A photoactivated DNA tetrahedron confined catalytic hairpin assembly nanodevice and its preparation method and application

Abstract

The present invention discloses a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice and its preparation method and application, which belongs to the field of biosensor technology. The nanodevice includes a DNA tetrahedron, a hairpin probe pair H1 and H2 for miRNA-21 determination, and a hairpin probe pair H3 and H4 for miRNA-155 determination; wherein, the four vertices of the DNA tetrahedron each have an arm chain, and the H1, H2, H3 and H4 are respectively connected to the four arm chains through base complementary pairing. The present invention introduces a light-sensitive group into the stem of the hairpins H1 and H3 for light regulation of their recognition ability of the target. It is found through experimental verification that the nanodevice prepared by the present invention can be used for miRNA imaging in living cells, providing an important tool for related biomedical research.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice and a preparation method and application thereof. Background Art

[0002] Enzyme-free nucleic acid amplification technology provides an effective platform for imaging low-abundance miRNA in living cells. Among them, catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) are common enzyme-free nucleic acid amplification technologies, both of which achieve signal amplification through chain displacement. However, these HCR circuits require the consumption of intracellular target RNA for signal amplification, which may interfere with the normal function of living cells or organs. CHA is composed of two partially complementary DNA hairpins and a trigger chain (target miRNA), and relies on the process of triggering the two DNA hairpins to form a hybrid duplex by the trigger chain (target miRNA), thereby releasing the target miRNA. The formation of this duplex does not consume but re-releases the target miRNA to repeatedly trigger the next reaction, thereby realizing the circulation of the target miRNA, so CHA is widely used in the field of analysis. Although CHA technology is sensitive, there are still some defects. For example, DNA hairpin nanodevices cannot freely cross the plasma membrane into living cells, and usually require transfection reagents or nanomaterials to enter cells; secondly, the kinetics of CHA depends on the diffusion rate and concentration of free DNA hairpins; thirdly, individual DNA hairpin nanodevices have poor biological stability and are easily degraded by nucleases. Therefore, there is an urgent need to develop a CHA system with good cell permeability and stability, and to improve performance such as kinetics and accuracy, so as to provide a better biological platform for imaging low-abundance miRNAs in living cells. Summary of the invention

[0003] The purpose of the present invention is to provide a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice and its preparation method and application to solve the problems existing in the above-mentioned prior art. The nanodevice can be used for miRNA imaging analysis in living cells, providing an important tool for miRNA-related biomedical research.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice, the nanodevice comprises a DNA tetrahedron, a hairpin probe pair H1 and H2 for miRNA-21 determination, and a hairpin probe pair H3 and H4 for miRNA-155 determination; wherein the four vertices of the DNA tetrahedron respectively have an arm chain, and the H1, H2, H3 and H4 are respectively connected to the four arm chains through base complementary pairing.

[0006] Preferably, the DNA tetrahedron is self-assembled from four oligonucleotide chains T1, T2, T3 and T4, and the sequences of the four oligonucleotide chains T1, T2, T3 and T4 are shown in SEQ ID NOs: 1-4, respectively.

[0007] Preferably, the nucleotide sequences of H1, H2, H3 and H4 are shown in SEQ ID NOs: 5-8, respectively.

[0008] Preferably, the 5' ends of H2 and H4 are modified with a quenching group, the middle part is modified with a fluorescent group, and a photocleavable group is embedded between the 8th and 9th bases at the 5' ends of H1 and H3.

[0009] Preferably, the quenching group includes BHQ1 and BHQ2, the fluorescent group includes FAM and Cy3, and the photocleavable group includes PC-Linker.

[0010] The present invention also provides a method for preparing the light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice, comprising the following steps:

[0011] The four oligonucleotide chains T1, T2, T3 and T4 were annealed and self-assembled in TE buffer to form a DNA tetrahedron;

[0012] The hairpin probe pairs H1 and H2 used for the miRNA-21 assay and the hairpin probe pairs H3 and H4 used for the miRNA-155 assay were quenched in TE buffer to form hairpin nanodevices;

[0013] The DNA tetrahedron and the hairpin nanodevice are subjected to hybridization reaction to obtain the light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice.

[0014] Preferably, the annealing conditions are: 95°C for 5 min, and naturally cooled to room temperature;

[0015] The quenching conditions are: 95°C for 5 min, then quickly transferred to an ice bath for 5 min;

[0016] The hybridization conditions are: hybridization reaction in a 37° C. water bath for 4 hours.

[0017] The present invention also provides the use of the light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice in preparing a product for monitoring the expression level of miRNA in living cells.

[0018] Preferably, the miRNAs include miRNA-21 and miRNA-155.

[0019] Preferably, the nucleotide sequence of the miRNA-21 is shown as SEQ ID NO:9, and the nucleotide sequence of the miRNA-155 is shown as SEQ ID NO:10.

[0020] The present invention discloses the following technical effects:

[0021] (1) The DNA tetrahedron provided by the present invention is widely used to monitor low-abundance biomolecules in cells due to its advantages such as simple sequence design, rapid assembly, and good cell penetrability.

[0022] (2) The present invention assembles two pairs of DNA hairpin nanodevices onto a DNA tetrahedron structure, restricts the spatial position of the DNA hairpin nanodevices, increases their local concentration, and thereby improves the kinetic efficiency of the CHA reaction; and utilizes the advantages of the DNA nanostructure to improve the cell permeability and stability of the DNA hairpin nanodevices.

[0023] (3) The present invention can realize simultaneous imaging of multiple miRNAs by assembling two pairs of DNA hairpin probes onto a DNA tetrahedron structure, which is of great significance for studying the function of miRNA and related biomedical applications.

[0024] (4) The present invention introduces a photosensitive group (PC-Linker) into the hairpin stem for controllable identification and imaging of intracellular miRNAs, thereby reducing interference responses at non-specific locations and non-specific times during the transportation of the nanodevice and improving the accuracy of the analysis.

[0025] (5) The present invention constructs a light-activated DNA tetrahedron confined catalytic hairpin self-assembly nanodevice, which is used for controllable fluorescence imaging of two miRNAs in living cells and can also be used to monitor the expression levels of miRNAs in different cells. Therefore, the constructed nanodevice has important value in the diagnosis of miRNA-related diseases and biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1is the characterization of TDN nanodevice; wherein, A is the agarose gel electrophoresis characterization of TDN nanodevice: lane 1 is single-stranded T1, lane 2 is a mixture of T1+T2 DNA chains, lane 3 is a mixture of T1+T2+T3 DNA chains, lane 4 is a mixture of T1+T2+T3+T4 DNA chains, lane 5 is a mixture of T1+T2+T3+T4+H1 DNA chains, lane 6 is a mixture of T1+T2+T3+T4+H1+H2 DNA chains, lane 7 is a mixture of T1+T2+T3+T4+H1+H2+H3DNA chains, and lane 8 is a mixture of T1+T2+T3+T4+H1+H2+H3+H4 DNA chains; B is a schematic diagram of the structure of TDN nanodevice;

[0028] Figure 2 The fluorescence emission spectra of the TDN nanodevice in response to miRNA-21 (50nmol / L) at different illumination times (A) and the corresponding fluorescence ratio diagram (B);

[0029] Figure 3 The fluorescence emission spectra of the TDN nanodevice in response to miRNA-155 (50 nmol / L) at different illumination times (A) and the corresponding fluorescence ratio diagram (B);

[0030] Figure 4 Gel electrophoresis analysis of CHA responses triggered by targets miRNA-21 (A) and miRNA-155 (B);

[0031] Figure 5 Fluorescence emission spectra of TDN nanodevice detecting miRNA-21 (50 nmol / L) (A) and miRNA-155 (50 nmol / L) (B) under different experimental conditions;

[0032] Figure 6 Fluorescence detection of single miRNA for TDN nanodevices; 1. Add miRNA-21 and use UV irradiation; 2. No target added, use UV irradiation; 3. Add miRNA-21, no UV irradiation; 4. No target added, no UV irradiation; 5. Add miRNA-155, use UV irradiation; 6. Add miRNA-155, no UV irradiation;

[0033] Figure 7 The fluorescence emission spectra (A) and standard curve (B) of TDN nanodevice for detecting different concentrations of miRNA-21;

[0034] Figure 8 The fluorescence emission spectra (A) and standard curve (B) of TDN nanodevice for detecting different concentrations of miRNA-155;

[0035] Fig. 9 Results of the selective investigation of TDN nanodevices on target miRNAs; A: miRNA-21; B: miRNA-155;

[0036] Fig.10 This is an activity analysis of MCF-7 cells treated with PBS, TDN nanodevices, and with or without UV irradiation; the concentration of TDN nanodevices is 200nmol / L;

[0037] Fig.11 This is the confocal imaging of the light-activated response of the TDN nanodevice in MCF-7 cells; "+" indicates irradiation with ultraviolet laser, and "-" indicates no ultraviolet light irradiation;

[0038] Fig.12 Laser confocal imaging of TDN nanodevices in 7702, HeLa, and MCF-7 cells;

[0039] Fig.13 Schematic diagram for TDN nanodevice construction (A) and imaging of miRNAs in living cells (B). DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Compared with traditional inorganic nanomaterials, DNA nanomaterials have the advantages of simple assembly, good stability, programmable addressable functions, adjustable nanosize, excellent cell uptake ability, etc., and have been widely used in the fields of biosensors, drug delivery and disease diagnosis. Among them, DNA tetrahedrons are widely used to monitor low-abundance biomolecules in cells due to their advantages such as simple sequence design, fast assembly and good cell penetration. Based on this, the present invention has developed a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice for imaging miRNAs in living cells. The specific principle (see Fig.13 ) is: assemble two pairs of DNA hairpin nanodevices onto a DNA tetrahedron structure, restrict the spatial position of the DNA hairpin nanodevices, increase their local concentration and thus improve the kinetic efficiency of the CHA reaction; and utilize the advantages of the DNA nanostructure to improve the cell penetration and stability of the DNA hairpin nanodevices. Light is adjustable in time, space, dose and wavelength, and has become a powerful tool that can regulate biological functions in time and space. Therefore, a photosensitive group (PC-Linker) is introduced into the hairpin stem for controllable identification and imaging of intracellular miRNAs, reducing the interference response at non-specific locations and non-specific times during the transport of the nanodevice, and improving the accuracy of the analysis. When the TDN nanodevice is activated by irradiating the photosensitive group with ultraviolet light, the target miRNA will trigger the CHA amplification process, achieving controllable analysis. Light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevices provide an important tool for miRNA-related biomedical research.

[0046] The oligonucleotide sequences used in the present invention are shown in Table 1 below.

[0047] Table 1 Oligonucleotide sequences

[0048]

[0049] In the above table, hairpin probes H2 and H4 are modified with quenching groups BHQ1 and BHQ2 at the 5' end, as well as quenching the corresponding fluorescent groups FAM and Cy3. A photocleavable group (PC-Linker) is embedded between the 8th and 9th bases at the 5' end of hairpin probes H1 and H3 to light-regulate their recognition ability of the target.

[0050] All sequences in the above table were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and purified by HPLC.

[0051] Example 1 Synthesis and Characterization of DNA Nanodevices

[0052] 1. Synthesis of photoactivated DNA tetrahedron confined catalytic hairpin assembly nanodevices

[0053] DNA tetrahedron is composed of oligonucleotide chains (T1, T2, T3, T4) mixed in TE buffer solution (20mmol / LTris, 150mmol / L NaCl, 5mmol / L MgCl2, pH 7.5) in equal proportions to make the concentration of each oligonucleotide 4μmol / L. The oligonucleotide chains are annealed. Annealing process: 95℃5min, naturally cool to room temperature. The obtained product is placed in a 4℃ refrigerator for subsequent use. At the same time, hairpins H1, H2, H3 and H4 are mixed in TE buffer solution with a final concentration of 4μmol / L. The hairpins are then quenched, quenching process: 95℃5min, quickly transferred to an ice bath for 5min. Then placed in a 4℃ refrigerator for subsequent use.

[0054] The prepared DNA tetrahedron and four quenched hairpin nanodevices were mixed in equal volume ratios and then hybridized in a 37°C water bath for 4 hours to obtain activated DNA tetrahedron confined catalytic hairpin assembly nanodevices (abbreviated as: TDN nanodevices).

[0055] 2. Agarose gel electrophoresis (AGE) detection of TDN nanodevices

[0056] The characterization of TDN nanodevices is performed as follows:

[0057] (1) Prepare 8 samples: oligonucleotide chain T1, a mixture of oligonucleotide chains T1+T2, a mixture of oligonucleotide chains T1+T2+T3, a mixture of oligonucleotide chains T1+T2+T3+T4, a mixture of oligonucleotide chains T1+T2+T3+T4+H1, a mixture of oligonucleotide chains T1+T2+T3+T4+H1+H2, a mixture of oligonucleotide chains T1+T2+T3+T4+H1+H2+H3, and a mixture of oligonucleotide chains T1+T2+T3+T4+H1+H2+H3. The concentration of each sample is 500 nmol / L.

[0058] (2) Add SYBR Green I (100×) dye to the above samples and react at 37°C for 20 min, then add 6× loading buffer and mix well. Take 10 μL of each of the above treated samples and add them to the agarose gel wells. The electrophoresis process uses 3% agarose gel and runs for 60 min in 0.5×TBE buffer solution at a working voltage of 100 V. Finally, the bands are observed and photographed using the Omega16ic gel imaging system.

[0059] like Figure 1 As shown in A in the figure, it is an agarose gel electrophoresis image. The results show that as the nucleic acid chains hybridize with each other, the molecular weight gradually increases, the migration rate of the sample gradually slows down, and lane 8 produces a band with a larger molecular weight, indicating the successful synthesis of the TDN nanodevice.

[0060] 3. AFM image scanning of TDN nanodevice

[0061] TDN nanodevices (2 μL, 20 nM) were added to the freshly cut mica sheets and kept adsorbed for 2-3 min, followed by the addition of 1×TAE-Mg 2+ Buffer (400 μL) was added and the sample was scanned on an atomic force microscope. The AFM experimental parameters were: the specific mode was Tapping Mode, the experimental environment was Tapping Mode in Air, and the nanodevice model was OTESPA.

[0062] like Figure 1 As shown in B, it is an AFM image of the nanodevice analyzed using an atomic force microscope. The figure shows the structural information of the TDN nanodevice, and also indicates that the DNA nanodevice was successfully synthesized.

[0063] Example 2 In vitro experimental verification of the feasibility of DNA nanodevices

[0064] 1. Study on the photoregulation performance of TDN nanodevices in buffer solution

[0065] The constructed TDN nanodevice introduced photocleavage groups as regulatory elements, and therefore the photoregulatory performance of the system was investigated.

[0066] The TDN nanodevice responded to miRNA-21 (50 nmol / L) and miRNA-155 (50 nmol / L) under different illumination times. Figure 2 and Figure 3As shown in the figure, the fluorescence emission spectrum and fluorescence ratio diagram are obtained. The results show that as the illumination time increases, the fluorescence of FAM and Cy3 at 518nm and 563nm gradually increases, indicating that the recognition and response of the TDN probe to the target is light-dependent, and also that the system is light-regulatable. The corresponding fluorescence ratio diagrams all show that the fluorescence of the system tends to be stable after 240s of illumination, so 240s (4min) is selected as the optimal illumination time for the system.

[0067] 2. Light control feasibility study

[0068] The feasibility of light-activated miRNA-catalyzed hairpin assembly was analyzed by gel electrophoresis. The steps are as follows:

[0069] First, five samples were prepared, namely: hairpin H1, hairpin H2, a mixture of hairpin H1+H2, a mixture of hairpin H1+H2+miRNA-21, and a mixture of hairpin H1+H2+miRNA-21, and subjected to ultraviolet irradiation for 4 minutes. The samples were reacted in a 37°C water bath for 3 hours, with H1 and H2 concentrations of 1 μmol / L and miRNA-21 concentration of 100 nmol / L (miRNA-155 group was operated in the same way). After the reaction, the treated samples were analyzed by electrophoresis using 4.5% agarose gel, and the operation was the same as the electrophoresis experimental steps of Example 1. The feasibility of the experimental principle was verified by fluorescence spectroscopy. The constructed TDN nanodevice was used to measure miRNA-21 (50 nmol / L) and miRNA-155 (50 nmol / L) in a buffer solution.

[0070] like Figure 4 As shown, it is an agarose gel electrophoresis image. These results prove that the light-activated CHA amplification designed in this experiment is feasible. Figure 4 Figure A verifies that miRNA-21 triggers the catalytic assembly reaction between H1 / H2. Lane 1 is hairpin H1, lane 2 is hairpin H2, lane 3 is a mixture of hairpin H1+H2, lane 4 is a mixture of hairpin H1+H2+miRNA-21, and lane 5 is a mixture of hairpin H1+H2+miRNA-21 irradiated with ultraviolet light for 4 minutes. At the same time, it can be found that no new bands are generated in lane 3 without the addition of target miRNA-21 and lane 4 without ultraviolet light irradiation, while lane 5 with the addition of target miRNA-21 and irradiation with ultraviolet light for 4 minutes produces a bright new band, indicating that a CHA reaction has occurred, which once again shows that the H1 / H2 designed by the present invention is dependent on light activation to undergo a CHA reaction. Figure 4 Middle B verifies that miRNA-21 triggers the catalytic assembly reaction between H1 / H2, and the results are the same as those of miRNA-21.

[0071] like Figure 5 As shown, it is a fluorescence emission spectrum diagram, and the results show that: before illumination, the sticky ends of hairpins H1 and H3 are closed, unable to recognize the target and open the signal hairpins H2 and H4, so it can be seen that the fluorescence signals of the orange curve and the blue curve are low. After illumination, when there is no target, the CHA reaction between the hairpin pairs cannot occur, resulting in the red curve The fluorescence signal is also very low. Only after illumination and in the presence of a target, the CHA reaction can occur between the hairpin pairs, and the system produces a strong fluorescence signal (purple curve). It shows that the TDN nanodevice can be activated by light and can achieve the detection of two miRNAs.

[0072] 3. Fluorescence detection of single miRNA

[0073] In order to investigate the detection performance of TDN nanodevices for single miRNA, TDN nanodevices were used for photoactivated controllable detection of miRNA-21 and miRNA-155 under the optimal UV irradiation time conditions.

[0074] like Figure 6 As shown, it is a fluorescence emission spectrum diagram. It can be seen from the figure that when the ultraviolet light is irradiated for 4min, in the absence of the target, the TDN nano device is in an inert state, and the fluorescence of FAM and Cy3 is weak (curve 2). When only miRNA-21 is added to the system, only the fluorescence of FAM is enhanced, and the fluorescence of Cy3 does not change significantly (curve 1). Similarly, when only miRNA-155 is added to the system, the fluorescence of Cy3 is significantly enhanced, and the fluorescence of FAM remains unchanged (curve 5). This shows that the TDN nano device can specifically bind to miRNA, and the constructed TDN nano device can be used for single miRNA determination. In addition, in the absence of ultraviolet light irradiation, whether the target miRNA is added or not, the TDN nano device is in an inert state, and the fluorescence of FAM and Cy3 is weak (curves 3, 4 and 6). This result once again shows that the TDN nano device constructed by the present invention is light-adjustable.

[0075] 4. Two miRNA fluorescence detection

[0076] In order to explore the sensitivity of this method, this experiment used fluorescence spectroscopy to measure the target miRNA. First, a series of target miRNA-21 and miRNA-155 solutions with different concentrations were prepared for use. Then, the reaction mixture containing TE buffer solution, TDN nanodevices (200nmol / L), and different concentrations (0, 0.01, 0.1, 0.5, 1, 5, 10, 20, 25, 40, 50, 100nmol / L) of miRNA-21 / miRNA-155 was irradiated with 365nm ultraviolet light for 4min, and then placed in a 37℃ water bath for incubation for 20min. After the reaction, fluorescence spectroscopy was measured. Three groups were measured in parallel.

[0077] like Figure 7 and Figure 8 As shown in FIG. 1 , the fluorescence emission spectra and standard curves of the TDN nanodevice for detecting different concentrations of miRNA-21 or miRNA-155 are shown. As the concentration of the target miRNA increases, the fluorescence intensity of FAM and Cy3 gradually increases (see FIG. Figure 7 Middle A and Figure 8 This is because the higher the target concentration, the more H1-H2 and H3-H4 duplex structures the TDN probe forms, and the more corresponding dye fluorescence is restored. In this experiment, the concentrations of miRNA-21 and miRNA-155 were linearly regressed using the fluorescence intensity values ​​at 518nm and 563nm, respectively. The results showed that when the concentrations of miRNA-21 and miRNA-155 were in the range of 0 to 20nmol / L, the ratio of dye fluorescence intensity showed a good linear relationship with the target concentration (see Figure 7 Middle B, Figure 8 (middle B).

[0078] The resulting linear equations are I FAM =0.0817C+1.0205(R 2 =0.9977), I Cy3 =0.0347C+1.0284(R 2 =0.9930), where I is the fluorescence intensity ratio of the corresponding dyes, and C is the concentration of the target miRNA (mol / L). According to the 3σ (where σ represents the standard deviation S0 / slope S of multiple blank samples) rule, the detection limits of miRNA-21 and miRNA-155 were 6.2 pmol / L and 2.8 pmol / L, respectively.

[0079] 5. Specificity investigation

[0080] In order to investigate the selectivity of the constructed TDN nanodevice in response to miRNA-21 and miRNA-155, the TDN nanodevice was used to perform fluorescence detection analysis of target miRNAs (miRNA-21 and miRNA-155) and other non-target miRNAs (miRNA-26a, miRNA-141 and miRNA-233) under the optimized optimal UV illumination experimental conditions, where the concentration of all miRNAs was 50nmol / L.

[0081] The results are as follows Fig. 9 As shown in the figure, it can be seen that when only the target miRNA-21 and miRNA-155 are present, the fluorescence intensity of FAM and Cy3 dyes in the system is significantly enhanced; when other non-target miRNAs are present, the fluorescence intensity of FAM and Cy3 dyes in the system is the same as that of the blank group and does not change significantly. This shows that the constructed TDN nanodevice is highly specific, and the target miRNA-21 and miRNA-155 can only bind to specific H1 and H3, and then open H2 and H4 to achieve the response of the corresponding dye fluorescence signal, while other non-target miRNAs cannot bind to H1 or H3, and cannot continue to open H2 and H4, so the corresponding dye fluorescence cannot be restored.

[0082] Example 3 Cell experiment verifies in vivo miRNA imaging of DNA nanodevices

[0083] 1. Cytotoxicity assay

[0084] In this experiment, the MTT method was used to investigate the toxicity of TDN nanodevices to cells. First, a suspension of human breast cancer epithelial cells MCF-7 was inoculated into a 96-well plate and incubated in a CO2 incubator for 24 hours to allow the cells to fully adhere to the wall. Then, PBS was added to the control group, and TDN nanodevices (200nmol / L) were added to the experimental group. Each group was repeated 3 times and incubated for another 24 hours. The illumination group was then irradiated with a 365nm ultraviolet lamp for 10 minutes and incubated for another 2 hours. Subsequently, MTT solution (0.5mg / mL) was added to each well and incubated for another 4 hours. When the formazan crystals were completely formed, dimethyl sulfoxide (DMSO) was added, and the well plate was shaken to completely dissolve the formazan crystals. The absorbance at 570nm was measured using an enzyme reader, and the absorbance at 630nm was used as a reference. The group with PBS added and no ultraviolet illumination was used as the complete control group. The formula for calculating cell viability is as follows:

[0085] Cell survival rate (%) = (OD treated / OD control )×100%, in the above formula, OD treated For the experimental group, OD control As the control group.

[0086] like Fig.10As shown, the constructed TDN nanodevice has low cytotoxicity and good biocompatibility, and is expected to be used for controllable imaging of photoactivated miRNA in living cells.

[0087] 2. Fluorescence imaging of intracellular miRNA

[0088] The MCF-7 cell suspension was inoculated into a 35mm confocal dish at the bottom and cultured for 24 hours to reach a density of 80% for later use. The fully adhered cells were washed 3 times with PBS, and DMEM medium containing 200nmol / L TDN nanodevices was added to continue incubation for 6 hours under light-proof conditions. Washed 3 times with PBS, the TDN nanodevices that did not enter the cells were removed, and irradiated with a 365nm ultraviolet lamp for 10 minutes. Fresh culture medium was added and incubated for 1.5 hours in the dark. Finally, DAPI was added for nuclear staining for 30 minutes, and fluorescence imaging was performed using an LSM710 laser confocal microscope. Imaging detection conditions: the excitation wavelength of the DAPI channel is 405nm, and the collection range is: 430-490nm; the excitation wavelength of the FAM channel is 488nm, and the collection range is: 490-540nm; the excitation wavelength of the Cy3 channel is 543nm, and the collection range is: 550-600nm.

[0089] like Fig.11 As shown, the results show that in the cell group irradiated with UV light for 10 min, strong fluorescence signals can be observed in the FAM and Cy3 channels, while in the cell group not irradiated with UV light, only weak fluorescence signals can be observed. This shows that the TDN nanodevice constructed by the present invention is still photoactivatable in living cells and can realize controllable imaging analysis of target miRNAs in living cells.

[0090] 3. Fluorescence imaging analysis of miRNAs expression in different cells

[0091] The expression levels of miRNA-21 and miRNA-155 in different cells vary. In order to further study the analytical ability of the constructed TDN nanodevice on the expression levels of miRNAs in different cells, it was used for fluorescence imaging analysis of miRNA in normal human liver cells (7702), human cervical cancer cells (HeLa) and human breast cancer epithelial cells (MCF-7).

[0092] like Fig.12As shown, the results show that almost no fluorescence signal is observed in normal hepatocyte 7702 cells, indicating that the expression levels of miRNA-21 and miRNA-155 in normal hepatocytes are low. Obvious green (FAM) and red (Cy3) fluorescence signals can be observed in HeLa and MCF-7 cells, and the fluorescence signal observed in MCF-7 cells is stronger than that in HeLa cells, indicating that the expression levels of miRNA-21 and miRNA-155 in MCF-7 are higher than those in HeLa cells. The above cell fluorescence imaging shows that the constructed TDN nanodevice can be used for imaging analysis of miRNA in different cells.

[0093] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice, characterized in that: The nanodevice comprises a DNA tetrahedron, a hairpin probe pair H1 and H2 for measuring miRNA-21, and a hairpin probe pair H3 and H4 for measuring miRNA-155; wherein each of the four vertices of the DNA tetrahedron has an arm chain, and H1, H2, H3 and H4 are connected to the four arm chains respectively through complementary base pairing; The DNA tetrahedron is self-assembled from four oligonucleotide chains T1, T2, T3 and T4, and the sequences of the four oligonucleotide chains T1, T2, T3 and T4 are shown in SEQ ID NOs: 1-4, respectively; The nucleotide sequences of H1, H2, H3 and H4 are shown in SEQ ID NOs: 5-8, respectively; The 5' ends of the H2 and the H4 are modified with a quenching group, the middle part is modified with a fluorescent group, and the photo-cleavable group is embedded between the 8th base and the 9th base of the 5' ends of the H1 and the H3.

2. The light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice according to claim 1, characterized in that: The quenching group includes BHQ1 and BHQ2, the fluorescent group includes FAM and Cy3, and the photocleavage group includes PC-Linker.

3. A method for preparing a light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: The four oligonucleotide chains T1, T2, T3 and T4 were annealed and self-assembled in TE buffer to form a DNA tetrahedron; The hairpin probe pairs H1 and H2 used for the miRNA-21 assay and the hairpin probe pairs H3 and H4 used for the miRNA-155 assay were quenched in TE buffer to form hairpin nanodevices; The DNA tetrahedron and the hairpin nanodevice are subjected to hybridization reaction to obtain the light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice.

4. The preparation method according to claim 3, characterized in that: The annealing conditions are: 95°C for 5 min, and then naturally cooled to room temperature; The quenching conditions are: 95°C for 5 min, then quickly transferred to an ice bath for 5 min; The hybridization conditions are as follows: hybridization reaction in a 37°C water bath for 4 h.

5. Use of the light-activated DNA tetrahedron confined catalytic hairpin assembly nanodevice as described in any one of claims 1 to 2 in the preparation of a product for monitoring the expression level of miRNAs in living cells.

6. The use according to claim 5, characterized in that The miRNAs include miRNA-21 and miRNA-155.

7. The use according to claim 6, characterized in that The nucleotide sequence of the miRNA-21 is shown in SEQ ID NO:9, and the nucleotide sequence of the miRNA-155 is shown in SEQ ID NO:10.

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