DNA framework nucleic acid and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to DNA framework nucleic acid and application thereof. According to the application, a dynamic DNA self-assembly system is constructed by taking DNA framework nucleic acid capable of forming FRET fluorescent pairs as a construction unit, so that fluorescence signal change is realized. The DNA framework nucleic acid has the intelligent characteristics of excellent biocompatibility, high signal-to-noise ratio and the like, so that the DNA framework nucleic acid shows strong capability of monitoring the biological process of the cell exosome. DNA framework nucleic acid is directly loaded into the EVs through instantaneous pores and electroporation, so that instant and efficient labeling of the EVs is achieved, tedious pretreatment, chemical modification and genetic engineering are not needed, and the native characteristics and functions of the EVs can be kept to the maximum extent. Based on the EV with a DNA framework nucleic acid label, long-term non-invasive whole-body high-resolution mode tracking of the in-vivo EV is realized, and dynamic biological distribution of the EV is disclosed in a real-time and in-situ quantitative manner.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a DNA framework nucleic acid and an application thereof. Background Art

[0002] DNA framework nucleic acids (FNAs) are a type of nucleic acid system with a specific framework structure constructed based on DNA nanotechnology. This type of structure is assembled from natural nucleic acids based on the principle of base complementary pairing, and has a uniform and stable topological structure, good biocompatibility and extremely low cytotoxicity (Zhu Dan, Zhang Chengwen, Wang Zichun, et al. Application of framework nucleic acids in molecular diagnosis and imaging of living cells [J]. Journal of Guangzhou University (Natural Science Edition), 2020.). Framework nucleic acids can directly pass through the cell membrane without the assistance of transfection reagents, effectively protecting the carried biological molecules from degradation by nucleases in the physiological environment for a long time, providing a new tool for molecular diagnosis and imaging of living cells.

[0003] The research on framework nucleic acids focuses on using the base pairing principle of DNA to construct nanostructures with specific shapes and sizes, such as DNA-DNA framework nucleic acids, cubes, etc. (Wang Hao, Long Xingru, Jiang David, et al. Application of framework nucleic acids in biomedicine and research progress on their biological properties under the guidance of nuclear medicine imaging [J]. Pharmaceutical Progress, 2024, 48(3): 178-185.). These structures are not only highly programmable and stable, but also can achieve precise spatial rearrangement of molecules and improve the efficiency of biomolecular signal transduction. The design and functionalization of framework nucleic acids can be achieved in many ways, including but not limited to precise spatial rearrangement of molecules through pricing and site-specific molecular modification (such as functional recognition units, fluorescent signal molecules, and biological reaction substrates, etc.) (Yin Fangfei. Design and application of framework nucleic acid molecular recognition probes [D]. University of Chinese Academy of Sciences (Shanghai Institute of Applied Physics, Chinese Academy of Sciences), 2021.). This addressable property with nanoscale resolution enables framework nucleic acids to achieve precise control of parameters such as the number, spacing and spatial conformation of ligands, and then study the binding characteristics and influencing factors of cell surface receptor ligands (Guo Linjie, Peng Hongzhen, Li Jiang, et al. Research progress on cell surface receptor ligand interactions based on framework nucleic acids [J]. Applied Chemistry, 2022.).

[0004] In CRISPR-Cas9 delivery and optical probe construction, framework nucleic acids also show their unique advantages. For example, framework nucleic acids can enter cells directly through the endocytosis pathway without the help of transfection reagents, and have good resistance to nuclease cleavage, which can maintain sensing or therapeutic properties in complex physiological environments. By encapsulating organic dyes into the hydrophobic cavity within the DNA framework, a series of fluorescent probes can be developed, which show excellent performance in super-resolution fluorescence imaging. Fluorescent nanoprobes constructed with framework nucleic acids can realize the simultaneous detection and analysis of multiple targets in complex samples (Huang Qiuling. Design and application of framework nucleic acid fluorescent nanoprobes [D]. University of Chinese Academy of Sciences (Shanghai Institute of Applied Physics, Chinese Academy of Sciences), 2021.). In addition, framework nucleic acids can also be used to construct more stable, efficient and highly biocompatible fluorescent biosensors, while introducing photoacoustic imaging to improve the biological tissue penetration depth and imaging contrast of nucleic acid probes (Peng Fangqi. Research on the application of framework nucleic acids in optical probe construction and CRISPR-Cas9 delivery [D]. Hunan University, 2021.).

[0005] DNA framework nucleic acid is a nano-assembled oligonucleotide with unique physical, chemical and biological properties. Its application in biomedicine and the progress of research on its biological properties under the guidance of nuclear medicine imaging show its great potential in disease diagnosis, treatment and biomedical research (Wang Hao, Long Xingru, Jiang David, et al. Application of framework nucleic acid in biomedicine and the progress of research on its biological properties under the guidance of nuclear medicine imaging [J]. Pharmaceutical Progress, 2024, 48(3): 178-185.).

[0006] Extracellular vesicles (EVs) are a class of tiny vesicles with lipid bilayer membrane structures secreted by cells, with sizes ranging from 30 to 5000 nm. They are formed through different mechanisms, including microvesicles, apoptotic bodies and exosomes (Huang Lin, Wang Dianbing, Gu Ning, et al. Extracellular vesicles and their application in disease diagnosis and treatment [J]. Science Bulletin, 2019, 64(19): 2025-2036.). Extracellular vesicles play an important role in intercellular communication. They can carry a variety of bioactive molecules including mRNA, microRNA, proteins, lipids, etc., and specifically recognize target cells for signal transduction, thereby affecting the function and phenotype of target cells (Zhang Sipan, Liu Zhihong. Extracellular vesicles: a new type of intercellular messenger [J]. Journal of Nephrology and Dialysis Kidney Transplantation, 2017.). Studies on extracellular vesicles have shown that they play an important role in a variety of physiological and pathological processes, such as inflammatory immune response, cell survival and apoptosis, angiogenesis, thrombosis, autophagy, etc. (Wang Jing, Chen Jianying. New progress in extracellular vesicle research [J]. Chinese Journal of Tissue Engineering Research, 2017.). In addition, extracellular vesicles are also considered to be one of the effective biomarkers for liquid biopsy, which can be used for non-invasive early diagnosis of diseases and efficacy monitoring (Yang Limin, Li Jinghong. Extracellular vesicle analysis method [J]. Biomedical Engineering Research, 2024, 43(1): 1-8. DOI: 10.19529 / j.cnki.1672-6278.2024.01.01.). With the deepening of research, the potential application of EVs in disease diagnosis, treatment and biomedical research has received widespread attention. The development of extracellular vesicle imaging technology is crucial to understanding its role in physiological and pathological processes.

[0007] Although framework nucleic acids have shown great application potential in many fields, they still face some challenges, such as structural stability, biocompatibility, and large-scale production. Future research needs to further optimize the design and synthesis methods of framework nucleic acids and improve their stability and functionality in complex biological environments to promote their practical applications in biomedicine, sensing, and computing. As a highly programmable and responsive tool, framework nucleic acids provide new possibilities for extracellular vesicle imaging. Summary of the invention

[0008] The present invention has discovered a DNA framework nucleic acid that is programmable, can bind to a specific nucleotide sequence, and can interact with extracellular vesicles (EVs) to form an EV-framework nucleic acid complex that can monitor EVs in real time. Based on this, the present invention has been completed.

[0009] In the first aspect, the present invention provides a DNA framework nucleic acid, which is composed of 6 sides composed of DNA chains, wherein at least one side is a single-stranded DNA and the rest are double-stranded DNA. When the single-stranded DNA in the DNA framework nucleic acid is complementary to two targeting nucleotides, any one sequence of the above two targeting nucleotides can be captured.

[0010] Furthermore, the double-stranded DNA region in the DNA framework nucleic acid is formed by local complementary pairing of nucleotides shown in SEQ ID NOs. 1 to 6.

[0011] Furthermore, the edge length of the framework nucleic acid is 1 to 50 bp, preferably 24 to 39 bp; preferably 40 to 45 bp; more preferably 27 bp.

[0012] Furthermore, the apparent lateral area of ​​the framework nucleic acid is 180 to 420 nm 2 , preferably 265~360nm 2 .

[0013] Furthermore, the targeting nucleotide refers to a targeting single-stranded nucleotide; preferably a short-stranded nucleotide, such as microRNA, small interfering RNA.

[0014] Furthermore, the microRNA is any microRNA, such as microRNA 23a, microRNA 125b, microRNA 145, microRNA 23 or microRNA 21; preferably microRNA 21 or microRNA 23; more preferably microRNA 21.

[0015] Furthermore, the nucleotide sequences of the two captured targeting nucleotides are microRNAs shown in SEQ ID NO.7 or SEQ ID NO.8.

[0016] In a specific embodiment of the present invention, the DNA chain carries one or more fluorescent groups when constituting a fluorescent probe, and the fluorescent group is located at any position of the DNA chain; preferably, the fluorescent group is located at a position where the spatial distance between the donor and the acceptor molecules is 6 to 12 nm and no fluorescence quenching will occur between the donor and acceptor fluorophores themselves.

[0017] Furthermore, the carried fluorescent group is a conjugated fluorescent group, such as CFP-YFP; CFP-dsRED; BFP-GFP; GFP-dsRED; YFP-dsRED; Cy3-Cy5; Alexa488-Alexa555; Alexa488-Cy3; FITC-Rhodamine (TRITC); YFP-TRITC; YFP-Cy; preferably Cy3 and Cy5.

[0018] Furthermore, the donor is an electron-accepting fluorophore, such as Cy5.

[0019] Furthermore, the acceptor is an electron-donating fluorophore, such as Cy3.

[0020] Furthermore, when the carried fluorescent groups are Cy3 and Cy5, it is preferred that the first strand of the DNA framework nucleic acid is labeled with Cy3, and the first strand of the second strand is labeled with Cy5.

[0021] Furthermore, the DNA sequences of strand 1 and strand 2 are shown as SEQ ID NOs. 1 and 2.

[0022] Furthermore, the targeted nucleotide refers to a targeted single-stranded nucleotide; the nucleotide is derived from the environment and extracellular vesicles.

[0023] In the second aspect, the present invention provides a DNA framework nucleic acid (EV-framework nucleic acid) complex of an extracellular vesicle, wherein the complex is composed of a single-stranded frame nucleic acid complementary to one or more nucleotides inside the EV, and the frame nucleic acid can capture any one of the two nucleotides inside the EV; the DNA double-stranded region in the DNA framework nucleic acid is formed by local complementary pairing of the nucleotides shown in SEQ ID NO.1 to 6; the nucleotides captured inside the EV have a nucleotide sequence such as microRNA shown in SEQ ID NO.7 or SEQ ID NO.8.

[0024] Furthermore, when the DNA chain constitutes a fluorescent probe, it carries a fluorescent group, and the fluorescent group is located at any position of the DNA chain; preferably, the spatial distance between the fluorescent group donor and acceptor molecules is 6 to 12 nm, and the position between the donor, acceptor and the fluorophore itself will not cause fluorescence quenching.

[0025] Furthermore, the carried fluorescent group is a conjugated fluorescent group, such as CFP-YFP; CFP-dsRED; BFP-GFP; GFP-dsRED; YFP-dsRED; Cy3-Cy5; Alexa488-Alexa555; Alexa488-Cy3; FITC-Rhodamine (TRITC); YFP-TRITC; YFP-Cy; preferably Cy3 and Cy5.

[0026] Furthermore, the donor is an electron-accepting fluorophore, such as Cy5.

[0027] Furthermore, the acceptor is an electron-donating fluorophore, such as Cy3.

[0028] Furthermore, when the carried fluorescent groups are Cy3 and Cy5, it is preferred that the No. 1 strand of the DNA framework nucleic acid is labeled with Cy3, and the No. 2 strand is labeled with Cy5.

[0029] Furthermore, the DNA sequences of strand 1 and strand 2 are shown as SEQ ID NOs. 1 and 2.

[0030] Furthermore, the extracellular vesicles are derived from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and / or bone marrow mesenchymal stem cells.

[0031] In a third aspect, the present invention provides a use of the DNA framework nucleic acid as described in the first aspect as a probe, wherein the probe is used to detect a targeted nucleotide.

[0032] Furthermore, the targeting nucleotide refers to a targeting single-stranded nucleotide; preferably a short-stranded nucleotide, such as microRNA, small interfering RNA.

[0033] Furthermore, the microRNA is any microRNA, such as microRNA 23a, microRNA 125b, microRNA 145, microRNA 23 or microRNA 21; preferably microRNA 21 or microRNA 23; more preferably microRNA 21.

[0034] In a fourth aspect, the present invention provides a use of a DNA framework nucleic acid as described in the first aspect as a targeted nucleotide molecule imaging agent, wherein when a single strand of the framework nucleic acid is complementary to a targeted nucleotide, the fluorescence of the framework nucleic acid itself may undergo specific spectral changes after the fluorescent group is excited by exogenous excitation light.

[0035] Furthermore, the targeted nucleotide molecule imaging agent is used for extracellular vesicle imaging, cell imaging and / or free nucleotide imaging in body fluids.

[0036] Furthermore, when the fluorescent groups are selected as Cy3 and Cy5, after being excited by the excitation light, the framework nucleic acid itself emits red fluorescence; the extracellular vesicles labeled with the framework nucleic acid emit yellow fluorescence.

[0037] Furthermore, the excitation light wavelength is 440-720nm, preferably 488-525nm; preferably 550-560nm; preferably 570-580nm; preferably 660-700nm.

[0038] Furthermore, the extracellular vesicles are derived from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and / or bone marrow mesenchymal stem cells.

[0039] Furthermore, the extracellular vesicle imaging is in vivo imaging and / or ex vivo imaging.

[0040] Beneficial Effects

[0041] This application constructs a dynamic DNA self-assembly system using DNA framework nucleic acids that can form FRET fluorescent pairs as building blocks to achieve changes in fluorescence signals. Due to the excellent biocompatibility and intelligent properties of DNA framework nucleic acids, such as high signal-to-noise ratio, they have demonstrated a powerful ability to monitor the biological processes of extracellular vesicles. DNA framework nucleic acids are directly loaded into EVs through electroporation through transient pores to achieve instant and efficient labeling of EVs without the need for cumbersome pretreatment, chemical modification, and genetic engineering, which can maximize the preservation of the native properties and functions of EVs. Based on EVs labeled with DNA framework nucleic acids, long-term non-invasive whole-body high-resolution mode tracking of EVs in vivo is achieved, and the dynamic biodistribution of EVs is revealed in a real-time and in situ quantitative manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Framework nucleic acid construction diagram.

[0043] Figure 2 Framework nucleic acids capture targeted nucleotides inside EVs.

[0044] Figure 3 Fluorescence spectra of the framework nucleic acid when not incubated with microRNA21.

[0045] Figure 4 Fluorescence spectroscopic spectrum of the incubation of framework nucleic acid and microRNA21, at which the molar ratio of microRNA21 to framework nucleic acid is 1.

[0046] Figure 5 Fluorescence spectroscopic spectrum of the incubation of framework nucleic acid and microRNA21, at which the molar ratio of microRNA21 to framework nucleic acid is 2.

[0047] Figure 6 Fluorescence spectroscopic spectrum of the incubation of framework nucleic acid and microRNA21, at which the molar ratio of microRNA21 to framework nucleic acid is 3.

[0048] Figure 7 Spectral statistics of framework nucleic acid incubated with different ratios of microRNA21.

[0049] Figure 8 Fluorescence spectra of the framework nucleic acid when not incubated with microRNA23.

[0050] Fig. 9 Fluorescence spectroscopic spectrum of the incubation of framework nucleic acid and microRNA23, at which the molar ratio of microRNA23 to framework nucleic acid is 1.

[0051] Fig.10 Fluorescence spectroscopic diagram when framework nucleic acid is incubated with microRNA 23, at which time the molar ratio of microRNA 23 to framework nucleic acid is 2.

[0052] Fig.11 Fluorescence spectroscopic diagram when framework nucleic acid is incubated with microRNA 23, at which time the molar ratio of microRNA 23 to framework nucleic acid is 3.

[0053] Fig.12 Fluorescence spectroscopic diagram when framework nucleic acid is incubated with microRNA 23, at which time the molar ratio of microRNA 23 to framework nucleic acid is 4.

[0054] Fig.13 Fluorescence spectroscopic diagram when framework nucleic acid is incubated with microRNA 23, at which time the molar ratio of microRNA 23 to framework nucleic acid is 5.

[0055] Fig.14 Spectral statistics of framework nucleic acid incubated with different ratios of microRNA23. DETAILED DESCRIPTION

[0056] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0057] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0058] Explanation of terms

[0059] Extracellular vesicles (EV / EVs): EVs are a heterogeneous group of membrane structures secreted by cells and wrapped in a lipid bilayer, containing tiny particles secreted by cells. EV vesicles vary in biological origin and size: exosomes are smaller vesicles with a diameter of about (50-150nm), formed in endosomes, which are organelles responsible for endocytosis and decomposition of substances inside the cell; microvesicles are larger vesicles with a diameter between 50-500nm and 1000nm, which are formed by the shedding of the cell membrane and can mature and separate through protrusions on the cell membrane; apoptotic bodies are larger vesicles formed during cell death, with a diameter greater than 1000nm, and when cells enter the process of apoptosis (programmed cell death), they release these vesicles. Extracellular vesicles are cell-derived nanosized vesicles that mediate intercellular communication by transporting bioactive molecules and therefore play important roles in various physiological and pathological conditions.

[0060] microRNA (miR): is a small, non-coding RNA molecule with a length of about 18 to 25 nucleotides. It binds to the target mRNA in a completely or incompletely complementary manner, resulting in target mRNA degradation or translation inhibition. It plays an important role in the post-transcriptional regulation of eukaryotic gene expression and participates in physiological processes such as individual growth and development. In recent years, studies have found that abnormal expression of microRNA may play an important role in the occurrence and development of tumors (such as proliferation, differentiation, and apoptosis).

[0061] miR-21: It is one of the earliest miRNA molecules discovered and confirmed in mammals. Currently, miR-21 has been found in various tissues and cells in 31 species. Like other miRNAs, miR-21 is encoded by the miR-21 gene, which is transcribed by RNA polymerase II in the cell nucleus to produce the initial transcript pri-miR-21, and then modified in two orderly steps to form mature miR-21.

[0062] miR-23: The miR-23 family plays an important role in cell differentiation, apoptosis, tumorigenesis, and drug action. The miR-23 family includes miR-23a, miR-23b, and miR-23c subtypes. miR-23 is involved in all stages of cell development, including cell proliferation, apoptosis, and mutation. miR-23a is a microRNA that has been widely studied in a variety of cancers. It plays a complex and diverse role in the occurrence, development, and treatment of tumors. miR-23a exhibits different functions in different types of cancer.

[0063] Flow NanoAnalyzer: It covers the blind spot of particle size detection below 200nm of traditional flow cytometer, including the characterization of natural biological nanoparticles such as nanoparticles and subcellular structures, bacteria, viruses, and extracellular vesicles (exosomes), opening a window to the nano world for flow detection technology. Through high-resolution, high-selectivity, and high-throughput detection of the particle size and distribution, particle concentration, and biochemical properties of single nanoparticles (7-1000nm), the nanoflow detector provides a powerful characterization tool for life science and biomedical research and the development of nanotechnology.

[0064] FRET (Fluorescence Resonance Energy Transfer): is a physical phenomenon involving energy transfer between a donor fluorophore and an acceptor fluorophore. When the donor fluorophore is in an excited state, it transfers energy to a neighboring acceptor fluorophore through non-radiative dipole-dipole interactions, causing the acceptor fluorophore to emit fluorescence while the fluorescence of the donor fluorophore is weakened or eliminated. The effectiveness of FRET depends on the distance between the donor and the acceptor, and is usually most effective in the range of 1 to 10 nanometers.

[0065] Mesenchymal stem cells (MSC): an important member of the stem cell family, belonging to adult stem cells. They originate from the mesoderm and ectoderm in the early stages of development. MSC was first discovered in Friedenstein's bone marrow in 1968, and has attracted increasing attention due to its multi-directional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation and self-replication. For example, under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc., and they still have multi-directional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology.

[0066] Example 1 Synthesis of DNA framework nucleic acid probe

[0067] A. Test Methods

[0068] (1) providing six DNA framework nucleic acid single strands (2uL) targeting specific microRNAs, including a single strand with a fluorescent group, dissolved in 1XTM buffer and mixed;

[0069] (2) After PCR reaction (95°C for 10 min), rapid cooling;

[0070] (3) DNA framework nucleic acid gel running verification: The gel was stained with gel red, and the DNA framework nucleic acid synthesis effect was imaged and analyzed using a gel imager.

[0071] B. DNA sequence

[0072] Six single strands of DNA framework nucleic acid targeting specific microRNA, including a single strand with a fluorescent group, when the fluorescent group is Cy3 and Cy5, the single strand DNA sequence is shown in Table 1.

[0073] Table 1. DNA framework nucleic acid fluorescent group single-stranded DNA sequence

[0074]

[0075] C. Test Results

[0076] like Figure 1 As shown in A, S1-S6 are constructed framework nucleic acid DNA sequences, and fluorescent groups are attached to the edges of the framework nucleic acids. The framework nucleic acids have a tracing function, and at this time the framework nucleic acids are in a "relaxed" state.

[0077] Example 2 DNA framework nucleic acid capture microRNA

[0078] A. Experimental Methods

[0079] The framework nucleic acid was prepared by selecting the sequence of Example 1. The DNA framework nucleic acid was labeled with the FRET fluorophore pair Cy3 / Cy5 as the donor and acceptor.

[0080] (1) The amount of microRNA added is in multiples with respect to the amount of framework nucleic acid;

[0081] (2) Add (1) to the buffer solution to prepare 120 μL of 1X PBS buffer system;

[0082] (3) Mix the system in (2) and place on a shaker incubated away from light.

[0083] The captured microRNAs are shown in Table 3, and the method is capable of capturing any one of the two microRNAs.

[0084] Table 2. RNA sequences of captured microRNAs

[0085]

[0086] B. Test Results

[0087] like Figure 1 As shown, after the framework nucleic acid captures the targeting nucleotide, the framework nucleic acid is in an "extended" state and its surface area increases.

[0088] Example 3 DNA framework nucleic acid enters extracellular vesicles derived from mesenchymal stem cells

[0089] DNA framework nucleic acid can be introduced into mesenchymal stem cell-derived extracellular vesicles by incubation at 37 degrees overnight / electroporation. The specific method is as follows:

[0090] A. Obtaining extracellular vesicles from mesenchymal stem cells

[0091] (1) Mesenchymal stem cells are cultured in a medium containing extracellular vesicles removed;

[0092] (2) performing continuous centrifugation to remove cells, apoptotic bodies, and cell debris, respectively;

[0093] (3) harvesting extracellular vesicles by centrifugation;

[0094] (4) The precipitate is resuspended and ultra-concentrated to remove contaminating proteins, thereby obtaining extracellular vesicles derived from mesenchymal stem cells.

[0095] B. Framework nucleic acid incubated at 37 degrees overnight into mesenchymal stem cell-derived extracellular vesicles

[0096] The synthesized DNA framework nucleic acid can be incubated overnight at 37 degrees and enter the mesenchymal stem cell-derived extracellular vesicles.

[0097] C. DNA framework nucleic acid electroporation into mesenchymal stem cell-derived extracellular vesicles

[0098] (1) The synthesized framework nucleic acid is concentrated using an ultrafiltration tube as needed to obtain a concentration of 15 uM;

[0099] (2) Precooling the electric rotating cup;

[0100] (3) Prepare electroporation solution (82 μL Nucloefector TM Solution+18μL Supplement), add 20μL of DNA framework nucleic acid and 2μL of extracellular vesicles with a concentration of 25μg / μL into the electroporation solution, and mix the system thoroughly;

[0101] (4) Transfer the liquid to an electric rotating cup;

[0102] (5) Idle running;

[0103] (6) Electroporation;

[0104] (7) Cooling after electroporation;

[0105] (8) transferring the liquid in the electroporation cup to an ultrafiltration tube, and ultrafiltration is performed to remove the DNA framework nucleic acid that has not entered the extracellular vesicles;

[0106] (9) collecting the product in the ultrafiltration tube, flushing the ultrafiltration tube, transferring the flushing solution to the product collection tube, and obtaining the DNA framework nucleic acid that is electrotransferred into the mesenchymal stem cell-derived extracellular vesicles.

[0107] D. Test results

[0108] like Figure 2 As shown, the framework nucleic acid can also capture the nucleic acid inside EV. The nanoflow cytometry results showed that the unlabeled extracellular vesicles had no fluorescent signal, while the framework nucleic acid labeled extracellular vesicles had fluorescent signals and were clearly grouped.

[0109] Example 4 Framework nucleic acid targeted nucleotide detection

[0110] The framework nucleic acid prepared in Example 1 and the target nucleotide captured in Example 2 were used for the test. Targeted detection was performed on microRNA 21 and microRNA 23 respectively, and the grouping was set as shown in Table 3:

[0111] Table 3. Targeted detection of microRNA 21 and microRNA 23

[0112]

[0113]

[0114] like Figure 3 As shown, when the framework nucleic acid is not incubated, its fluorescence intensity is measured. When excited at 520 nm, the framework nucleic acid has emission peaks at 560 nm and 670 nm.

[0115] like Figure 4 As shown, the microRNA21 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA21 to the framework nucleic acid is 1. The fluorescence image of microRNA-21 after binding with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after binding that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0116] like Figure 5As shown, the microRNA21 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA21 to the framework nucleic acid is 2. The fluorescence image collected after microRNA-21 is combined with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after combination that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0117] like Figure 6 As shown, the microRNA21 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA21 to the framework nucleic acid is 3. The fluorescence image of microRNA-21 after binding with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after binding that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0118] like Figure 7 As shown, the framework nucleic acid fluorescence images of microRNA-21 collected when the framework nucleic acid of microRNA-21 is 1, 2, and 3 are compared. The ratio of framework nucleic acid fluorescence emission Cy3 / Cy5 corresponds to the ratio of microRNA-21 / frame nucleic acid, and a bar graph is drawn, as shown in Figure 7 As shown, the Cy3 / Cy5 value increases with the increase of the microRNA-21 framework nucleic acid ratio. The results of this real-time fluorescence data acquisition and microRNA-21 framework nucleic acid value analysis show that the framework nucleic acid detector can quantify microRNA21.

[0119] like Figure 8 As shown, when the framework nucleic acid is not incubated, its fluorescence intensity is measured. The framework nucleic acid is excited at 520 nm and has emission peaks at 560 nm and 670 nm.

[0120] like Fig. 9 As shown, the microRNA23 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA23 to the framework nucleic acid is 1. The fluorescence image collected after microRNA-23 is combined with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after combination that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0121] like Fig.10As shown, the microRNA 23 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA 23 to the framework nucleic acid is 2. The fluorescence image collected after microRNA-23 is combined with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after the combination that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0122] like Fig.11 As shown, the microRNA 23 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA 23 to the framework nucleic acid is 3. The fluorescence image of microRNA-23 after binding with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after binding that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0123] like Fig.12 As shown, the microRNA 23 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA 23 to the framework nucleic acid is 4. The fluorescence image collected after microRNA-23 is combined with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after combination that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0124] like Fig.13 As shown, the microRNA 23 reagent and the framework nucleic acid are mixed, and the amount ratio of microRNA 23 to the framework nucleic acid is 5. The fluorescence image collected after microRNA-23 is combined with the framework nucleic acid is collected, and the fluorescence intensity of Cy3 and Cy5 is calculated. It can be seen from the fluorescence spectrum image after combination that the Cy3 value increases with the addition of microRNA. The Cy5 value decreases with the addition of microRNA.

[0125] like Fig.14 As shown, the framework nucleic acid fluorescence images of microRNA-23 collected when the microRNA-23 framework nucleic acid is 1, 2, 3, 4, and 5 are compared. The ratio of framework nucleic acid fluorescence emission Cy3 / Cy5 corresponds to the microRNA-23 framework nucleic acid ratio, and a bar graph is drawn. The Cy3 / Cy5 value increases with the increase of the microRNA-23 framework nucleic acid ratio. The results of this real-time fluorescence data collection and microRNA-23 framework nucleic acid value analysis show that the framework nucleic acid detector can quantify microRNA23.

Claims

1. A DNA framework nucleic acid, the DNA framework nucleic acid is composed of 6 sides composed of DNA chains, wherein: At least one side is a single-stranded DNA, and the rest are double-stranded DNA. The double-stranded DNA region in the DNA framework nucleic acid is formed by local complementary pairing of nucleotides shown in SEQ ID NO.1 to 6; when the single-stranded DNA in the DNA framework nucleic acid is complementary to two targeting nucleotides, any one sequence of the above two targeting nucleotides can be captured; the nucleotide sequences of the two captured targeting nucleotides are microRNAs shown in SEQ ID NO.7 or SEQ ID NO.

8.

2. The DNA framework nucleic acid according to claim 1, wherein the framework nucleic acid edge length is 27 bp, 39 bp and / or 45 bp; The apparent lateral area of ​​the framework nucleic acid is selected from 180 to 420 nm 2 .

3. The DNA framework nucleic acid according to claim 1, wherein the targeting nucleotide refers to a targeting single-stranded nucleotide; the single-stranded nucleotide is a short-chain nucleotide, such as microRNA, small interfering RNA; wherein, The microRNA is any microRNA, such as microRNA 23a, microRNA 125b, microRNA 145, microRNA 23 or microRNA 21; preferably microRNA 21 or microRNA 23; more preferably microRNA 21.

4. The targeted nucleotide according to claim 3, wherein the nucleotide is derived from the environment and extracellular vesicles; and the extracellular vesicles are derived from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and / or bone marrow mesenchymal stem cells.

5. The DNA framework nucleic acid as described in claim 1, wherein the DNA chain carries one or more fluorescent groups when constituting a fluorescent probe, and the fluorescent groups are located at any position of the DNA chain; preferably, the fluorescent groups are located at a position where the spatial distance between the donor and the acceptor molecules is 6 to 12 nm and where no fluorescence quenching will occur between the donor and acceptor fluorophores themselves.

6. The DNA chain as claimed in claim 5 carries one or more fluorescent groups when constituting a fluorescent probe, wherein the carried fluorescent groups are conjugated fluorescent groups, such as CFP-YFP; CFP-dsRED; BFP-GFP; GFP-dsRED; YFP-dsRED; Cy3-Cy5; Alexa488-Alexa555; Alexa488-Cy3; FITC-Rhodamine (TRITC); YFP-TRITC; YFP-Cy.

7. The method of claim 6 wherein the fluorescent group carried is Cy3 or Cy5, wherein preferably, the first strand of the DNA framework nucleic acid is labeled with Cy3, and the first strand of the second strand is labeled with Cy5; wherein the DNA sequences of the first strand and the second strand are as shown in SEQ ID NOs. 1 and 2; wherein when the fluorescent groups are Cy3 or Cy5, after being excited by an excitation light, the framework nucleic acid itself is red fluorescent; and the extracellular vesicles labeled by the framework nucleic acid are yellow fluorescent; and wherein the wavelength of the excitation light is 440-720 nm.

8. A DNA framework nucleic acid (EV-framework nucleic acid) complex of an extracellular vesicle, wherein the complex is composed of a single strand of the framework nucleic acid as described in claim 1 and complementary to one or more nucleotides inside the EV, and the framework nucleic acid can capture any one of the two nucleotides inside the EV; the DNA double-stranded region in the DNA framework nucleic acid is formed by local complementary pairing of the nucleotides shown in SEQ ID NO.1 to 6; the nucleotides inside the captured EV have a nucleotide sequence such as microRNA shown in SEQ ID NO.7 or SEQ ID NO.

8.

9. Use of the DNA framework nucleic acid as claimed in claim 1 as a probe for detecting a targeted nucleotide.

10. A use of the DNA framework nucleic acid as claimed in claim 1 as a targeted nucleotide molecular imaging agent, wherein when the single strand of the framework nucleic acid is complementary to the targeted nucleotide, the fluorescence of the framework nucleic acid itself can undergo specific spectral changes after the fluorescent group is excited by exogenous excitation light; the targeted nucleotide molecular imaging agent is used for extracellular vesicle imaging, cell imaging and / or free nucleotide imaging in body fluids.

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