miR-21 is shown in SEQ ID NO.3.

[0021] Furthermore, the mitochondrial-specific DNA tetrahedron DT is assembled from four single-stranded DNAs, wherein the four single-stranded DNAs are DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4; the nucleotide sequence of DNA S1 is shown in SEQ ID NO.4; the nucleotide sequence of DNA S2 is shown in SEQ ID NO.5; the nucleotide sequence of DNAS3 is shown in SEQ ID NO.6; and the nucleotide sequence of DNAS4 is shown in SEQ ID NO.7.

[0022] The second aspect of the present application provides a method for preparing a hierarchical programmable DNA nanodevice, comprising the following steps:

[0023] (1) mixing four single-stranded DNAs in an assembly buffer, wherein the four single-stranded DNAs are single-stranded DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4, heating and then rapidly cooling to assemble into a DNA tetrahedron;

[0024] (2) DNA tetrahedrons are mixed with cH1 and cH2 in an equimolar ratio in an assembly buffer and maintained at a certain temperature to produce a hierarchical programmable DNA nanodevice.

[0025] The third aspect of the present application provides an application of a hierarchical programmable DNA nanodevice HPDN in detecting mitochondrial miRNA imaging in tumor cells.

[0026] Furthermore, the hierarchical programmable DNA nanodevice HPDN can achieve spatiotemporally selective mitochondrial miRNA imaging under the overexpression of human apurinic / apyrimidinic endonuclease APE1 in the cytoplasm and artificially controllable ultraviolet light irradiation.

[0027] The fourth aspect of the present application provides a method for imaging mitochondrial miRNA in tumor cells using a hierarchical programmable DNA nanodevice, comprising the following steps:

[0028] (1) Co-incubating HPDN with tumor cells;

[0029] (2) HPDN is activated under specific conditions. The signal output of the HPDN system includes a three-step reaction process: human apurinic / apyrimidinic nuclease APE1 in the cytoplasm activates cH2; ultraviolet light UV in the mitochondria activates cH1; and the target miR-21 triggers signal amplification transduction recognition;

[0030] (3) Imaging of mitochondrial miRNA is achieved by enhancing the fluorescence signal.

[0031] Further, in step (2), the method for detecting the target miR-21 by HPDN comprises the following steps:

[0032] In the presence of APE1, the AP site in the nucleic acid is recognized and cleaved, resulting in the conversion of hairpin cH2 to H2;

[0033] In mitochondria, UV irradiation breaks the PC linker, resulting in the conversion of hairpin cH1 to H1;

[0034] The target miR-21 initiates a hybridization strand displacement reaction between hairpins H1 and H2, generating an amplified fluorescent signal.

[0035] Furthermore, in step (2), the specific conditions include: the concentration of APE1 is 5 U / mL; the UV irradiation time is 6 minutes; the concentration of miR-21 is 20 nM; and the detection limit of the fluorescence signal is 96.2 pM.

[0036] Furthermore, in step (2), the method further comprises quantifying the fluorescent signal using flow cytometry analysis.

[0037] In a fifth aspect, the present application provides a kit containing a hierarchical programmable DNA nanodevice, the kit comprising APE1 for activating the hierarchical programmable DNA nanodevice HPDN; an ultraviolet light source for activating HPDN; and a device for detecting fluorescence signals.

[0038] Beneficial effects: The present invention provides a hierarchical programmable DNA nanodevice (HPDN), which can achieve spatiotemporal selective imaging of mitochondrial miRNA in tumor cells. Through sophisticated design, HPDN integrates functions such as mitochondrial targeted delivery, specific activation and signal amplification, providing a new strategy for the detection and imaging of mitochondrial miRNA in tumor cells. Compared with traditional miRNA detection methods, HPDN is easier to prepare and operate, does not require complex operating procedures and a large number of samples, and reduces the difficulty and cost of the experiment.

[0039] Compared with the prior art, the present invention has the following advantages: (1) Spatiotemporal selective imaging: The hierarchical programmable DNA nanodevice (HPDN) provided by the present invention can achieve spatiotemporal selective imaging of mitochondrial miRNA in tumor cells. By precisely controlling APE1 activation and UV light activation, the HPDN system can be hierarchically activated in the cytoplasm and mitochondria of tumor cells to achieve in situ imaging of miRNA.

[0040] (2) High-sensitivity detection: HPDN exhibits excellent detection sensitivity, with a detection limit of 96.2pM, which is much lower than traditional methods, which is of great significance for early tumor diagnosis and treatment monitoring. High-specificity recognition: HPDN has high specificity for the target miR-21 and can effectively distinguish the target miRNA from other control miRNAs, reducing false positive results and improving the accuracy of detection.

[0041] (3) Signal amplification mechanism: HPDN uses hybridization strand displacement reaction to achieve signal amplification. When the target miR-21 is present, Cy5 and BHQ2 separate to generate an amplified fluorescent signal, which enhances the detectability of the signal. Real-time dynamic monitoring: HPDN can achieve real-time dynamic monitoring of miRNA in cells, providing a new tool for studying the role of miRNA in tumor development.

[0042] (4) Targeted delivery: By modifying TPP, HPDN can be delivered to mitochondria, reducing nonspecific binding to other organelles and improving imaging accuracy. Controllable activation: The activation of HPDN depends on the intelligent "dual stimulation" of endogenous APE1 and exogenous UV light, providing a controllable activation mechanism, making the imaging process more flexible and precise.

[0043] (5) Biocompatibility: HPDN is made of biocompatible materials, which reduces toxicity to cells and is suitable for biomedical research and clinical applications. Multimodal analysis: HPDN can not only be used for fluorescence imaging, but also for quantitative analysis by flow cytometry, providing a multimodal analysis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figure 1 Schematic diagram of the hierarchical programmable DNA nanodevice (HPDN) of the present invention used for spatiotemporal selective imaging of mitochondrial miRNA in tumor cells.

[0046] Figure 2 This is a feasibility analysis diagram of the use of HPDN in the present invention for miR-21 detection. Figure 2 A is an electrophoresis characterization diagram of the assembly of HPDN of the present invention. Lane 1: S1; Lane 2: S1+S2; Lane 3: S1+S2+S3; Lane 4: S1+S2+S3+S4 (DT); Lane 5: cH1; Lane 6: cH2; Lane 7: DT+cH1; Lane 8: DT+cH2. Figure 2B is an electrophoretic characterization diagram of the HPDN of the present invention in response to different combinations of targets (APE1, UV and miR-21). Figure 2 C is the fluorescence spectra of the HPDN of the present invention in response to different combinations of targets (APE1, UV and miR-21). Figure 2 D Kinetic analysis of the HPDN of the present invention in response to different combinations of targets (APE1, UV and miR-21) at different times.

[0047] Figure 3 This is a performance analysis diagram of the HPDN of the present invention for miR-21 detection. Figure 3 A is a fluorescence spectrum diagram of the HPDN of the present invention in response to different concentrations of miR-21. Figure 3 B is the fluorescence spectrum of the HPDN of the present invention in response to different concentrations of APE1. Figure 3 C is a histogram of the fluorescence intensity of the HPDN of the present invention in response to different miRNAs (miR-21, Let-7d, miR-155, miR-221 and miR-224). Figure 3 D is a histogram of the fluorescence intensity of the HPDN of the present invention in response to different nucleases (APE1, Exo I, EcoR I, Kpn I and Not I). Figure 3 E is the fluorescence spectra of the HPDN of the present invention in response to different UV irradiation times. Figure 3 F is a histogram of the fluorescence intensity of the HPDN of the present invention and the control nanodevices (m-HPDN, n-HPDN and o-HPDN) in response to different combinations of targets (APE1, UV and miR-21).

[0048] Figure 4 This is a diagram showing the imaging analysis of mitochondrial miR-21 in living cells using the HPDN of the present invention. Figure 4 A is a confocal imaging result diagram of the co-localization of HPDN and Mito-tracker green in MCF-7 cells of the present invention. Figure 4 B is a schematic diagram of incubation of MCF-7 of the present invention with HPDN, m-HPDN, n-HPDN and o-HPDN respectively. Figure 4 C is a confocal imaging result diagram of the MCF-7 of the present invention after incubation with different nanodevices; Figure 4 D is a flow cytometry analysis result diagram of the MCF-7 of the present invention after incubation with different nanodevices. Figure 4 E is the fluorescence quantitative result of the present invention corresponding to Figure D. a: HPDN; b: m-HPDN; c: n-HPDN; d: o-HPDN. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] In a first aspect, an embodiment of the present application provides a hierarchical programmable DNA nanodevice, which is composed of the following modules:

[0057] Mitochondrial-specific DNA tetrahedron DT;

[0058] two caged HCR hairpin probes, cH1 and cH2;

[0059] DNA tetrahedrons serve as structural backbones and nanocarriers to anchor hairpin probes cH1 and cH2 and modify TPP for targeted delivery into mitochondria;

[0060] Hairpin cH1 was designed by introducing an AP site, which was located at the eighth nucleotide from the 5' end;

[0061] The hairpin cH2 has a short 7-base tail added to the 3' end via a PC linker and is labeled with fluorescein Cy5 and quencher BHQ2 on the opposite sides of its stem.

[0062] Signal amplification transduction recognition triggered by target miR-21; the nucleotide sequence of the hairpin probe cH1 is shown in SEQ ID NO.1; the nucleotide sequence of the hairpin probe cH2 is shown in SEQ ID NO.2; the nucleotide sequence of the target miR-21 is shown in SEQ ID NO.3.

[0063] In some embodiments, the mitochondrial-specific DNA tetrahedron DT is assembled from four single-stranded DNAs, wherein the four single-stranded DNAs are DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4; the nucleotide sequence of DNA S1 is shown in SEQ ID NO.4; the nucleotide sequence of DNA S2 is shown in SEQ ID NO.5; the nucleotide sequence of DNA S3 is shown in SEQ ID NO.6; and the nucleotide sequence of DNA S4 is shown in SEQ ID NO.7.

[0064] A second aspect of the present application provides a method for preparing a hierarchical programmable DNA nanodevice, comprising the following steps:

[0065] (1) four single-stranded DNAs are mixed in an assembly buffer, the four single-stranded DNAs are single-stranded DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4, and then heated and rapidly cooled to assemble into a DNA tetrahedron;

[0066] (2) DNA tetrahedrons are mixed with cH1 and cH2 in an equimolar ratio in an assembly buffer and maintained at a certain temperature to produce a hierarchical programmable DNA nanodevice.

[0067] A third aspect of the embodiments of the present application provides an application of a hierarchical programmable DNA nanodevice HPDN in detecting mitochondrial miRNA in tumor cells.

[0068] In some embodiments, the hierarchical programmable DNA nanodevice HPDN can achieve spatiotemporally selective mitochondrial miRNA imaging under the overexpression of human apurinic / apyrimidinic nuclease APE1 in the cytoplasm and artificially controllable ultraviolet light irradiation.

[0069] A fourth aspect of the present application provides a method for imaging mitochondrial miRNA in tumor cells using a hierarchical programmable DNA nanodevice, comprising the following steps:

[0070] (1) Co-incubating HPDN with tumor cells;

[0071] (2) HPDN is activated under specific conditions. The signal output of the HPDN system includes a three-step reaction process: human apurinic / apyrimidinic nuclease APE1 in the cytoplasm activates cH2; ultraviolet light UV in the mitochondria activates cH1; and the target miR-21 triggers signal amplification transduction recognition;

[0072] (3) Imaging of mitochondrial miRNA is achieved by enhancing the fluorescence signal.

[0073] In some embodiments, in step (2), the method for detecting the target miR-21 by HPDN comprises the following steps:

[0074] In the presence of APE1, the AP site in the nucleic acid is recognized and cleaved, resulting in the conversion of hairpin cH2 to H2;

[0075] In mitochondria, UV irradiation breaks the PC linker, resulting in the conversion of hairpin cH1 to H1;

[0076] The target miR-21 initiates a hybridization strand displacement reaction between hairpins H1 and H2, generating an amplified fluorescent signal.

[0077] In some embodiments, in step (2), the specific conditions include: the concentration of APE1 is 5 U / mL; the UV irradiation time is 6 minutes; the concentration of miR-21 is 20 nM; and the detection limit of the fluorescent signal is 96.2 pM.

[0078] In some embodiments, in step (2), the method further comprises quantifying the fluorescent signal using flow cytometry analysis.

[0079] A fifth aspect of an embodiment of the present application provides a kit containing a hierarchical programmable DNA nanodevice, the kit comprising APE1 for activating the hierarchical programmable DNA nanodevice HPDN; an ultraviolet light source for activating HPDN; and a device for detecting a fluorescent signal.

[0080] Example 1

[0081] A hierarchical programmable DNA nanodevice of the present invention is composed of the following modules:

[0082] Mitochondrial-specific DNA tetrahedron DT;

[0083] two caged HCR hairpin probes, cH1 and cH2;

[0084] DNA tetrahedrons serve as structural backbones and nanocarriers to anchor hairpin probes cH1 and cH2 and modify TPP for targeted delivery into mitochondria;

[0085] Hairpin cH1 was designed by introducing an AP site, which was located at the eighth nucleotide from the 5 end;

[0086] Hairpin cH2 has a short 7-base tail added to the 3' end via a PC linker and is labeled with fluorescein Cy5 and quencher BHQ2 on the opposite side of its stem;

[0087] Signal amplification transduction recognition triggered by target miR-21; the nucleotide sequence of the hairpin probe cH1 is shown in SEQ ID NO.1; the nucleotide sequence of the hairpin probe cH2 is shown in SEQ ID NO.2; the nucleotide sequence of the target miR-21 is shown in SEQ ID NO.3.

[0088] The signal output of the HPDN system includes a three-step reaction process: human apurinic / apyrimidinic nuclease APE1 activates cH2 in the cytoplasm; ultraviolet light UV activates cH1 in the mitochondria; and signal amplification transduction triggered by the target miR-21 is used for in situ imaging of mitochondrial miRNA.

[0089] The mitochondrial-specific DNA tetrahedron DT is assembled from four single-stranded DNAs, namely DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4; the nucleotide sequence of DNA S1 is shown in SEQ ID NO.4; the nucleotide sequence of DNA S2 is shown in SEQ ID NO.5; the nucleotide sequence of DNA S3 is shown in SEQ ID NO.6; and the nucleotide sequence of DNA S4 is shown in SEQ ID NO.7.

[0090] Example 2

[0091] A method for preparing a hierarchical programmable DNA nanodevice of the present invention comprises the following steps:

[0092] (1) four single-stranded DNAs are mixed in an assembly buffer, the four single-stranded DNAs are single-stranded DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4, and then heated and rapidly cooled to assemble into a DNA tetrahedron;

[0093] (2) DNA tetrahedrons are mixed with cH1 and cH2 in an equimolar ratio in an assembly buffer and maintained at a certain temperature to produce a hierarchical programmable DNA nanodevice.

[0094] The hierarchical programmable DNA nanodevice HPDN of the present invention is used for detecting mitochondrial miRNA in tumor cells. The hierarchical programmable DNA nanodevice HPDN can realize spatiotemporally selective mitochondrial miRNA imaging under the irradiation of human apurinic / apyrimidinic nuclease APE1 overexpressed in the cytoplasm and artificially controllable ultraviolet light.

[0095] Example 3

[0096] A method for imaging mitochondrial miRNA in tumor cells using a hierarchical programmable DNA nanodevice of the present invention comprises the following steps:

[0097] (1) Co-incubating HPDN with tumor cells;

[0098] (2) Activating HPDN under specific conditions, including APE1 activation in the cytoplasm, UV activation in the mitochondria, and recognition of the target miR-21; the method for using HPDN to detect the target miR-21 includes the following steps:

[0099] In the presence of APE1, the AP site in the nucleic acid is recognized and cleaved, resulting in the conversion of hairpin cH2 to H2;

[0100] In mitochondria, UV irradiation breaks the PC linker, resulting in the conversion of hairpin cH1 to H1;

[0101] The target miR-21 initiates a hybridization strand displacement reaction between hairpins H1 and H2, generating an amplified fluorescent signal. The specific conditions include: the concentration of APE1 is 5 U / mL; the UV irradiation time is 6 minutes; the concentration of miR-21 is 20 nM; the detection limit of the fluorescent signal is 96.2 pM. The method also includes using flow cytometry analysis to quantify the fluorescent signal.

[0102] (3) Imaging of mitochondrial miRNA is achieved by enhancing the fluorescence signal.

[0103] The kit containing the hierarchical programmable DNA nano device of the present invention comprises APE1 for activating the hierarchical programmable DNA nano device HPDN; an ultraviolet light source for activating HPDN; and a device for detecting fluorescence signals.

[0104] Test Example 1

[0105] 1. Experimental part

[0106] 1.1 Materials

[0107] All oligonucleotides were synthesized and purified by Shanghai Shenggong Biotechnology Co., Ltd. (Shanghai, China). Triphenylphosphine (TPP)-modified oligonucleotides were provided by Bao Bioengineering Co., Ltd. (Dalian, China). APE1 was purchased from New England Biotechnology Co., Ltd. (Beijing, China). Exo I, Kpn I, Not I, and EcoR I were purchased from Yisheng Biotechnology Co., Ltd. (Shanghai, China). Phosphate buffered saline (PBS), cell culture medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin, and trypsin solution were provided by Wuhan Saiweier Biotechnology Co., Ltd. (Wuhan, China). Hoechst 33342 and Mito-tracker Green were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China). All solutions were prepared with ultrapure water (resistivity ≥18.2 MΩcm).

[0108] 1.2 Instruments

[0109] Gel imaging was performed on a ChemiScope 6200 (Shanghai, China). Fluorescence measurements were performed on a multi-function microplate reader (Bern, Switzerland). Cell imaging was performed on a Nikon confocal laser scanning microscope (Tokyo, Japan). Flow cytometric analysis was performed on an Agilent flow cytometer (California, USA).

[0110] 1.3 Probe preparation

[0111] Four single-stranded DNAs (S1, S2, S3 and S4) were mixed in assembly buffer (20mM Tris, 10mM MgCl2, 150mM NaCl, pH 8.0), heated at 95°C for 5min, rapidly cooled to 4°C, and kept for 30min to assemble into DNA tetrahedrons. Then, DNA tetrahedrons were mixed with cH1 and cH2 in an equimolar ratio in assembly buffer and kept at 37°C for another 30min to obtain DNA nanodevices (HPDN).

[0112] 1.4 Gel electrophoresis

[0113] Different reaction samples were mixed with 2 μL 6× DNA loading buffer and 1 μL 100× SYBR Gold dye. After being pipetted evenly, 10 μL of the mixed sample was added to the agarose gel wells and incubated at a constant voltage of 100 V for about 60 min in 1× TBE. The ChemiScope 6200 system was used to take pictures.

[0114] 1.5 Fluorescence detection

[0115] To measure the detection sensitivity, 100nM HPDN was incubated with different concentrations of miR-21 in the presence of APE1 (5U / mL) and 365nm UV (6min); 100nM HPDN was incubated with different concentrations of APE1 in the presence of 365nm UV (6min) and miR-21 (20nM). In addition, 100nM HPDN was illuminated with UV for different times in the presence of APE1 (5U / mL) and miR-21 (20nM). To investigate the specificity, 100nM HPDN was incubated with miR-21 (20mM) and other control miRNAs; 100nM HPDN was incubated with APE1 (5U / mL) and other control enzymes. All samples were incubated at 37°C for 60min. The excitation wavelength was 615nm and the collection range was 650-750nm. All experiments were repeated three times independently.

[0116] 1.6 Cell culture

[0117] Human breast cancer cells (MCF-7) were cultured in DMEM medium containing 10% fetal bovine serum and incubated in a cell culture incubator at 37°C with 5% CO2.

[0118] 1.7 Confocal laser fluorescence imaging analysis

[0119] MCF-7 cells were evenly plated in a 35 mm glass-bottomed culture dish and placed in a cell culture incubator overnight to allow them to adhere completely to the wall. They were washed twice with PBS and incubated in DMEM containing 250 nM HPDN for 4 h. UV illumination was then performed for 6 min and incubated for 90 min. The cells were washed three times with PBS and stained with 0.05 mg / mL Hoechst 33342 and 100 nM Mito-tracker Green for 15 min. The excitation and emission wavelengths are described as follows. Hoechst 33342: Ex = 405 nm, Em = 425-470 nm; Mito-traker Green: Ex = 488 nm, Em = 500-550 nm; Cy5: Ex = 640 nm, Em = 663-738 nm.

[0120] 1.8 Flow cytometric analysis

[0121] MCF-7 cells were seeded in 12-well plates for 24 hours, incubated with DMEM containing different DNA probes for 4 hours, then UV-irradiated for 6 minutes and incubated for 90 minutes. After washing the cells twice with PBS, the cells were digested with cell digestion solution, centrifuged at 2000 rpm for 4 minutes at 4°C, the supernatant was discarded, washed once with PBS, resuspended in 200 μL PBS, and the fluorescence signal was collected by flow cytometry.

[0122] 2. Results

[0123] 2.1 Working Mechanism

[0124] The present invention develops a hierarchical programmable DNA nanodevice (HPDN) for spatiotemporal selective imaging of mitochondrial miRNA in tumor cells, such as Figure 1As shown in Figure 2. HPDN consists of two modules: a mitochondrial-specific DNA tetrahedron (DT) and two caged HCR hairpin probes (cH1 and cH2). The signal output of the HPDN system includes a three-step reaction process: activation of cH2 by human apurinic / apyrimidinic endonuclease (APE1) in the cytoplasm; activation of cH1 by ultraviolet light (UV) in the mitochondria; and signal amplification transduction triggered by the target miR-21 for in situ imaging of mitochondrial miRNA. In detail, DT serves as a structural backbone and nanocarrier to anchor the hairpin probes cH1 and cH2, and to modify TPP for targeted delivery into mitochondria. Hairpin cH1 is designed by introducing an APsite, which is located at the eighth nucleotide at the 5 end. Hairpin cH2 is tailed with a short 7-base at the 3 end by a PC linker, and is labeled with fluorescein (Cy5) and a quencher (BHQ2) on the opposite side of its stem. In the presence of cytoplasmic APE1, the AP site in the nucleic acid is rapidly recognized and cleaved, resulting in the conversion of hairpin cH2 to H2. After entering the mitochondria, ultraviolet light irradiation causes the PC linker to break, resulting in the conversion of hairpin cH1 to H1. Subsequently, the target miR-21 initiates the hybridization chain displacement reaction between hairpins H1 and H2, at which time Cy5 and BHQ2 separate from each other, thereby generating an amplified fluorescence signal. Based on the overexpression of APE1 in the tumor cytoplasm and artificially controllable ultraviolet light irradiation, the HPDN system can be hierarchically activated in the tumor cell cytoplasm and mitochondria, thereby achieving spatiotemporal selective mitochondrial miRNA imaging. Figure 1 Schematic diagram of the hierarchical programmable DNA nanodevice (HPDN) of the present invention used for spatiotemporal selective imaging of mitochondrial miRNA in tumor cells.

[0125] Test Example 2

[0126] Feasibility verification

[0127] The present invention uses agarose gel electrophoresis to investigate the assembly process of HPDN. Figure 2 As shown in A, with the increase of single-stranded DNA, the migration rate of the band gradually slowed down (lanes 1-4), and a single and bright band appeared in lane 4, indicating that the DNA tetrahedron was successfully constructed. When the DNA tetrahedron was mixed with cH1 (lane 5) or H2 (lane 6), products with larger molecular weight were observed in lanes 7 and 8, respectively. When the DNA tetrahedron was incubated with cH1 and cH2 at the same time, products with further increased molecular weight were observed in lane 9, indicating the successful preparation of HPDN. Next, the present invention investigated the detection ability of HPDN for the target miR-21 under different conditions. Figure 2As shown in Figure B, in the absence of one or more of APE1, UV and miR-21, the electrophoretic bands of HPDN did not change significantly (lanes bh); when APE1, UV and miR-21 were present at the same time, an electrophoretic band with a significantly slower mobility was observed (lane a), indicating that HPDN assembled into products with larger molecular weight. Subsequently, the present invention investigated the response performance of HPDN in the presence of different targets through a fluorescence spectroscopy system. Only when APE1, UV and miR-21 were present at the same time, HPDN showed a significantly enhanced fluorescence signal ( Figure 2 C). As time goes by, the fluorescence intensity of HPDN gradually increases and reaches saturation at about 60 minutes ( Figure 2 D). Therefore, the present invention selects 60 min as the optimal reaction time in the in vitro experiment.

[0128] Figure 2 .( Figure 2 A) Assembly electrophoresis characterization of HPDN. Lane 1: S1; Lane 2: S1+S2; Lane 3: S1+S2+S3; Lane 4: S1+S2+S3+S4 (DT); Lane 5: cH1; Lane 6: cH2; Lane 7: DT+cH1; Lane 8: DT+cH2. ( Figure 2 B) Electrophoretic characterization of HPDN in response to different combinations of targets (APE1, UV and miR-21). Figure 2 C) Fluorescence spectra of HPDN in response to different combinations of targets (APE1, UV and miR-21).

[0129] Test Example 3

[0130] Sensitivity and selectivity analysis

[0131] The present invention evaluated the detection sensitivity of HPDN to the target miR-21. Figure 3 As shown in A, as the concentration of miR-21 increased from 0 nM to 50 nM, the fluorescence intensity of the system gradually increased and reached a plateau. According to the standard curve corresponding to the low target concentration and the fluorescence intensity, the calculated detection limit was 96.2 pM, indicating that HPDN has excellent target detection performance. Next, the present invention investigated the fluorescence changes of HPDN in the presence of different concentrations of APE1. The results are shown in Figure 3 As shown in Figure B, as the concentration of APE1 increased from 0U / mL to 2U / mL, the fluorescence intensity of HPDN gradually increased, and had a good linear relationship at low concentrations. The present invention selected several control miRNAs to study the detection specificity of D-FNAN for the target miR-21. The results are shown in Figure 2. Figure 3 As shown in C, compared with the control miRNA, HPDN produced a significantly enhanced fluorescence signal for miR-21, indicating that HPDN has good detection specificity.

[0132] Subsequently, the present invention incubated HPDN with several control nucleases. Figure 3 As shown in Figure D, compared with APE1, none of the control nucleases produced significantly enhanced fluorescence signals, indicating that HPDN can respond to APE1 for accurate detection of target miRNA. At the same time, the fluorescence response of HPDN was positively correlated with the UV irradiation dose and reached saturation after about 6 minutes ( Figure 3 E). In addition, the present invention designed three control nanodevices (m-HPDN, n-HPDN and o-HPDN) to evaluate the determinants of HPDN's ability to respond to targets. Among them, the design of m-HPDN is consistent with the structure of HPDN, but lacks PC linker, so that it cannot be broken by UV light; the design of n-HPDN is consistent with the structure of HPDN, but lacks AP site, so that it cannot be recognized and cut by APE1; the cH1 sequence in o-HPDN is designed with mutant bases, so that it loses the ability to bind miR-21. The results are as follows Figure 3 As shown in Figure F, compared with HPDN, m-HPDN, n-HPDN and o-HPDN still have only low fluorescence intensity in the presence of APE1, UV and miR-21, indicating that HPDN can detect and analyze the target miR-21 under the stimulation of APE1 and UV.

[0133] Figure 3 This is a performance analysis diagram of the HPDN of the present invention for miR-21 detection. Figure 3 A is a fluorescence spectrum diagram of the HPDN of the present invention in response to different concentrations of miR-21. Figure 3 B is the fluorescence spectrum of the HPDN of the present invention in response to different concentrations of APE1. Figure 3 C is a histogram of the fluorescence intensity of the HPDN of the present invention in response to different miRNAs (miR-21, Let-7d, miR-155, miR-221 and miR-224). Figure 3 D is a histogram of the fluorescence intensity of the HPDN of the present invention in response to different nucleases (APE1, Exo I, EcoR I, Kpn I and Not I). Figure 3 E is the fluorescence spectra of the HPDN of the present invention in response to different UV irradiation times. Figure 3 F is a histogram of the fluorescence intensity of the HPDN of the present invention and the control nanodevices (m-HPDN, n-HPDN and o-HPDN) in response to different combinations of targets (APE1, UV and miR-21).

[0134] Test Example 4

[0135] Imaging analysis of miR-21 in mitochondria

[0136] First, the present invention used a laser confocal microscope to investigate the fluorescence imaging effect of MCF-7 cells after incubation with HPDN (only labeled with Cy5) for different time periods. Figure 4 As shown in A, over time, the red fluorescence of HPDN gradually coincided with the green fluorescence of Mito-tracker, and the Pearson correlation coefficient increased from 0.42 to 0.88, indicating that HPDN can be well localized in mitochondria. Next, the present invention introduced several control probes (m-HPDN, n-HPDN and o-HPDN) to study the imaging performance of HPDN in response to APE1 and UV activation for miR-21 in mitochondria ( Figure 4 B) Figure 4 As shown in Figure C, after incubation with HPDN, MCF-7 cells showed obvious fluorescence signals (group a). In comparison, MCF-7 cells treated with m-HPDN (group b) or o-HPDN (group d) had very low fluorescence signals, demonstrating the miRNA imaging ability of HPDN under APE1 / UV activation. In addition, no obvious fluorescence signal was observed after MCF-7 cells were incubated with n-HPDN (group c), indicating that the signal of HPDN did come from the specific activation of miR-21. The imaging results are consistent with the flow cytometry analysis, showing that the fluorescence intensity of MCF-7 cells incubated with HPDN was 3.65 times, 3.48 times, and 4.67 times higher than that of cells incubated with m-HPDN, n-HPDN, and o-HPDN, respectively ( Figure 4 D, E). These results reveal that the HPDN system can be controlled by the intelligent “dual stimulation” of endogenous APE1 and exogenous light, thereby imaging and analyzing miR-21 in mitochondria.

[0137] Figure 4 This is a diagram showing the imaging analysis of mitochondrial miR-21 in living cells using the HPDN of the present invention. Figure 4 A is a confocal imaging result diagram of the co-localization of HPDN and Mito-tracker green in MCF-7 cells of the present invention. Figure 4 B is a schematic diagram of incubation of MCF-7 of the present invention with HPDN, m-HPDN, n-HPDN and o-HPDN respectively. Figure 4 C is a confocal imaging result diagram of the MCF-7 of the present invention after incubation with different nanodevices; Figure 4 D is a flow cytometry analysis result diagram of the MCF-7 of the present invention after incubation with different nanodevices. Figure 4 E is the fluorescence quantitative result of the present invention corresponding to Figure D. a: HPDN; b: m-HPDN; c: n-HPDN; d: o-HPDN.

[0138] 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 hierarchical programmable DNA nanodevice, characterized in that: The hierarchical programmable DNA nanodevice consists of the following modules: Mitochondrial-specific DNA tetrahedron DT; Two caged HCR hairpin probes cH1 and cH2; The DNA tetrahedron serves as a structural backbone and nanocarrier, anchoring the hairpin probes cH1 and cH2, and modifying TPP for targeted delivery into mitochondria; The hairpin cH1 is designed by introducing an AP site, which is located at the eighth nucleotide from the 5 end; The hairpin cH2 has a short 7-base tail added to the 3' end via a PC linker, and is labeled with fluorescein Cy5 and quencher BHQ2 on the opposite side of its stem; Signal amplification transduction recognition triggered by target miR-21; the nucleotide sequence of the hairpin probe cH1 is shown in SEQ ID NO.1; the nucleotide sequence of the hairpin probe cH2 is shown in SEQ ID NO.2; the nucleotide sequence of the target miR-21 is shown in SEQ ID NO.

3.

2. The hierarchical programmable DNA nanodevice according to claim 1, characterized in that: The mitochondrial-specific DNA tetrahedron DT is assembled from four single-stranded DNAs, wherein the four single-stranded DNAs are DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4; the nucleotide sequence of DNA S1 is shown in SEQ ID NO.4; the nucleotide sequence of DNA S2 is shown in SEQ ID NO.5; the nucleotide sequence of DNA S3 is shown in SEQ ID NO.6; and the nucleotide sequence of DNA S4 is shown in SEQ ID NO.

7.

3. The method for preparing the hierarchical programmable DNA nanodevice according to claim 1, characterized in that The steps include: (1) mixing the four single-stranded DNAs of claim 2 in an assembly buffer, wherein the four single-stranded DNAs are single-stranded DNA S1, single-stranded DNA S2, single-stranded DNA S3 and single-stranded DNA S4, heating and then rapidly cooling to assemble into a DNA tetrahedron; (2) DNA tetrahedrons are mixed with cH1 and cH2 in an equimolar ratio in an assembly buffer and maintained at a certain temperature to produce a hierarchical programmable DNA nanodevice.

4. Use of the hierarchical programmable DNA nanodevice HPDN according to claim 1 in detecting mitochondrial miRNA imaging in tumor cells.

5. The use according to claim 4, characterized in that: The hierarchical programmable DNA nanodevice HPDN can achieve spatiotemporally selective mitochondrial miRNA imaging under the irradiation of human apurinic / apyrimidinic nuclease APE1 overexpressed in the cytoplasm and artificially controllable ultraviolet light.

6. A method for imaging mitochondrial miRNA in tumor cells using the hierarchical programmable DNA nanodevice according to any one of claims 1 to 4, characterized in that The steps include: (1) Co-incubating the hierarchical programmable DNA nanodevice HPDN with tumor cells; (2) Activating the hierarchical programmable DNA nanodevice HPDN under specific conditions, the signal output of the HPDN system includes a three-step reaction process: human apurinic / apyrimidinic nuclease APE1 in the cytoplasm activates cH2; ultraviolet light UV in the mitochondria activates cH1; target miR-21 triggers signal amplification transduction recognition; (3) Imaging of mitochondrial miRNA is achieved by enhancing the fluorescence signal.

7. The method according to claim 6, characterized in that: In step (2), the method for detecting the target miR-21 by HPDN comprises the following steps: In the presence of human apurinic / apyrimidinic endonuclease APE1 in the cytoplasm, the AP site in the nucleic acid is recognized and cleaved, resulting in the conversion of hairpin cH2 to H2; In mitochondria, ultraviolet light UV irradiation breaks the PC linker, causing the hairpin cH1 to convert to H1; The target miR-21 initiates a hybridization strand displacement reaction between hairpins H1 and H2, generating an amplified fluorescent signal.

8. The method according to claim 6, characterized in that: In step (2), the specific conditions include: the concentration of APE1 is 5 U / mL; the UV irradiation time is 6 minutes; the concentration of miR-21 is 20 nM; and the detection limit of the fluorescence signal is 96.2 pM.

9. The method according to claim 6, characterized in that: In step (2), the method further comprises quantifying the fluorescent signal using flow cytometry analysis.

10. A kit containing the hierarchical programmable DNA nanodevice according to any one of claims 1 to 3, characterized in that: The kit comprises APE1 for activating the hierarchical programmable DNA nanodevice HPDN; an ultraviolet light source for activating the HPDN; and a device for detecting a fluorescent signal.

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