DNA (deoxyribonucleic acid) branch prepared from DNA sequence, multi-labeled DNA branch, preparation method and application

Abstract

The invention provides a DNA branch prepared from a DNA sequence, a multi-labeled DNA branch, a preparation method and application, and belongs to the technical field of multi-fluorescence imaging. According to the invention, long-chain DNA is designed and synthesized, ssDNA and DNA branches are successfully prepared by using an enzyme digestion means, then a probe G43-3 'FAM (43nt) is designed according to the sequence of the ssDNA of the trunk length, and the probe G43-3' FAM (43nt) is hybridized with the trunk to construct the DNA branches. According to the invention, the reverse primer is subjected to Spacer modification and transformation, and then different DNA tags are arranged at the tail end of the DNA branch to be connected with corresponding connecting DNA, so that the multi-labeled DNA branch is successfully prepared. A quenching segment BHQ3 is designed according to a fluorescent probe G43-3 'FAM, a fluorophore is quenched after exposure, then exposure of a next fluorophore is carried out, and color chromatography and multiple detection are realized on streptavidin magnetic beads.

Description

Technical Field

[0001] The present invention relates to the technical field of multiple fluorescence imaging, and in particular to a DNA tree branch prepared by using a DNA sequence, a multi-labeled DNA tree branch, a preparation method and an application thereof. Background Art

[0002] In order to label and locate biomolecules in the spatial environment of complex cells and tissues, fluorescent labeling and imaging methods (such as fluorescence microscopy) have always been an important task in life science research. It has three advantages: (1) relatively high spatial resolution; (2) use of fluorescent groups with different spectra for multi-target labeling and detection; (3) widely available specific labeling reagents (such as proteins and nucleic acids).

[0003] As a universal spatial omics tool, the unique advantages of fluorescence microscopy are also its greatest fatal weaknesses. First, the relatively wide emission spectrum of fluorescent groups limits multiple imaging; second, the resolution of traditional optical microscopes is limited by light diffraction, which prevents the microscope from resolving individual molecules in cells and tissues; third, optical composites require adhesives to label molecules, which creates problems in specificity and labeling efficiency.

[0004] To circumvent the limitations of light diffraction in fluorescence microscopy, researchers have invented super-resolution techniques. Super-resolution techniques allow researchers to image with unprecedented precision below the classical diffraction limit of light. In the process of implementing single-molecule localization, molecules are "switched" between a non-fluorescent dark state (or off state) and a fluorescent bright state (or on state) so that their positions can be selected and detected with sub-diffraction accuracy to obtain super-resolution images. A study reported a sub-diffraction imaging method that generates point accumulation through energy collisions, called the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT). In this imaging method, the state switching between on and off is achieved by the dissociation and binding of a fluorescent group-labeled oligonucleotide (called the "imaging" strand, less than 10 nt in length) to its corresponding complementary strand (called the "docking" strand), and the transient binding of the imaging strand to the docking strand produces a noticeable flash. In summary, DNA-PAINT is an easy-to-implement super-resolution microscopy method that does not require fluorescent groups with specific photophysical properties, but its high background signal and slow imaging speed limit its widespread use, both of which can be attributed to the presence of a large number of unbound fluorescent groups in the solution.

[0005] Exchange-PAINT is committed to achieving super-resolution imaging. To achieve this goal, the concentration of imaging chains in Exchange-PAINT needs to be increased to ensure that most targets can be identified during image acquisition. However, the increase in the concentration of imaging chains in the solution will also increase the background fluorescence, which will have a certain impact on image resolution and quality.

[0006] The limitation of spectral overlap can be overcome by utilizing the programmability of DNA nanostructures, but in practice, these multiplex detection methods are difficult to be widely adopted due to the complexity of these DNA designs. Makino et al. reported a color-changing fluorescent barcode (CCFB) method based on strand displacement reaction. This method relies on simple linear nucleic acid design to achieve multiple labeling. The CCFB complex is composed of a fluorescent group-modified acyclic D-threoninol nucleic acids (D-aTNA) chain and its reverse complementary template chain. Each TNA fluorescent chain is modified with a fluorescent group and a quenching group at both ends, and then through reverse complementation, a linear double-stranded DNA can be formed. D-aTNA can form a stable double-stranded structure with the complementary D-aTNA chain, but cannot stably hybridize with the complementary DNA or RNA chain, thus greatly reducing the background interference caused by intracellular nucleic acids. In the initial state of the complex, only the terminal fluorescent group of the DNA chain emits fluorescence, and other fluorescent groups inside are quenched due to their proximity to the quenching group of the adjacent oligonucleotide. Subsequently, the quencher strand hybridizes to the complementary region, and the terminal oligonucleotide is then removed by a strand displacement reaction, quenching the fluorescence of the first fluorescent group and allowing the second fluorescent group to fluoresce. As the strand displacement occurs repeatedly, the other fluorescent groups change color. The emission color of each spot is observed by fluorescence microscopy. Multiple target molecules are identified by decoding the color sequence from the acquired image. However, the DNA-based method requires the design of many probes with different sequences. As the complexity of DNA barcodes increases, the inevitable question is: Is the encoding strategy prone to errors? The structural complexity of existing DNA assembly platforms may still pose certain challenges to the design of highly multiplexed orthogonal systems. In practice, the feasibility of simultaneous signal amplification of multiple targets remains to be proven.

[0007] At present, the molecular pathological information characteristics of tumors have been studied in depth through single-cell analysis, RNA sequencing and other genomic methods, but these technologies cannot provide spatial distribution information between cell populations and lack spatial visualization. Traditional in situ analysis platforms represented by immunohistochemical staining and fluorescent in situ hybridization staining can achieve spatial visualization, but have defects in multiplicity and sensitivity. Due to the spectral limitations of the fluorescent groups of the markers, the fluorescence color development of traditional in situ analysis platforms is usually less than 5 (generally red, orange and green fluorescent markers are used), which is not conducive to more multiple fluorescence color development and multi-target analysis. Therefore, solving the fluorescence color development problem of different DNA targets is crucial to achieve multiple fluorescence imaging. Summary of the invention

[0008] The purpose of the present invention is to provide a DNA tree branch prepared by using DNA sequence, a multi-labeled DNA tree branch, a preparation method and an application thereof, which can realize multiple fluorescence imaging.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a DNA sequence for preparing DNA branches, wherein the DNA sequence contains a biotin-labeled F / R primer binding sequence and multiple Nt.BstNBI specific nicking enzyme cleavage sites, wherein the DNA sequence is shown in SEQ ID NO.1; the F / R primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0011] The present invention also provides a DNA branch prepared from the above DNA sequence.

[0012] The present invention also provides a method for preparing DNA branches, comprising the following steps:

[0013] The DNA sequence is used as a template, and the F / R primers with biotin label are used to amplify the DNA sequence to obtain dsDNA. The dsDNA is cleaved by Nt.BstNBI specific nicking enzyme to obtain biotin-labeled single-stranded product ssDNA; the ssDNA is hybridized with the fluorescent probe G43-3'FAM to obtain DNA branches.

[0014] Preferably, the sequence of the fluorescent probe G43-3'FAM is shown as SEQ ID NO.4.

[0015] The present invention also provides a DNA branch or an application of a DNA branch obtained by a method for preparing a DNA branch in multiple fluorescence imaging, wherein the application method is:

[0016] The fluorescent group on the DNA branch is hybridized with the quenching fragment BHQ3 to achieve fluorescence quenching after exposure, and then the next fluorescent group is exposed;

[0017] The quenching fragment BHQ3 is the quenching fragment of the fluorescent probe G43-3'FAM, and its sequence is shown in SEQ ID NO.20.

[0018] The present invention also provides a multi-labeled DNA tree branch, which is prepared from the above-mentioned DNA sequence.

[0019] The present invention also provides a method for preparing a multi-labeled DNA branch, comprising the following steps:

[0020] (1) Modifying the reverse primer in the F / R primer with Spacer18 to obtain SMA-R-Spacer;

[0021] (2) PCR amplifying the DNA sequence using the F / R primer modified by Spacer18 to obtain a PCR product with sticky ends; hybridizing the biotin-modified DNA tag and its corresponding connecting DNA with the PCR product to obtain a multi-stage hybridization product; the multi-stage hybridization product is digested with Nt.BstNBI specific nicking enzyme and then hybridized with the fluorescent probe G43-3'FAM to obtain a multi-labeled DNA branch.

[0022] Preferably, the sequence of the SMA-R-Spacer is shown in SEQ ID NO.7; the biotin-modified DNA tag is one or more of bio-seq1 to bio-seq6, and its sequence is shown in SEQ ID NO.8 to SEQ ID NO.13; its corresponding linker DNA is linker-bio1 to linker-bio6, and its sequence is shown in SEQ ID NO.14 to SEQ ID NO.19.

[0023] The present invention also provides an application of a multi-labeled DNA branch or a multi-labeled DNA branch obtained by a method for preparing the multi-labeled DNA branch in multiple fluorescence imaging, wherein the application method is:

[0024] The fluorescent groups on the multi-labeled DNA branches are hybridized with the quenching fragment BHQ3 to achieve fluorescence quenching after exposure, and then the next fluorescent group is exposed;

[0025] The quenching fragment BHQ3 is the quenching fragment of the fluorescent probe G43-3'FAM, and its sequence is shown in SEQ ID NO.20.

[0026] The DNA branches and multi-labeled DNA branches of the present invention have the following beneficial effects compared with the prior art:

[0027] (1) The present invention designs and synthesizes long-chain DNA, successfully prepares ssDNA and DNA branches by enzyme cutting, and then designs probe G43-3'FAM (43nt) according to the sequence of the trunk long ssDNA, and hybridizes with the trunk to construct DNA branches. The obtained DNA branches have good enzyme stability and reliability, overcome the interference of non-target substances, show excellent performance in signal stability and selectivity, and provide a solid experimental basis for cell experiments. The biotinylated DNA branches were characterized, and the biotinylated DNA branches were successfully fixed on streptavidin magnetic beads as effective detection targets. In addition, the streptavidin magnetic beads only bind to the biotinylated DNA branches, and do not adsorb to the oligonucleotide probes and DNA chains, avoiding false positive results caused by the mutual adsorption of the oligonucleotide probes and streptavidin magnetic beads. (2) In order to achieve binding with different antibody-DNA complexes, the present invention successfully prepared multi-labeled DNA branches by modifying the reverse primer with Spacer, and then setting different DNA tags at the end of the DNA branches to connect with the corresponding connecting DNA. Under the action of different DNA tags, the linked DNA can specifically hybridize with the DNA tags complementary to the nucleic acid sequence, laying the foundation for multiple labeling. (3) The present invention designs a quenching fragment BHQ3 based on the fluorescent probe G43-3'FAM, and constructs a DNA tree branch "colorimetric" colorimetric method. The fluorescent group is quenched after exposure, and then the next fluorescent group is exposed, realizing "colorimetric" colorimetric and multiple detection on streptavidin magnetic beads. The fluorescence results show that the designed quenching fragment BHQ3 can be stably assembled with the probe G43-3'FAM and undergo energy resonance transfer to quench the fluorescence, realizing dual imaging of the fluorescence signal, overcoming the interference of non-target analytes in the system, and the method has excellent signal stability and selectivity, and has potential application and analysis potential in realizing multiple visualization of target analytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 labor.

[0029] Figure 1 This is a schematic diagram of the formation of biotin-labeled ssDNA in Example 1;

[0030] Figure 2 This is a schematic diagram of the preparation of DNA branches in Example 1;

[0031] Figure 3The preparation and characterization of the DNA trunk and branches in Example 1; A is the agarose gel electrophoresis of the product after PCR amplification of the plasmid (2%, w / w); b is the agarose gel electrophoresis of the DNA trunk (2%, w / w); c is the comparison of the absorbance of the DNA trunk and dsDNA at 260nm, ****p<0.0001; d is the agarose gel electrophoresis of the DNA branches (2%, w / w);

[0032] Figure 4 Characterization of the biotinylated DNA dendrites in Example 2; a is a non-denaturing polyacrylamide gel electrophoresis of streptavidin-CONDNA* binding (8%, w / w); b is an agarose gel electrophoresis of the DNA dendrite-streptavidin complex;

[0033] Figure 5 This is the fluorescence imaging of the biotinylated DNA branches on the streptavidin magnetic beads in Example 2; a is the fluorescence detection image of the unbiotinylated DNA branches; b is the fluorescence detection image of the biotinylated DNA branches;

[0034] Figure 6 The fluorescence detection diagram of the mixed magnetic bead system incubated with the biotinylated DNA branches in Example 2; the streptavidin magnetic beads / (streptavidin magnetic beads + control group magnetic beads) were mixed in the ratio of 0, 20%, 50%, 70%, 90%, and 100% (control group: CholPEG-Biotin) to block the biotin binding sites on the surface of the streptavidin magnetic beads to obtain magnetic beads;

[0035] Figure 7 The normality test and statistical analysis of the fluorescent streptavidin magnetic beads data at different ratios in Example 2 are performed; A is a normality test graph of the 20% group; b is a normality test graph of the 50% group; c is a normality test graph of the 70% group; d is a normality test graph of the 90% group; e is a normality test graph of the 100% group; f is a bar chart analysis graph of the 20%, 50%, 70%, 90%, and 100% groups;

[0036] Figure 8 It is the normality test analysis result in Example 2;

[0037] Fig. 9 Comparison of fluorescence of DNA branches and CONDNA*-G43-3'FAM on the surface of A549 cells in Example 3; a is the fluorescence imaging of A549 cells incubated with PBS (blank group), CONDNA*-G43-3'FAM (69 bp group), and DNA branches (293 bp group), scale bar: 100 μm; b is the statistical fluorescence intensity of the corresponding pictures, *p<0.05, ****p<0.0001;

[0038] Fig.10Comparison of the fluorescence of DNA branches and CONDNA*-G43-3'FAM on the surface of Mahlavu cells in Example 3; a is the fluorescence imaging of Mahlavu cells incubated with PBS (blank group), CONDNA*-G43-3'FAM (69 bp group), and DNA branches (293 bp group), scale bar: 100 μm; b is the statistical fluorescence intensity of the corresponding pictures, ***p<0.001, ****p<0.0001;

[0039] Fig.11 Schematic diagram of the rapid isothermal nucleic acid reaction in Example 4

[0040] Fig.12 is the real-time fluorescence curve caused by the short-chain CONDNA* and DNA trunk at a concentration of 0.5 μM in Example 4; a is the fluorescence imaging diagram of the short-chain CONDNA* and DNA trunk under the gel imager; b is the real-time fluorescence curve diagram of the reaction system of the short-chain CONDNA* and DNA trunk (1, 2, 3 represent the DNA trunk reaction system; 4, 5, 6 represent the short-chain CONDNA* reaction system; 7 represents the PBS control group);

[0041] Fig.13 The system of the rapid isothermal nucleic acid reaction of short-chain CONDNA* and DNA trunks at different concentrations in Example 4; a is the fluorescence signal appearance diagram of the reaction system of short-chain CONDNA* and DNA trunks at different concentrations; b is the statistical fluorescence intensity data in the corresponding tubule system (1, 2, 3 represent the reaction systems of DNA trunks at 0.500μM, 0.050μM, and 0.005μM, respectively; 4, 5, 6 represent the reaction systems of short-chain CONDNA* at 0.500μM, 0.050μM, and 0.005μM, respectively; 7 represents the PBS control system);

[0042] Fig.14 The selective characterization of the antibody-DNA trunk on different tumor cells in Example 4; a is the fluorescence signal appearance diagram of the antibody-DNA trunk after rapid isothermal nucleic acid reaction; b is the data statistics of the system fluorescence intensity of the corresponding tubules (1 represents GPC-1-DNA trunk, 2 represents EpCAM-DNA trunk, 3 represents PANC-1 marked by GPC-1-DNA trunk, 4 represents A549 marked by EpCAM-DNA trunk, 5 represents leukocytes marked by GPC-1-DNA trunk, 6 represents leukocytes marked by EpCAM-DNA trunk, 7 represents Probe-Nt.BstNBI, and 8 represents PBS);

[0043] ****p<0.0001;

[0044] Fig.15Schematic diagram of the combination of the sticky end, DNA tag and connecting DNA in Example 5;

[0045] Fig.16 This is the non-denaturing polyacrylamide gel electrophoresis diagram in Example 5 (12%, w / w);

[0046] Fig.17 It is an agarose gel electrophoresis diagram of the multi-level structure hybridization product in Example 5 (2%, w / w); Lane 1 represents the product after PCR reaction with SMA-R-Spacer primer; Lane 2 represents the product formed by hybridization of linker-bio1 on the basis of Lane 1; Lane 3 represents the tertiary structure product formed after hybridization of bio-seq1 on the basis of Lane 2;

[0047] Fig.18 It is an agarose gel electrophoresis diagram of the hybridization product of the multi-level structure fluorescent probe in Example 5 (2%, w / w); Lane 1 represents the product after PCR reaction with SMA-R-Spacer primer; Lane 2 represents the tertiary structure product formed by hybridization of linker-bio1 and bio-seq1 on the basis of Lane 1; Lane 3 represents the product after digestion by Nt.BstNBI specific nicking enzyme on the basis of Lane 2; Lane 4 represents the hybridization of probe G43-3'FAM on the basis of Lane 3; 3 lanes are run in parallel for each sample;

[0048] Fig.19 It is a fluorescence image of the DNA branches in Example 5 incubated with streptavidin magnetic beads under the action of DNA tags and linked DNA; the blank group represents the incubation of the streptavidin magnetic beads-PBS system with the DNA branches; the experimental group represents the incubation of the streptavidin magnetic beads-bio-seq1 with the DNA branches; the control group 1 represents the incubation of the streptavidin magnetic beads-bio-seq2 with the DNA branches; the control group 2 represents the incubation of the streptavidin magnetic beads-bio-seq3 with the DNA branches; scale bar: 100 μm;

[0049] Fig. 20 The fluorescence display diagram of the streptavidin magnetic beads labeled with different DNA tags in Example 5 after incubation and washing with DNA branches; 20%, 40%, 70%, and 100% respectively represent the proportions of the streptavidin magnetic beads labeled with bio-seq1 in the streptavidin magnetic beads labeled with bio-seq1 and bio-seq2; scale bar: 100 μm;

[0050] Fig.21The normality test and statistical analysis of the data of streptavidin magnetic beads labeled with different DNA tags at different proportions in Example 5; a is a normality test graph of the 20% group; b is a normality test graph of the 40% group; c is a normality test graph of the 70% group; d is a normality test graph of the 100% group; e is a 3D bar graph of the 20%, 40%, 70%, and 100% groups; f is a data graph of the 20%, 40%, 70%, and 100% groups; ***p<0.001, ****p<0.0001;

[0051] Fig. 22 The specific principle of the "color stacking" color development technology in Example 6; a is the principle of hybridization of the target detection object with the DNA branch and quenching of the fluorescence signal after being labeled with different antibodies-DNA; b is the principle of "color stacking" color development labeling;

[0052] Fig.23 The characterization of the quenched fluorescence signal in Example 6; a is the quenching effect of the quenching fragment BHQ3 at different concentrations on the fluorescence intensity of the probe G43-3'FAM (FAM) detected by the microplate reader, the final concentration of FAM is 1.0μM (control), (1) 0.5μM BHQ3; (2) 1.0μM BHQ3; (3) 1.5μM BHQ3, ****p<0.0001; b is a bar graph comparing the signal intensity before and after fluorescence quenching of the gel imaging instrument characterization system; c is a 3D waterfall graph of the fluorescence spectrophotometer characterizing the quenching of FAM fluorescence intensity by the quenching fragment BHQ3 at different concentrations; d is a 3D bar graph of the fluorescence spectrophotometer characterizing the quenching of FAM fluorescence intensity by the quenching fragment BHQ3 at different concentrations;

[0053] Fig.24 Characterization of the fluorescence signal of the DNA branches in Example 6; a is a diagram of the DNA branches and their quenching under ultraviolet light; (b is a comparison of the signal intensity before and after the fluorescence quenching of the DNA branches, ****p<0.0001; (c is an electrophoresis diagram of the DNA branches quenched (2%, w / w, before staining with 3×GelRedTM nucleic acid dye); (d is an electrophoresis diagram of the DNA branches quenched (2%, w / w, after staining with 3×GelRedTM nucleic acid dye)

[0054] Fig.25 This is a characterization diagram of DNA tree fluorescence quenching in Example 6; 50% and 90% represent the proportion of positive events in the mixed magnetic bead system; scale bar: 100 μm;

[0055] Fig.26The fluorescence quenching characterization of the antibody-DNA branches on the surface of A549 lung cancer cells in Example 6; a is a fluorescence quenching diagram of DNA branches on the surface of A549 cells, scale: 100 μm; b is the statistical fluorescence intensity of the corresponding pictures, the blank group means that no DNA branches are added; the fluorescence group means that the DNA branches are combined with EpCAM antibodies through streptavidin; the quenching group means that the quenching fragment BHQ3 is added on the basis of the fluorescence group;

[0056] Fig. 27 This is the “chromatographic” application of the DNA branches on streptavidin magnetic beads in Example 6. DETAILED DESCRIPTION

[0057] The embodiments of the present invention are described in detail below, and the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. If specific techniques or conditions are not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0058] The DNA sequences used in the present invention are shown in Table 1:

[0059]

[0060]

[0061] Example 1

[0062] In Example 1 of the present invention, DNA branches with fluorescent groups were prepared. The specific method is as follows:

[0063] (1) Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize a 293 nt DNA sequence containing an F / R primer binding sequence and multiple Nt.BstNBI specific nicking enzyme cleavage site sequences (as shown in SEQ ID NO.1), and then the target DNA was synthesized and loaded into a plasmid, and introduced into Escherichia coli for amplification and preservation. (2) The plasmid in Escherichia coli was extracted using a plasmid extraction kit (TIANGEN, DP103-02). The extracted plasmid was cloned using primers SAM-F (as shown in SEQ ID NO.2) and biotin-labeled SAM-R * (as shown in SEQ ID NO.3) Amplification was performed according to the PCR system in Table 2 and the PCR reaction procedure in Table 3 to obtain a PCR product. The enzyme digestion results were detected by 2% agarose gel electrophoresis and UV spectrophotometer (see Figure 3 ).Depend on Figure 3a It can be seen that after PCR amplification of the extracted plasmid, its PCR product (dsDNA) is consistent with the PCR product (Positive) of the plasmid constructed by the biological company in terms of content and purity, and its band size is about 293bp.

[0064] Table 2 PCR reaction system

[0065]

[0066]

[0067] Table 3 PCR reaction program

[0068]

[0069] (3) Take the PCR product and prepare 50 μL of the restriction enzyme digestion reaction system in Table 4 in a 0.2 mL PCR reaction tube.

[0070] Table 4 Nt.BstNBI specific nicking enzyme digestion system

[0071] Element volume Final concentration PCR products 44 Nt.BstNBI specific nicking enzyme 1 10units 10×NEBuffer 5 1×

[0072] The above system was mixed, and the enzyme digestion was performed at 55°C for 1 h in a PCR thermal cycler, and the reaction was terminated by inactivation at 80°C for 20 min to obtain a biotin-labeled single-stranded product ssDNA (see Figure 1 ), ssDNA can be used as a DNA trunk to construct DNA branches. 2% agarose gel electrophoresis and UV spectrophotometer are used to detect the enzyme digestion results (see Figure 3 ).

[0073] Nt.BstNBI is a site-specific nicking enzyme that only cleaves one strand of a double-stranded DNA substrate.

[0074] The nicking enzyme creates a nicking site at the 3' end of the 4th base after the recognition sequence. The recognition site is as follows:

[0075] 5'...GAGTCNNNNN...3'(SEQ ID NO.21);

[0076] 3'...CTCAGNNNNN...5' (SEQ ID NO. 22).

[0077] This 293bp DNA sequence contains multiple repeated base sequences and multiple restriction sites. The Nt.BstNBI specific nicking enzyme recognizes the restriction sites on the complementary chain and cuts them to form many short fragments. The Tm values ​​of many short fragments (17.2℃ and 29.7℃) are lower than the reaction temperature (55℃), and they fall off from the main chain to obtain single-stranded product ssDNA.

[0078] Depend on Figure 3 b shows that the bases of the DNA trunk are exposed, and the topological structure is more sparse than that of dsDNA. The resistance encountered during migration increases, and the band is closer to the top. The interaction between the electrons in the DNA molecule is the structural basis of ultraviolet absorption. When deoxyribonucleic acid is completely hydrolyzed to generate deoxyribose, phosphate and nitrogenous bases, purine bases and pyrimidine bases both contain conjugated double bonds and have the property of absorbing ultraviolet light. The maximum absorption wavelength is around 260nm. Figure 3 c It can be seen that the ultraviolet absorption density of the DNA trunk at 260nm is greater than that of dsDNA, and the difference is statistically significant (****p<0.0001). These two results show that the PCR product obtained after enzyme digestion is ssDNA, which can be used as a DNA trunk. (4) Denature the biotin-labeled ssDNA liquid at 95℃ for 2 minutes to avoid intrachain polymerization to form secondary or higher-order structures, and then prepare the following mixed solution in a 0.2mL PCR reaction tube: Take 7μL of the denatured single-stranded product ssDNA and 1μL of the probe G43-3'FAM (10μM, as shown in SEQ ID NO.4) and react at 95℃ for 2min and 40℃ for 30min. Many G43-3'FAM with fluorescent groups hybridize and bind to the trunk to construct DNA branches (see Figure 2 ). 2% agarose gel electrophoresis to detect DNA dendrites (see Figure 3 ).Depend on Figure 3 As shown in d, after the successfully prepared DNA trunk was hybridized with the probe G43-3'FAM, the hybridization band (theoretical molecular weight is 493bp) was higher than that of dsDNA and DNA trunk, and the molecular weight shifted upward, indicating that the probe G43-3'FAM was successfully hybridized with the DNA trunk.

[0079] Example 2

[0080] In Example 2 of the present invention, a biotinylated 69 nt short-chain CONDNA* was designed and synthesized to bind to streptavidin to verify the accuracy and stability of DNA tree labeling. The specific method is as follows:

[0081] (1) Construct a DNA branch and streptavidin hybridization system. Prepare the following mixture in a 0.2 mL PCR reaction tube: 8 μL of DNA branch mixture, 1 μL of streptavidin (about 1.5 μM), and react at room temperature for 1 h. 2% agarose gel electrophoresis was used to verify whether streptavidin and DNA branches were successfully bound. (See Figure 4 ).

[0082] (2) Construction of streptavidin-biotin conjugates and selection of appropriate content

[0083] ① Prepare the following mixture in a 0.2 mL PCR reaction tube:

[0084] Table 5 Construction of streptavidin and CONDNA* conjugates and selection of appropriate content

[0085]

[0086] 8% polyacrylamide gel electrophoresis analysis was performed using 1×TBE buffer and 80V voltage for 40 min. 3×GelRedTM nucleic acid dye was mixed with the sample and then loaded. After the electrophoresis was completed, the gel was taken out and placed under a gel imager for exposure and observation. (See Figure 4 )

[0087] Depend on Figure 4 a It can be seen that when 0.5 μL of short-chain CONDNA* (10 μM) reacts with 4 μL of streptavidin (about 1.5 μM), 3 or 4 CONDNA* bind to streptavidin. This result proves that when the amount of CONDNA* increases, it can fully occupy 3 or 4 subunits of streptavidin. Subsequently, the biotinylated DNA branches are bound to streptavidin and determined by gel electrophoresis. Figure 4 b The results show that the molecular weight of the DNA branches after hybridization with streptavidin is significantly higher than that of the DNA branches, indicating that streptavidin, DNA trunks, and probe G43-3'FAM are successfully hybridized. Unlike direct labeling of DNA with biotin, the biotin on the DNA branches comes from the end-labeled biotin primers and undergoes the high temperature process of PCR. Figure 4 b The results also show that biotinylated primers will not be inactivated or denatured during the PCR reaction, and their products can stably hybridize with streptavidin.

[0088] The biotinylated DNA branches and the non-biotinylated DNA branches were characterized using the Olympus whole slide scanning system SLIDEVIEW VS200. Figure 5 .Depend on Figure 5It can be seen that after the unbiotinylated DNA branches were incubated with streptavidin magnetic beads, no fluorescence signal was observed; however, after the biotinylated DNA branches were incubated with streptavidin magnetic beads, obvious fluorescence signals were observed. This result shows that the stability of the binding between biotinylated DNA branches and streptavidin can be the basis for qualitative analysis and detection. (3) Pretreatment of streptavidin magnetic beads: Take 10 μL of streptavidin magnetic beads from each group in a 1.5 mL centrifuge tube and centrifuge 3 times with 200 μL of buffer (1× PBS buffer prepared with 100 mM sodium chloride buffer), 5 min / time (12,500 rpm) to eliminate the possible influence of the reagents in the storage solution on the subsequent molecular spatial hybridization. Take 20 μL of Chol-PEG-Biotin (10 μM) to block the biotin sites on the surface of streptavidin magnetic beads as control magnetic beads, wash them three times by centrifugation with 200 μL of 100 mM sodium chloride buffer, 5 min / time (12,500 rpm), and mix them in a 1.5 mL centrifuge tube according to the following ratio.

[0089] Table 6 Volume of magnetic beads added in different streptavidin magnetic bead systems

[0090] Grouping / Ratio (μL) 0 20％ 50％ 70％ 90％ 100％ Streptavidin magnetic beads 0 10 25 35 45 50 Control Magnetic Beads 50 40 25 15 5 0

[0091] (4) Covalent coupling of streptavidin magnetic beads and biotinylated DNA branches (protected from light): 100 μL of biotinylated DNA branches were added to the 0, 20%, 50%, 70%, 90%, and 100% streptavidin magnetic bead systems in sequence. The centrifuge tubes were wrapped in tin foil and coupled overnight at 4°C (12-18 h) in a dark place. The coupled streptavidin magnetic beads were washed three times by centrifugation with 200 μL of 100 mM sodium chloride buffer, 5 min / time (12,500 rpm); the streptavidin magnetic beads were resuspended in 20 μL of 100 mM sodium chloride buffer. 10 μL of the streptavidin magnetic beads were dropped onto the adhesive slide, and the microscope cover slip was gently covered from one side to avoid bubbles; the slide was sealed with nail polish, and the sample information was marked.

[0092] (5) Fluorescence detection: The probe G43-3'FAM with FAM fluorescent group was excited with 520 nm excitation light; the fluorescence intensity of streptavidin magnetic beads was detected by Olympus whole slide scanning system SLIDEVIEWVS200 and other methods. The results are shown in Figure 6 .Depend on Figure 6 It can be seen that in the mixed magnetic bead system, the fluorescent streptavidin magnetic beads increase with the increase of the initial added streptavidin magnetic beads. The data of the 20%, 50%, 70%, 90%, and 100% groups were tested for normality and analyzed using QQ graphs. The results are shown in Figure 7 , 8 .Depend on Figure 7It can be seen that the data of the 20%, 50%, 70%, 90%, and 100% groups all have a high degree of coincidence with the straight line, and from Figure 7 It can be observed in f that as the amount of streptavidin magnetic beads added increases, the number of fluorescent streptavidin magnetic beads in the mixed magnetic bead system increases. Figure 8 It can also be seen that: 20%, 50%, 70%, 90%, and 100% are not significant (p>0.05), which means that the data obtained in the 20%, 50%, 70%, 90%, and 100% groups all have normal characteristics, proving that the biotinylated DNA branches have reliable and stable strong anti-interference ability. The present invention successfully prepared and verified the DNA trunk and DNA assembly-DNA branches. The feasibility of biotinylated DNA branches in avidin-biotin action was characterized, and the biotinylated DNA branches were successfully fixed on streptavidin magnetic beads as an effective detection target. The above results prove that streptavidin magnetic beads only bind to biotinylated DNA branches, and do not adsorb to oligonucleotide probes and DNA chains, avoiding false positive results caused by mutual adsorption of oligonucleotide probes and streptavidin magnetic beads. After incubation of biotinylated DNA branches with streptavidin magnetic beads, the fluorescence signal is positively correlated with the ratio of the added ones within a certain range. This result proves the stability of the constructed DNA branches in vitro. These results indicate that DNA branches have good enzyme stability and reliability, overcome the interference of non-target substances, and show superior performance in signal stability and selectivity, providing a solid experimental basis for cell experiments.

[0093] Example 3

[0094] Example 3 of the present invention provides a method for labeling and applying DNA branches at the cellular level. The present invention uses the biotin method to connect antibodies and DNA branches to construct antibody-DNA branches. The antibody part can bind to the marker protein on the cell, and the DNA branches are connected to the probe G43-3'FAM through base complementarity to achieve antibody DNA branch labeling and signal amplification. The specific method includes:

[0095] (1) Fluorescence signal amplification detection of DNA branches on tumor cells: Biotinylated 69nt short-chain CONDNA* was used as a control for parallel comparison. Group setting: blank group (PBS), 69bp group (CONDNA*-G43-3'FAM), 293bp group (DNA branches). Construction of CONDNA*-G43-3'FAM: CONDNA* (69nt, 10μM) and probe G43-3'FAM (10μM) were hybridized and combined at room temperature for 30min in a 1:1 ratio. The DNA branches were constructed using the method of Example 1. The same probe was used for CONDNA*-G43-3'FAM and the amount of probe added for the construction of both was kept consistent. (2) Incubation of tumor cells with antibody EpCAM: 20μL of EpCAM antibody (10μg / mL) was added and incubated with A549 and Mahlavu cells overnight at 4°C. After completion, the antibody was removed and the cells were centrifuged and washed 3 times with PBS buffer, 5min / time (1,000rpm). (3) Take 40 μL of CONDNA*-G43-3'FAM and DNA dendrites and add them to A549 and Mahlavu cells that have been incubated with antibodies. Mix them with a mini vortex and add 10 μL of streptavidin (100 μg / mL, approximately 1.5 μM). Incubate at room temperature for 3 h. Remove the incubation system and wash three times with PBS buffer by centrifugation, 5 min / time (1,000 rpm). (4) Nuclear staining: Add 200 μL of Hoechst33258 (10 μg / mL) solution to each group and incubate at room temperature in the dark for 30 min. After incubation, wash three times with PBS buffer by centrifugation, 5 min / time (1,000 rpm). (5) Seal the slides and observe under VS200. The results are shown in the figure. Fig. 9 , Fig.10 .

[0096] Depend on Fig. 9 , Fig.10 It can be seen that PBS did not observe fluorescence signals on the cell surface, while CONDNA*-G43-3'FAM showed weak fluorescence signals on the cell surface. In contrast, cells co-incubated with DNA branches showed bright green fluorescence in the FITC channel. The comparison results show that DNA branches have the function of signal amplification, which is due to the many sites of repeated hybridization probe G43-3'FAM on the DNA branches, further improving the sensitivity of detecting targets.

[0097] The present invention uses streptavidin as a carrier to couple CONDNA*-G43-3'FAM, DNA branches and antibodies, successfully evaluates the difference in fluorescence signals of antibody-CONDNA* conjugates on different cells, and verifies the ability of the constructed antibody-CONDNA* conjugates to stably image and select specific cells. The assembly-DNA branches successfully achieves sensitive detection of fluorescence imaging at the level of multiple tumor cells, improving the sensitivity of fluorescence detection and imaging. The above results show that DNA branches have a significant ability to amplify signals.

[0098] Example 4

[0099] Example 4 of the present invention uses a detection probe (isothermal MGB-2) to construct a signal amplification detection experiment based on the cleavage characteristics of the Nt.BstNBI specific nicking enzyme and the sequence of the DNA trunk. The specific method is as follows:

[0100] The detection probe (isothermal MGB-2, 17nt) used is different from the probe G43-3'FAM in that one end is modified with a FAM fluorescent group and the other end is modified with an MGB quenching group. The isothermal MGB-2 is shorter in length. When the MGB group is present at one end, the fluorescence signal of the FAM group at the other end is blocked, that is, this bidirectionally modified probe has no fluorescence signal under excitation light. However, once the probe is cleaved by enzymes, the MGB group and the FAM group are freed into the solution and will show green fluorescence under excitation light. As more and more probes are cleaved by enzymes, the fluorescence intensity will gradually increase. Short-chain CONDNA* and DNA trunks can hybridize with this probe. The principle of this rapid isothermal nucleic acid reaction is as follows Fig.11 .

[0101] Depend on Fig.11 It can be seen that at 55°C, the Nt.BstNBI specific nicking enzyme recognizes the hybridized isothermal MGB-2 probe and cuts it, forming two shorter base fragments that fall off the DNA trunk and excite fluorescence. The DNA trunk exposes the probe binding site again, and the probe in the solution binds to the DNA trunk again and continues to be cut to show fluorescence. This reaction is repeated many times, and the fluorescent signal gradually accumulates, achieving signal amplification and target detection.

[0102] (1) Fluorescence signal amplification of DNA trunk-isothermal probe

[0103] The DNA trunk and short-chain CONDNA* (69 nt) were diluted to 0.5, 0.05, and 0.005 μM, respectively, and mixed with an equal amount of the probe isothermal MGB-2 (10 μM). The fluorescence signal amplification after the rapid isothermal nucleic acid reaction was observed at these three concentrations. Each group was repeated 3 times, with PBS as the control.

[0104] (2) Rapid isothermal nucleic acid reaction and gel imaging detection

[0105] Prepare the mixture in 0.2mL eight-link reaction tube according to the following reaction system, mix thoroughly on a mini vortexer, and place on a PCR thermal cycler for rapid isothermal nucleic acid reaction. The reaction conditions are 55℃, 60min; 80℃, 20min.

[0106] Table 7 Rapid isothermal nucleic acid reaction system

[0107]

[0108] After the rapid isothermal nucleic acid reaction, the fluorescence changes of the system were observed under a gel imager, and photos were taken and the fluorescence values ​​were recorded. A bar graph was drawn in GraphPad Prism 9.0 based on the values ​​to analyze whether there was a significant difference in fluorescence intensity between the DNA trunk and the short-chain CONDNA* after binding the same amount of probes. The results are shown in Fig.12 , 13 .

[0109] Depend on Fig.12 It can be seen that the DNA trunk reaction system at a concentration of 0.5 μM was repeated three times (tubes 1, 2, and 3) and all showed very strong fluorescence signals. In contrast, the fluorescence intensity in the short-chain CONDNA* reaction system at a concentration of 0.5 μM (tubes 4, 5, and 6) was weaker. The above results show that the DNA trunk target sequence successfully binds to multiple fluorescent probes and can achieve the amplification of the fluorescence signal.

[0110] Depend on Fig.13 It can be seen that as the concentration of the target sequence decreases, the fluorescence signals of the hybridization systems of short-chain CONDNA* (tubes 4, 5, 6) and DNA trunks (tubes 1, 2, 3) are weakened. Further comparison of the fluorescence changes of the isothermal MGB-2 reaction system of short-chain CONDNA* (tubes 4, 5, 6) and DNA trunks (tubes 1, 2, 3) with the probe under three concentrations shows that the fluorescence signal of the hybridization system of short-chain CONDNA* (tubes 4, 5, 6) and DNA trunks (tubes 1, 2, 3) is weakened. Fig.13 b It was observed that the fluorescence intensity of the DNA trunk system (tubes 1, 2, and 3) was stronger than that of the short-chain CONDNA* system (tubes 4, 5, and 6) under the premise of excluding the background fluorescence interference in the control PBS system (tube 7). These experimental results prove that the number of DNA trunk molecules in the system is the key to determining the number of binding probe molecules, and also prove that the DNA trunk has more binding sites than the short-chain CONDNA*, which can stably bind to the probe in the system.

[0111] (2) Selective labeling of DNA trunks and fluorescence signal amplification detection

[0112] According to the specificity of the cell surface expressed protein binding to the corresponding antibody, the corresponding protein is not expressed on the surface of leukocytes, and even under the action of avidin-biotin, it does not bind to the DNA trunk hybridization system, thereby judging the presentation of the system fluorescence at the cellular level.

[0113] Fix cells: PANC-1, A549, and leukocytes were fixed with 4% cell tissue fixative for 30 min at room temperature, and then washed three times by centrifugation with PBS buffer, 5 min / time (1,000 rpm).

[0114] Cell blocking: 450 μL of 5% BSA blocking agent was mixed with 50 μL of salmon sperm DNA (10 mg / mL) as a blocking agent, and PANC-1, A549, and leukocytes were blocked with shaking at 37°C for 1 hour; 500 μL of 1× PBS buffer was centrifuged and washed 3 times, 5 min / time (1,000 rpm).

[0115] Construction of antibody-DNA trunk conjugates: 20 μL of EpCAM antibody (10 μg / mL) and 2 μL of DNA trunk were mixed with 3 μL of streptavidin (100 μg / mL, approximately 1.5 μM) and incubated at room temperature for 30 min; 20 μL of GPC-1 antibody (2 μg / mL) and 2 μL of DNA trunk were mixed with 3 μL of streptavidin (100 μg / mL, approximately 1.5 μM) and incubated at room temperature for 30 min.

[0116] Incubation of cells with antibody-DNA trunk conjugates: The blocked and constructed antibody-DNA trunk conjugates (i.e., PANC-1 and GPC-1-DNA trunk conjugates, A549 and EpCAM-DNA trunk conjugates, and leukocytes and GPC-1 and EpCAM-DNA trunk conjugates, respectively) were incubated at room temperature for 1 hour. After the incubation, the cells were washed three times by centrifugation with 1× PBS buffer, 5 min / time (1,000 rpm) to remove excess DNA-DNA complexes and antibody-DNA trunk complexes. The cells were resuspended in 42 μL of 1× PBS buffer.

[0117] The two constructed antibody-DNA trunks (GPC-1 antibody-DNA trunks, EpCAM antibody-DNA trunks) were incubated with PANC-1 (tube 3), A549 tumor cells (tube 4), and leukocytes (tubes 5 and 6), respectively, and the probe G43-3'FAM (tube 7) and PBS (tube 8) reaction systems were used as blanks to eliminate background fluorescence interference.

[0118] Rapid isothermal nucleic acid reaction amplification fluorescence signal: PANC-1, A549, and leukocytes labeled with antibody-DNA trunk conjugates were incubated with 2 μL of probe isothermal MGB-2 (10 μM) at room temperature for 30 min, and then 5 μL of 10×NEBuffer and 1 μL of Nt.BstNBI specific nicking enzyme (500 units) were added in turn and incubated at 55°C for 1 h. The fluorescence of the system was observed under ultraviolet light. The results are shown in Fig.14 .Depend on Fig.14 It can be seen that, without incubation with tumor cells, GPC-1 antibody, EpCAM antibody and DNA trunk conjugates observed strong fluorescence signals under rapid isothermal nucleic acid reaction (tubes 1 and 2). Data statistics of the fluorescence intensity of the tumor cell group (tubes 3 and 4) and the control group (tubes 5 and 6) system showed that the fluorescence intensity of PANC-1 (tube 3) and A549 (tube 4) tumor cells was significantly higher than that of leukocytes (tubes 5 and 6), that is, the fluorescence of the leukocyte system that did not express GPC-1 and EpCAM proteins was significantly weaker than that of the corresponding PANC-1 and A549 tumor cell systems, and the difference was statistically significant. The above results show that DNA trunks can selectively target and bind to tumor cells and amplify the fluorescence signal. The fluorescence intensity of tubes 1 and 2 was weaker than that of the reaction system of tubes 3 and 4. Considering that the spatial volume of cells and cell-to-cell adhesion have certain steric interference on the constructed antibody-DNA trunk binding. The present invention combines the DNA trunk hybridization system with the rapid isothermal nucleic acid reaction to successfully verify the signal amplification function of the antibody-DNA trunk hybridization system and the selective labeling of tumor cells. By introducing oligonucleotide probes, the detection of target molecules is converted into the detection of DNA fragments, and the sensitivity of the detection is effectively improved. On the basis of the DNA trunk, the relevant probes can be stably hybridized to construct DNA branches, thereby realizing the amplification of the fluorescent signal. At the same time, in the rapid isothermal nucleic acid detection method, based on the easy availability of nicking endonucleases and the simple requirements for probe sequences, this reaction is expected to become a universal method for detecting proteins and various biological molecules, providing a new means for drug screening, initial clinical diagnosis and biological detection.

[0119] Example 5

[0120] In Example 5 of the present invention, the reverse primer is modified with Spacer18 to form a PCR product with a sticky end to construct a multi-labeled DNA tree branch, which specifically includes the following steps:

[0121] In order to achieve visualization of multiple targets, the reverse primer was modified with Spacer18 to form a PCR product with sticky ends for multiple labeling (see sequence SEQ ID NO.7). Spacer18 is a linker chain composed of 6 consecutive ethylene glycols (CH2CH2O-). Two Spacer18s were modified consecutively in the middle of the primer, and the extension reaction of the DNA polymerase was terminated to form a PCR product with sticky ends. Then, 6 biotin-modified DNA tags and their corresponding connecting DNAs (see sequences SEQ ID NO.8 to SEQ ID NO.19) were designed to hybridize with DNA branches to achieve multiple labeling, see Fig.15 .

[0122] (1) Hybridization of PCR products with sticky ends and DNA tags: ① Thoroughly melt 2×MasterMix in an ice bath, mix well, and centrifuge quickly to collect the solution at the bottom of the tube. ② Prepare 50 μL of reaction system in a 0.2 mL PCR reaction tube:

[0123] Table 8 PCR reaction system of SMA-R-Spacer primers

[0124]

[0125]

[0126] ③ Quickly centrifuge to collect the reaction solution at the bottom of the tube. ④ Perform the following procedures on the PCR thermal cycler:

[0127] Table 9 PCR reaction program of SMA-R-Spacer primer

[0128]

[0129] ⑤ Prepare the following system in a 0.2 mL PCR reaction tube and react at 95°C for 2 min and 56°C for 30 min.

[0130] Table 10 Multi-stage hybridization system of PCR products of SMA-R-Spacer primers

[0131]

[0132] ⑥ First, use 12% polyacrylamide gel to verify the hybridization of linker-bio1 and bio-seq1 (see the results Fig.16 ), electrophoresis conditions: 12% non-denaturing polyacrylamide gel, 1×TBE electrophoresis buffer, 120V constant voltage, electrophoresis for 45min. 2% agarose gel electrophoresis for multiple hybridization: electrophoresis conditions were 1×TAE, 150V, 60min.

[0133] Depend on Fig.16 It can be seen that in the hybridization lane, a band with an obviously upward molecular weight can be observed, and the electrophoresis speed is very slow, indicating that a stable hybrid is formed compared with lanes bio-seq1 and linker-bio1.

[0134] Next, by adding the positive counter ion Na + To promote the hybridization of multi-level structures, the hybridization products of each level of structure were run in parallel in three lanes to eliminate the errors in the electrophoresis process. The product after PCR reaction with SMA-R-Spacer primers (1) was verified by 2% agarose gel electrophoresis, followed by hybridization of the linker-bio1 complex on the basis of the PCR product (2), and then the tertiary structure product after combining with bio-seq1 on the basis of the complex (3). Fig.17 As shown, when linker-bio1 was added, the molecular weight increased and the electrophoresis speed slowed down, that is, the corresponding band of (2) was produced, indicating that DNA reaction assembly occurred in the presence of linker-bio1.

[0135] The addition of linker-bio1 and bio-seq1 produced DNA assembly products with larger molecular weight (3), indicating that linker-bio1 and bio-seq1 successfully hybridized on the basis of SMA-R-Spacer-PCR products. On the other hand, the introduction of Spacer reduced the steric hindrance of the DNA assembly process and provided space for DNA assembly to assemble and connect DNA.

[0136] (2) Construction of multi-labeled DNA branches

[0137] Take 44 μL of the multi-stage hybridization product prepared in Table 10, add 5 μL of 10×NEBuffer buffer and 1 μL of Nt.BstNBI specific nicking enzyme. React at 55°C for 60 minutes, and react at 80°C for 20 minutes to inactivate the enzyme activity. Take 14 μL of the obtained product and hybridize with 0.5 μL of probe G43-3'FAM (50 μM), react at 95°C for 2 minutes, and react at 48°C for 30 minutes. Electrophoresis of 2% agarose gel in 1×TAE buffer at a constant voltage of 150V for 60 minutes, the results are shown in Fig.18 .Depend on Fig.18It can be seen that the molecular weight of lane 1 (the product of the PCR reaction with SMA-R-Spacer primer) shifted significantly after hybridization with linker-bio1 and bio-seq1 (lane 2). After the product of lane 2 was digested with Nt.BstNBI specific nicking enzyme, the electrophoresis band speed slowed down and the molecular weight shifted up (lane 3). The product of lane 3 was hybridized with probe G43-3'FAM, and the electrophoresis speed was very slow and the molecular weight increased (lane 4). The above results show that DNA tags, linker DNA and probe G43-3'FAM were successfully assembled on the basis of sticky-end PCR products, that is, the formation of multi-labeled DNA dendrites.

[0138] (3) Selective detection of multi-labeled DNA branches

[0139] In order to observe the selectivity of DNA branches under the action of ligated DNA and DNA tags (bio-seq1, bio-seq2, bio-seq3), this paper set up experimental group, control group and blank group for verification. Blank group: streptavidin magnetic beads incubated with PBS; experimental group: streptavidin magnetic beads incubated with bio-seq1; control group 1: streptavidin magnetic beads incubated with bio-seq2; control group 2: streptavidin magnetic beads incubated with bio-seq3. ① Take 100μL of streptavidin magnetic beads in each group in a 1.5mL centrifuge tube and wash them three times with 200μL of buffer (10mM sodium chloride buffer prepared with 1× PBS buffer), 5min / time (12,500rpm) to eliminate the possible influence of the reagents in the storage solution on the subsequent molecular spatial hybridization. ②According to the group arrangement, take 20μL of 1×PBS and incubate with streptavidin magnetic beads, 20μL of bio-seq1 (10μM) and incubate with streptavidin magnetic beads, 20μL of bio-seq2 (10μM) and incubate with streptavidin magnetic beads, and 20μL of bio-seq3 (10μM) and incubate with streptavidin magnetic beads, and shake at room temperature for 1h. Then, centrifuge and wash 3 times with 200μL of 100mM sodium chloride buffer, 5min / time (12,500rpm). The above groups were blocked overnight at 4℃ with 1mg / mL salmon sperm DNA prepared with 5% BSA, and centrifuged and washed 3 times with 200μL of 100mM sodium chloride buffer, 5min / time (12,500rpm).

[0140] ③Mix the magnetic beads according to the following ratio:

[0141] Table 11 Table of different proportions of magnetic beads added volume under the action of multiple DNA tags

[0142]

[0143] ④Each group was co-incubated with 150μL of constructed multi-labeled DNA branches and added to a 1.5mL centrifuge tube and mixed; coupled at 4℃ overnight (12-18h); washed 3 times by centrifugation with 200μL of 100mM sodium chloride buffer, 5min / time (12,500rpm), and streptavidin magnetic beads were resuspended in 20μL of 100mM sodium chloride buffer. ⑤Take 10μL of sample suspension and drop it on the adhesion slide, and gently cover the microscope cover glass from one side to avoid bubbles; seal the slide with nail polish, mark the sample information, and observe under VS200. The results are shown in Figures 19 to 21 .

[0144] Depend on Fig.19 It can be seen that in the presence of DNA linker-bio1, the DNA branches can hybridize with the DNA tag bio-seq1, and a strong fluorescence signal is observed on the surface of the streptavidin magnetic beads; while the DNA tags (bio-seq2, bio-seq3) do not hybridize with the DNA linker-bio1, and no fluorescence signal is detected on the surface of the corresponding streptavidin magnetic beads. The above results show that bio-seq1 only hybridizes with linker-bio1, and the selective labeling of DNA branches at the level of streptavidin magnetic beads can be achieved. Next, the selectivity of DNA branches on streptavidin magnetic beads under the action of different DNA tags and linker DNA was further studied. According to Table 11, the bio-seq1 group streptavidin magnetic beads (experimental group) and the bio-seq2 group streptavidin magnetic beads (control group) were mixed in a ratio of 20%, 40%, 70%, and 100%, respectively, and then incubated with the prepared DNA branches and the DNA linker-bio1, respectively, and coupled at 4°C overnight (12-18h). Fig. 20 The results showed that the proportion of magnetic beads showing fluorescence in the 20%, 40%, 70%, and 100% groups was positively correlated with the number of magnetic beads added to the experimental group within a certain range. This indicates that selective labeling of DNA branches on streptavidin magnetic beads can be achieved by connecting DNA linker-bio1 and the corresponding DNA tag bio-seq1. As can be seen from Table 12, the results of the SW test on the data showed that: 20%, 40%, 70%, and 100% did not show significant differences (p>0.05), which means that 20%, 40%, 70%, and 100% all have normal characteristics, and the data can be used for scientific research and analysis. At the same time, a normal QQ probability analysis was performed on the proportion of positive magnetic beads. Fig.21 The points of the 20%, 40%, 70%, and 100% groups are all close to the straight line, indicating that the sample conforms to the normal distribution. Fig.21 It was observed that as the proportion of streptavidin magnetic beads increased, the probability of positive events in the system also increased. Fig.21The ratio of fluorescent magnetic beads increased within a certain range as the ratio of streptavidin magnetic beads added increased. The difference in fluorescence intensity between the 40% and 70% groups was statistically significant (***p<0.001). The difference in fluorescence intensity between the 100% and 70% groups was statistically significant (****p<0.0001). The above results show that the constructed multi-labeled DNA dendrites have stable labeling and selection properties in an in vitro environment and can serve as the basis for quantitative analysis.

[0145] Table 12 Normality test analysis results

[0146]

[0147] The present invention successfully prepared multi-labeled DNA branches by modifying the reverse primer with Spacer. Under the action of different DNA tags, the connected DNA can specifically hybridize with the DNA tags complementary to the nucleic acid sequence. When mixed streptavidin magnetic beads with different ratios are incubated with the DNA branches, it is observed that the magnetic beads showing fluorescence have the same degree of complexation with the added ratio within a certain range. The above results prove that the multi-labeled DNA branches constructed in this chapter can achieve selective characterization on the surface of streptavidin magnetic beads, laying the foundation for the next step of multi-labeling.

[0148] Example 6

[0149] In the process of multiple fluorescence imaging, if multiple fluorescent groups coexist, the non-exposed fluorescent groups may be excited by the excitation light of the similar wavelength band, interfering with the exposed fluorescent groups, causing cross-color and confusion, and limiting the accuracy of multiple color development and exposure. Therefore, if the fluorescent group is quenched after exposure, and then the next fluorescent group is exposed, cross-color and confusion can be effectively eliminated, which will be a feasible way to perform multiple color development and exposure. Example 6 of the present invention establishes a "color stacking" color development technology, and its principle is as follows Fig. 22 As shown. Fig. 22 It can be seen that first, different antibodies are connected to corresponding DNA tags and bind to different antigens. Then, the DNA branches are combined with the corresponding antibodies through the corresponding connecting DNA to achieve fluorescent labeling. For example, after antigen A is labeled with antibody-DNA branches, it shows fluorescence and is photographed and recorded; then the quenching fragment BHQ3 is added to quench the DNA branches. After washing, the second DNA branch is added and other antigens are labeled, and photographed and recorded. Each target is combined with the DNA branch in turn to achieve fluorescent color development and take pictures, which can achieve multiple labeling. Through multiple rounds of color development and quenching, each target in the target detection object takes turns to color and emit light. Finally, the fluorescence superposition of multiple targets is obtained through the software. It is expected to eventually achieve 5-6 color visualization analysis and apply it to multiple fluorescence imaging. The specific method is as follows:

[0150] The corresponding quenching fragment BHQ3 (see sequence SEQ ID NO. 20) was designed based on the probe G43-3'FAM. When the two DNA segments hybridize, the green fluorescent group and the quenching group come close to each other, and resonance energy transfer occurs to quench the fluorescence.

[0151] (1) Detection of the fluorescence intensity changes of G43-3'FAM under the action of different concentrations of the quenching fragment BHQ3 by ELISA

[0152] ① Prepare the following mixed solution according to the following volume:

[0153] Table 13 Reaction system of different concentrations of quenching fragment BHQ3 and probe G43-3'FAM

[0154]

[0155] Note: The initial concentration of the probe G43-3'FAM is 10μM, and the final concentration is 0.1μM; the initial concentration of the quenching fragment BHQ3 is 10μM, and the final concentrations of the system are 0.05, 0.10, and 0.15μM, respectively. ② According to the above reaction system, measure the fluorescence intensity of 6 replicates. ③ Set the excitation wavelength to 480nm, the emission wavelength to 520nm and other parameters on the microplate reader, remove the lid of the 96-well microplate and place it in the microplate reader for testing. ④ After the measurement is completed, save the pop-up data, copy it and draw a bar graph in GraphPad Prism 9.0 based on the numerical value, and analyze whether there is a significant difference between the fluorescence intensity and the initial probe G43-3'FAM. The results are shown in Fig.23 a, b. As shown in Figures 23a and b, the fluorescence intensity of G43-3'FAM decreases with the increase of the concentration of the quenching fragment BHQ3. From Figure b, it can be observed that the fluorescence intensity decreases with the increase of the concentration of the quenching fragment BHQ3. The difference between the FAM+BHQ3(3) group and the Control group is statistically significant (****p<0.0001). (2) Fluorescence signal quenching detection by fluorescence spectrophotometer: ① Prepare reaction systems of different concentrations according to the following volumes:

[0156] Table 14 Reaction system of different concentrations of quenching fragment BHQ3 and probe G43-3'FAM

[0157]

[0158] Note: The initial concentration of the probe G43-3'FAM is 10μM, and the final concentration is 0.1μM; the initial concentration of the quenching fragment BHQ3 is 10μM, and the final concentrations of the system are 0.05, 0.10, and 0.15μM, respectively.

[0159] ② Turn on the machine to preheat the xenon lamp for about 30 minutes, and keep the reaction system away from light for 30 minutes. You can proceed at the same time, set the excitation wavelength to 480nm, the emission wavelength to 520nm and other parameters on the instrument, and establish a detection method. Use a four-sided light-transmissive cuvette filled with deionized water to run a stable baseline, and then detect the fluorescence intensity value of the prepared solution at 520nm in turn (see the same results in 23c). Note: Before taking out the cuvette from the light chamber, click the stop button first to reduce the error caused by external light sources when opening and closing the light chamber. When measuring a new sample, click the reset button to eliminate the interference caused by the previous sample measurement. ③ Save the data and draw the fluorescence intensity change curve in Origin 2018 and Plot2 software (see the results). Fig.23 d). Fig.23 As shown in Figures c and d, the fluorescence intensity at 520 nm in the system decreases with the increase of the concentration of the quenching fragment BHQ3. The above results indicate that the designed oligonucleotide quenching fragment BHQ3 can stably assemble with the probe G43-3'FAM and undergo energy resonance transfer to quench the fluorescence. (3) The quenching effect of the quenching fragment BHQ3 at different concentrations was characterized by a gel imaging instrument: ① Prepare the following mixed solution in a 0.2 mL PCR reaction tube:

[0160] Table 15 Reaction system of different concentrations of quenching fragment BHQ3 and probe G43-3'FAM

[0161]

[0162]

[0163] Note: The initial concentration of the probe G43-3'FAM is 10μM, and the final concentration is 0.1μM; the initial concentration of the quenching fragment BHQ3 is 10μM, and the final concentrations of the system are 0.05, 0.10, and 0.15μM, respectively. ② Protect reaction 3 from light and observe the fluorescence changes of the system, take pictures, and record the fluorescence values. ③ Draw a bar graph in GraphPad Prism 9.0 based on the values, and analyze whether there is a significant difference between the fluorescence intensity and the initial probe G43-3'FAM after adding the quenching fragment BHQ3. (4) Fluorescence quenching of DNA branches: Prepare the following mixed solution in a 0.2mL PCR reaction tube: 8μL of DNA branches (Example 1) and 1μL of quenching fragment BHQ3 (10μM) are reacted at room temperature for 30 minutes. Then, qualitative and quantitative analysis was performed using 2% agarose gel electrophoresis and a gel imager. The results are shown in Fig.24 . Fig.24The results showed that the DNA branch system itself had significant fluorescence. After adding the quenching fragment BHQ3 to this system, the fluorescence signal was quenched through energy resonance transfer. The DNA branch and DNA branch + BHQ3 were exposed and observed by gel imaging, and the fluorescence of the system was statistically analyzed. Prism9 analysis showed that the fluorescence value before and after the addition of the quenching fragment BHQ3 was significantly different (****p<0.0001), which proved that the DNA branch and the quenching fragment BHQ3 had DNA hybridization and the fluorescence signal was quenched. At the same time, 2% agarose gel electrophoresis was used to characterize the quenching effect of the DNA branch. Fig.24 As shown in Figure c, the DNA branch lane without 3×GelRedTM nucleic acid dye staining has obvious fluorescence, but after adding the quenching fragment BHQ3, the fluorescence of the assembled band in the lane (DNA branch + BHQ3) is significantly weakened, which indicates that the quenching fragment BHQ3 has hybridized with the probe G43-3'FAM. The fluorescence brightness of the band around 50 bp also shows that the quenching fragment BHQ3 has a significant quenching effect on the fluorescence of the probe G43-3'FAM. The above results prove the stability of the quenching of the fluorescence signal of the DNA branch. (5) Fluorescence quenching of DNA branches on streptavidin magnetic beads: Take 20 μL of Chol-PEG-Biotin (10 μM) to block the biotin binding sites on the surface of streptavidin magnetic beads, and the resulting magnetic beads are used as the control group; the ratio of streptavidin magnetic beads to the total number of streptavidin magnetic beads and control group magnetic beads is 50% and 90% respectively, and then the mixture is mixed; finally, the mixed magnetic bead system is incubated with the constructed biotinylated DNA branch overnight. The fluorescence information was characterized using Olympus's whole slide scanning system SLIDEVIEWVS200, and then 10 μM of the quenching fragment BHQ3 was added to the above magnetic bead system to cover the streptavidin magnetic beads, incubated in the dark for 30 minutes, and the fluorescence image was collected again. Fig.25 It was observed that the fluorescence signal of streptavidin magnetic beads in the 50% group and the 90% group was obvious in the initial state, but the fluorescence was quenched 30 minutes after the addition of the quenching fragment BHQ3, which indicated that the quenching fragment BHQ3 could stably quench the fluorescence signal at the magnetic bead level. (6) Fluorescence quenching of DNA branches on tumor cells: ① A549 / ATCC epithelial cells were subcultured, digested, and then plated: a blank group, a fluorescence group, and a quenching group were set up, with 5 replicates in each group; according to the size of the slide, it was placed in a 24-well plate for culture, and 10 μL of culture solution was first dropped into each well, and then the slide was placed on the droplet and pressed tightly, so that the slide and the culture dish were bonded together by the tension of the culture solution to prevent the slide from floating up when the cell suspension was added, resulting in a double-layer cell patch. A549 cells were plated at 4×10 4②Fix and seal the cells, and construct DNA branches with reference to Example 1. ③Take 160 μL of DNA branches in the fluorescence group, add them to the washed slide in ①, then add 20 μL of streptavidin (100 μg / mL, about 1.5 μM), incubate at room temperature for 3 hours, remove the incubation system, wash the slide 3 times with PBS buffer, 5 minutes / time; add 200 μL of quenching fragment BHQ3 (10 μM) to the fluorescence group, incubate at room temperature for 30 minutes, wash the slide 3 times with PBS buffer, 5 minutes / time; the blank group is directly incubated with PBS. ④Cell nuclear staining: add 200 μL of Hoechst 33258 (10 μg / mL) solution to each group, incubate at room temperature in the dark for 30 minutes to stain the cell nucleus, then wash the slide 3 times with PBS buffer, 5 minutes / time, take the slide and seal it. The fluorescence was characterized using Olympus's full slide scanning system SLIDEVIEWVS200. Fig.26It can be observed that the fluorescence signal of the cells in the fluorescence group was significant after incubation with the DNA branches, and the fluorescence signal of the cells in the quenching group was significantly quenched after adding the quenching fragment BHQ3. This shows that the quenching fragment BHQ3 can also quench the fluorescence signal at the cellular level. However, the quenching efficiency of the DNA branches fixed on the cells is lower than that in the solution system. This difference may be due to incomplete hybridization caused by electrostatic interactions and spatial effects on the cells. (7) Chromatographic application of DNA branches on streptavidin magnetic beads: ① Pretreatment of streptavidin magnetic beads: Take two 50μL streptavidin magnetic beads in two 1.5mL centrifuge tubes and wash them three times with 200μL buffer (100mM sodium chloride buffer prepared with 1×PBS buffer), 5min / time (12,500rpm). Then, add 20 μL of 5% BSA blocking solution to fully cover the surface of the carrier to block other binding sites of streptavidin magnetic beads, shake at room temperature for 30 minutes, centrifuge and wash 3 times with 200 μL of 100mM sodium chloride buffer, 5min / time (12,500rpm), and discard 5% BSA. ② Add 100 μL of biotinylated DNA branches to the magnetic beads in one centrifuge tube, labeled as N1, and incubate at 4℃ overnight (12-18h). Then, centrifuge and wash 3 times with 200 μL of 100mM sodium chloride buffer, 5min / time (12,500rpm). Resuspend to 50 μL with 100mM sodium chloride buffer. Add the same volume of water to the magnetic beads in another centrifuge tube, labeled as N2. Take out 6 μL of magnetic beads from tube N1 and 14 μL of magnetic beads from tube N2, mix the two (N1:N2=3:7), and add 100mM sodium chloride buffer to a volume of 50 μL. 10 μL of sample suspension was dropped onto the adhesion slide, and the VS200 was used to collect images. ③ After image collection, 50 μL of quenching fragment BHQ3 (10 μM) was added to the mixed system and incubated at room temperature for 30 min; 200 μL of 100 mM sodium chloride buffer was centrifuged and washed 3 times, 5 min / time (12,500 rpm), and the buffer was added to maintain the volume at 50 μL. 10 μL of sample suspension was dropped onto the adhesion slide, and the VS200 was used to collect images. ④ Then 100 μL of biotinylated DNA branches was added to the mixed system, and incubated at 4°C overnight (12-18 h). 200 μL of 100 mM sodium chloride buffer was centrifuged and washed 3 times, 5 min / time (12,500 rpm). The buffer was added to maintain the volume at 50 μL. 10 μL of sample suspension was dropped onto the adhesion slide, and the VS200 was used to collect images. ⑤ Repeat step ③ to analyze the above collected images. The results are shown in Fig. 27 .Depend on Fig. 27It can be seen that in the first round of color development (ON), the observed proportion of fluorescent streptavidin magnetic beads n1 accounted for about 30%. After the quenching fragment was added, the fluorescence of the system was significantly quenched (OFF). This shows that at this time, about 30% of the streptavidin magnetic beads in the system have successfully assembled their surface DNA branches with the quenching fragment BHQ3, and energy resonance transfer occurs, resulting in significant fluorescence quenching. Then, in the second round of color development (ON2), the observed fluorescent streptavidin magnetic beads (n2) are consistent with the added N2 proportion (about 70%); with the addition of the quenching fragment, no fluorescent signal is observed on the surface of the fluorescent streptavidin magnetic beads (OFF2). This shows that the streptavidin magnetic beads after quenching in the previous round have no effect on the next round of color development and the DNA branches are only bound to the surface of the added N2 streptavidin magnetic beads. The above results show the feasibility of the DNA branch "color stacking" color development technology. In the process of sampling and taking pictures, the number of streptavidin magnetic beads in the system is compared with the initial total number of streptavidin magnetic beads, and it will be reduced with each sampling and taking pictures. Therefore, the present invention performs a normality test on the collected fluorescent streptavidin magnetic beads to study whether the quantitative data analysis has normal distribution characteristics. Because the sample size of the research data is all less than or equal to 50, the SW test is used. As can be seen from Table 16, neither ON nor ON2 shows significance (p>0.05), which means that the data have normal characteristics; and the absolute value of the skewness is less than 3, which means that although the data is not absolutely normal, it is basically acceptable as a normal distribution, and the results can be used for scientific research and analysis.

[0164] Table 16 Normality test analysis results of “color matching” color data

[0165]

[0166] The present invention utilizes energy resonance transfer to achieve quenching of fluorescence signals in a free state and on a carrier, and successfully constructs a new strategy for assembly reaction based on DNA branches combined with quenching fragment BHQ3. The assembled DNA branches-BHQ3 successfully achieves quenching of fluorescence signals. Fluorescence results show that the designed quenching fragment BHQ3 can be stably assembled with the probe G43-3'FAM and undergo energy resonance transfer to quench fluorescence. The streptavidin magnetic beads incubated with DNA branches have obvious fluorescence signals in the initial state, but the fluorescence is quenched 30 minutes after the addition of the quenching fragment BHQ3, which indicates that the quenching fragment BHQ3 can stably quench the fluorescence signal at the magnetic bead level. However, the quenching efficiency of DNA branches fixed on cells is lower than that in the solution and the streptavidin magnetic bead system, and this difference may be due to incomplete hybridization caused by electrostatic interaction and spatial effects on cells. In addition, through the "color stacking" color development method, the present invention realizes dual imaging of fluorescence signals on streptavidin magnetic beads, overcomes the interference of non-target analytes in the system, and enables the method to show excellent performance in terms of signal stability and selectivity. In summary, the DNA branches constructed in the present invention have potential applications and analytical potentials in achieving multiple visualization of target analytes through the "color stacking" color development method.

[0167] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A DNA sequence for preparing DNA branches, characterized in that: The DNA sequence contains a biotin-labeled F / R primer binding sequence and multiple Nt.BstNBI specific nicking enzyme cleavage sites, and the DNA sequence is shown in SEQ ID NO.1; the F / R primers are shown in SEQ ID NO.2 and SEQ ID NO.

3.

2. A DNA branch, characterized in that: The DNA branches are prepared from the DNA sequence of claim 1.

3. A method for preparing the DNA branches according to claim 2, characterized in that: The steps include: The DNA sequence is used as a template, and the F / R primers with biotin label are used to amplify the DNA sequence to obtain dsDNA. The dsDNA is cleaved by Nt.BstNBI specific nicking enzyme to obtain biotin-labeled single-stranded product ssDNA; the ssDNA is hybridized with the fluorescent probe G43-3'FAM to obtain DNA branches.

4. The method for preparing DNA branches according to claim 3, characterized in that: The sequence of the fluorescent probe G43-3'FAM is shown in SEQ ID NO.

4.

5. Use of the DNA branches obtained by the preparation method of the DNA branches according to claim 2 or claim 3 in multiple fluorescence imaging, characterized in that: The method of application is: The fluorescent group on the DNA branch is hybridized with the quenching fragment BHQ3 to achieve fluorescence quenching after exposure, and then the next fluorescent group is exposed; The quenching fragment BHQ3 is the quenching fragment of the fluorescent probe G43-3'FAM, and its sequence is shown in SEQ ID NO.

20.

6. A multi-labeled DNA dendrite, characterized in that: The multi-labeled DNA branches are prepared from the DNA sequence of claim 1.

7. A method for preparing the multi-labeled DNA dendrites according to claim 6, characterized in that: The steps include: (1) Modifying the reverse primer in the F / R primer with Spacer18 to obtain SMA-R-Spacer; (2) PCR amplifying the DNA sequence using the F / R primer modified by Spacer18 to obtain a PCR product with sticky ends; hybridizing the biotin-modified DNA tag and its corresponding connecting DNA with the PCR product to obtain a multi-stage hybridization product; the multi-stage hybridization product is digested with Nt.BstNBI specific nicking enzyme and then hybridized with the fluorescent probe G43-3'FAM to obtain a multi-labeled DNA branch.

8. The method for preparing multi-labeled DNA branches according to claim 7, characterized in that: The sequence of the SMA-R-Spacer is shown in SEQ ID NO.7; the biotin-modified DNA tag is one or more of bio-seq1 to bio-seq6, and its sequence is shown in SEQ ID NO.8 to SEQ ID NO.13; its corresponding linker DNA is linker-bio1 to linker-bio6, and its sequence is shown in SEQ ID NO.14 to SEQ ID NO.

19.

9. Use of the multi-labeled DNA tree branches obtained by the method for preparing the multi-labeled DNA tree branches according to claim 6 or claim 7 in multiplex fluorescence imaging, characterized in that: The method of application is: The fluorescent groups on the multi-labeled DNA branches are hybridized with the quenching fragment BHQ3 to achieve fluorescence quenching after exposure, and then the next fluorescent group is exposed; The quenching fragment BHQ3 is the quenching fragment of the fluorescent probe G43-3'FAM, and its sequence is shown in SEQ ID NO.20.

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