A method for detecting abnormal activation of subcellular proteins in a single cell based on DNA tetrahedron probe

Abstract

This invention discloses a single-cell detection method for subcellular protein aberration activation based on DNA tetrahedral probes, belonging to the field of biotechnology. This invention utilizes the programmability and addressable modification characteristics of complementary base pairing in DNA tetrahedrons to quantitatively modify target molecules and fluorescent signal molecules at a single vertex, ensuring well-defined probe function and uniform performance. Through precise structural design at the nanometer scale, the structure exhibits uniform rigidity, achieving size matching and high affinity recognition with the target protein. The probe system provided by this invention can simultaneously detect proteins at multiple subcellular sites closely related to tumor development and progression at the single-cell level, comprehensively assessing cellular heterogeneity and abnormal protein expression states. Furthermore, DNA tetrahedral probes offer advantages such as simple synthesis, programmable sequences, precise and controllable modification sites, good water solubility, and high biocompatibility, providing a flexible and scalable new tool for precise tumor diagnosis, pathological mechanism research, and dynamic monitoring.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a single-cell detection method for subcellular protein abnormal activation based on a DNA tetrahedral probe. Background Technology

[0002] Aberrant expression, subcellular localization dysregulation, and dynamic translocation of subcellular proteins are core molecular characteristics of the development and progression of major diseases such as malignant tumors and neurodegenerative diseases. Achieving in-situ, multi-target simultaneous labeling and quantitative analysis of abnormally activated proteins in different subcellular regions, including the cell membrane, cytoplasm, and organelles, at the single-cell level is a crucial prerequisite for elucidating disease mechanisms and supporting precision diagnosis and treatment.

[0003] Current mainstream molecular detection technologies mostly focus on the gene level, identifying gene mutations, amplifications, or fusions through methods such as PCR and gene sequencing. These methods can only indirectly infer the functional state of proteins and cannot directly reflect the true subcellular location of proteins or the heterogeneity of expression between cells. Traditional protein detection methods, such as immunohistochemistry and Western blotting, rely on fixed tissues or lysed samples, which not only loses the dynamic information of living cells but also makes it difficult to achieve precise subcellular quantification at the single-cell level, failing to meet the needs of refined pathological research and clinical diagnosis.

[0004] The interaction between nanoprobes and cells at the single-cell level is a core factor determining in-situ detection performance. The cellular uptake efficiency, intracellular transport pathway, and subcellular localization accuracy of the probe directly determine the accuracy and reliability of the detection results. Traditional inorganic nanoparticles (such as silica and iron oxide) and polymer microspheres, with their randomly distributed surface modification sites and limited controllability of structural parameters, struggle to achieve precise arrangement of functional groups at the molecular scale. This results in low size matching with target proteins, limiting targeted recognition accuracy. Furthermore, probe construction is often customized, making it difficult to quickly adapt to the detection needs of different subcellular targets. Structured DNA nanotechnology, represented by DNA tetrahedra, offers a novel approach to solving this problem. Unlike traditional carriers, DNA tetrahedra, relying on the precise programmability of complementary base pairing, allows for precise control of structural size, surface charge, and modification sites at the nanometer scale. This enables site-specific and quantitative controllable modification of functional molecules, allowing for precise regulation of cellular uptake and intracellular transport processes through structural optimization, while also possessing high modular design capabilities and flexible scalability. Its natural nanoscale size, matching the molecular weight of proteins, along with excellent water solubility and biocompatibility, allows for efficient penetration of cell membranes and precise targeting of specific subcellular structures. Furthermore, the recognition units for different targets can be flexibly replaced according to detection needs, rapidly constructing specific probes for abnormally activated proteins in different subcellular regions such as the cell membrane, cytoplasm, and organelles, forming a universal single-cell protein detection probe platform. Combined with mature single-cell analysis technologies such as confocal microscopy and flow cytometry, it enables in-situ spatial localization and precise quantification of proteins at the single-cell level, providing reliable technical support for subcellular level pathological research and disease detection.

[0005] Existing detection probes based on DNA nanostructures are mostly customized designs targeting single targets. On the one hand, they lack standardized and rapidly scalable modular construction systems, making it difficult to flexibly adapt to the detection needs of abnormal proteins in different subcellular regions. On the other hand, they mostly focus only on the binding function of target molecules and do not fully utilize the programmable characteristics of DNA structures to systematically optimize endocytosis and subcellular transport processes. Intracellular delivery efficiency and positioning accuracy are unstable, making it difficult to form a standardized and reproducible quantitative identification process with single-cell analysis technologies such as confocal imaging and flow cytometry.

[0006] Based on this, those skilled in the art are dedicated to developing a modular subcellular targeted fluorescent probe platform based on DNA tetrahedrons to achieve single-cell in situ detection and quantitative analysis of abnormal proteins in different subcellular regions. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a single-cell detection method for subcellular protein abnormal activation based on DNA tetrahedral probes.

[0008] To achieve the above objectives, the present invention provides a single-cell detection method for subcellular protein abnormal activation based on a DNA tetrahedral probe.

[0009] Furthermore, a method for preparing a DNA tetrahedral fluorescent nanoprobe with modular subcellular targeting function includes the following steps: Step 1: Four DNA single strands S1, S2, S3 and S4, each containing three complementary regions, are self-assembled into a rigid tetrahedral backbone by the base complementary pairing principle, thus obtaining a DNA tetrahedral nanostructure; at least one of the four single strands of DNA has a functional modification site; the nucleotide sequences of S1, S2, S3 and S4 are shown in SEQ ID NO:1-4 respectively. Step 2: Couple the target molecule to the functional modification site via an active group reaction; the active group reaction is an amide reaction or click chemistry; the target molecule is an aptamer or affinity ligand for tumor-related proteins of the cell membrane, cytoplasm, or endoplasmic reticulum, and the target molecule can be replaced according to the detection target to adapt to the protein detection needs of different subcellular regions; Fluorescent signal molecules are covalently linked to the same functional modification site or another functional modification site on a different chain via reactive groups, or loaded with fluorescent signal molecules via hydrophobic or electrostatic interactions, to obtain crude probe products. Step 3: Purify the crude probe product obtained in Step 2 by polyacrylamide gel electrophoresis or size exclusion chromatography to remove unassembled single strands, assembly intermediates, and unreacted modifications. Detect the probe's hydration size and zeta potential using dynamic light scattering (DLS). Confirm the success rate of fluorescent labeling using UV-Vis absorption spectroscopy and fluorescence spectroscopy, allowing for precise control of the labeled copy number of the fluorescent molecule. Store the obtained target probe at -20°C in the dark. The particle size of the DNA tetrahedral probe can be precisely controlled within the range of 5-20 nm by adjusting the number of bases in each arm.

[0010] Furthermore, the targeting molecule can be selected from aptamers, nanobodies, small molecule ligands, or specific affinity proteins according to the target site; the fluorescent dye can be selected from FITC, Cy series, Alexa Fluor series, etc., but it is necessary to ensure that the emission spectra of different probes do not overlap (maximum emission peaks are separated by >30nm).

[0011] Furthermore, in step one, the terminal regions of the four single-stranded DNA strands are modified with phosphate thioesters or 2'-O-methyl groups to resist nuclease degradation and prolong the action time and imaging window of the probe in living cells.

[0012] Furthermore, in step one, four DNA single strands in equimolar ratio are dissolved in TM buffer, heated to 95°C and held for 5 minutes, and then rapidly cooled to 4°C to form a DNA tetrahedral nanostructure.

[0013] Furthermore, in step two, the functionalization modification site is a reserved extension sequence or active group at the 5' or 3' end of at least one selected single strand of DNA, wherein the active group is a carboxyl, amino, or azide group.

[0014] Furthermore, in step two, when the fluorescent signal molecule is an organic fluorescent dye or a quantum dot, it is covalently linked through a reaction of similar active groups; when the fluorescent signal molecule is a small molecule fluorescent dye, it is loaded into the internal cavity of the tetrahedral framework through hydrophobic or electrostatic interactions.

[0015] Furthermore, in step two, at least one single chain of the tetrahedral backbone is also connected to a cell-penetrating peptide (such as TAT) and / or a pH-responsive polymer (such as imidazole-modified polylysine) to regulate the cell uptake efficiency and intracellular transport pathway of the probe and promote the probe's escape from the lysosome.

[0016] Furthermore, in step two, the functional modification site is a reserved extension sequence or active group at the 5' or 3' end of at least one selected single strand of DNA, wherein the active group is a carboxyl, amino, or azide group.

[0017] Furthermore, a single-cell detection method for abnormal activation of subcellular proteins includes the following steps: S1. The prepared DNA tetrahedral fluorescent nanoprobes are mixed with the cells to be tested and incubated. S2. Detect the fluorescence signal of the cells to be tested using a laser confocal microscope or flow cytometer; S3. Based on the location distribution and / or fluorescence intensity of the fluorescence signal, determine the in situ spatial location and / or single-cell quantitative analysis of the abnormal activation of each subcellular protein in the test cell.

[0018] Furthermore, in step S1, the cells to be tested are tumor cell lines, normal cell controls, solid tumor cells, or disease model cells.

[0019] Furthermore, in step S3, fluorescence detection is performed using a laser confocal microscope, and fluorescence images of different probes are acquired using different channels, with cell nuclei labeled with DAPI or Hoechst; the colocalization coefficient and relative intensity of the fluorescence signal are calculated using image analysis software to determine the expression and spatial distribution characteristics of abnormal activation of various subcellular proteins.

[0020] Furthermore, when using laser confocal microscopy to detect fluorescence signals, the incubation method for the cells to be tested is to seed the cells to be tested in a confocal culture dish, and after the cells adhere to the dish, add a culture medium containing the DNA tetrahedral nanofluorescent probe, incubate at 37°C for 4-6 hours, and wash with PBS after incubation. Furthermore, in step S3, fluorescence detection is performed using flow cytometry to measure the fluorescence intensity of the corresponding channels. At least 10,000 cells are collected for each sample, and the proportion of different protein expression subpopulations is calculated through scatter plot gating analysis.

[0021] Furthermore, when using flow cytometry to detect fluorescence signals, the incubation method for the test cells is to seed the test cells in a cell culture plate, add culture medium containing the DNA tetrahedral nanofluorescent probe, incubate at 37°C for 4 hours, wash with PBS after incubation, digest with trypsin and resuspend in PBS to obtain a single-cell suspension; In a preferred embodiment 1 of the present invention, the preparation process of the DNA tetrahedral nanoprobe targeting EGFR is described in detail.

[0022] In a preferred embodiment 2 of the present invention, the preparation process of a small molecule TKI-modified DNA tetrahedral nanoprobe targeting the cytoplasmic BCR-ABL fusion protein is described in detail.

[0023] In a preferred embodiment 3 of the present invention, the preparation process of an antibody-modified DNA tetrahedral nanoprobe targeting the endoplasmic reticulum tumor stress protein GRP78 is described in detail.

[0024] In a preferred embodiment 4 of the present invention, the preparation process of a DNA tetrahedral nanoprobe having EGFR targeting, fluorescent labeling, and TAT membrane-penetrating peptide functions is described in detail.

[0025] In a preferred embodiment 5 of the present invention, the preparation process of a small molecule TKI-modified DNA tetrahedral nanoprobe that targets the cytoplasmic BCR-ABL fusion protein and also has lysosomal escape function is described in detail.

[0026] In a preferred embodiment 6 of the present invention, the preparation process of the probe Tetra-GRP78-Cy5-PS is described in detail.

[0027] In a preferred embodiment 7 of the present invention, the preparation process of a DNA tetrahedral nanoprobe targeting the Golgi matrix protein GM130 is described in detail.

[0028] In a preferred embodiment 8 of the present invention, the preparation process of a small molecule modified DNA tetrahedral nanoprobe targeting a cytoplasmic KRAS mutant protein is described in detail.

[0029] In a preferred embodiment 9 of the present invention, the process of targeted and specific detection of subcellular target proteins by DNA tetrahedral nanofluorescent probes is described in detail.

[0030] In a preferred embodiment 10 of the present invention, the quantitative analysis process of subcellular target proteins by DNA tetrahedral nanofluorescent probes is described in detail.

[0031] In a preferred embodiment 11 of the present invention, the process of verifying the subcellular localization accuracy of the DNA tetrahedral fluorescent nanoprobe for cytoplasmic target proteins is described in detail.

[0032] The principle of this invention is as follows: This invention leverages the programmability and addressable modification properties of complementary base pairing in DNA tetrahedrons to quantitatively modify target molecules and fluorescent signaling molecules at a single vertex, ensuring well-defined probe function and uniform performance. Through precise structural design at the nanometer scale, the structure exhibits uniform rigidity, achieving size matching and high-affinity recognition with the target protein. Furthermore, by precisely controlling the distribution of surface charge and functional groups through sequence design, the endocytosis pathway is systematically optimized. Combined with transmembrane peptides and pH-responsive functional modifications, lysosomal escape is promoted, and backbone chemical modifications resist nuclease degradation, extending the probe's interaction time and imaging window within living cells. Confocal imaging enables in-situ spatial localization, while flow cytometry achieves high-throughput single-cell quantification, forming a complete single-cell protein analysis workflow.

[0033] Technical effects: 1. This invention overcomes the shortcomings of traditional molecular probes, such as poor water solubility and low cell entry efficiency. At the same time, it makes up for the shortcomings of ordinary nanoparticles, such as single surface modification sites and lack of programmability. It breaks through the indirect limitation of gene detection and directly realizes single-cell in situ analysis at the protein level, accurately reflecting the subcellular localization of proteins and cellular heterogeneity. 2. This invention adopts a modular and universal platform design, which can be adapted to the protein detection needs of different subcellular regions by quickly replacing the targeting unit, without the need to redesign the probe backbone, thus greatly shortening the development cycle; 3. The present invention is compatible with both imaging and flow cytometry technologies, which can meet the needs of in situ pathological research and also support high-throughput cell detection.

[0034] 4. This invention significantly improves the recognition and binding efficiency and specificity of target proteins and reduces non-specific adsorption through precise molecular-scale assembly and size matching; at the same time, the standardized self-assembly process ensures the uniformity of probe batches, and greatly improves the repeatability and reliability of detection results. 5. This invention, through systematic regulation of endocytosis and subcellular transport pathways, combined with systematic optimization of intracellular transport, can achieve precise delivery of targets deep within the cytoplasm and organelles, significantly improving subcellular localization accuracy and supporting long-term dynamic imaging monitoring of live cells.

[0035] 6. The modular DNA tetrahedral fluorescent nanoprobe system designed in this invention can simultaneously detect proteins at multiple subcellular sites closely related to tumor development and progression (such as cell membrane EGFR / HER2 / EpCAM, cytoplasmic KRAS / PIK3CA / BCR-ABL, endoplasmic reticulum GRP78, etc.) at the single-cell level, comprehensively assessing cellular heterogeneity and abnormal protein expression. Furthermore, the DNA tetrahedral probes possess advantages such as simple synthesis, programmable sequences, precise and controllable modification sites, good water solubility, and high biocompatibility, providing a flexible and scalable new tool for precise tumor diagnosis, pathological mechanism research, and dynamic monitoring. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the subcellular protein abnormal activation recognition based on DNA nanoprobes according to the present invention.

[0037] Figure 2 The difference in fluorescence intensity is shown in HCC827 cells with EGFR mutation in Example 9.

[0038] Figure 3 The image shows the co-localization of the DNA-targeting probe and BCR-ABL protein in K562 cells in Example 11. In the image, a is the nuclear fluorescence image, b is the fluorescence image of the DNA-targeting probe, c is the fluorescence image of the BCR-ABL protein, and d is the co-localization image. The Pearson co-localization coefficient between the nanoprobe and the BCR-ABL protein is 0.7. Detailed Implementation

[0039] The following describes several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0040] Example 1: Preparation of DNA tetrahedral nanoprobes targeting EGFR

[0041] 1. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA strands (named S1, S2, S3, and S4, with nucleotide sequences as shown in SEQ ID NO: 1-4) were designed, each strand being 62-63 bases in length, with each edge containing approximately 20 base pairs. These four single-stranded DNA strands self-assembled into a tetrahedral framework with six double-stranded edges through complementary domains designed in their respective sequences, following the base-pairing principle. The four single-stranded DNA strands (S1, S2, S3, and S4) were dissolved in an equimolar ratio (1 μM each) in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0), with a total volume of 100 μL. The mixture was heated to 95°C and held for 5 minutes, then rapidly annealed to 4°C (cooling rate 0.1°C / s) to form a DNA tetrahedral nanostructure. Assembly efficiency was verified by 8% non-denaturing polyacrylamide gel electrophoresis (PAGE), with a single migration band observed, confirming successful tetrahedral assembly.

[0042] 2. Specific Site Functionalization: An extension sequence (5'-AAAAA-3') is reserved at the 5' end of the S1 strand as the aptamer hybridization site. The 3' end of the EGFR-targeting aptamer (5' end labeled with FITC) is designed with a fragment complementary to the S1 strand extension sequence (5'-TTTTT-3'). The FITC-labeled EGFR aptamer and the assembled DNA tetrahedron are mixed at a 1:1 molar ratio, with a total volume of 200 μL, and hybridization is carried out at 37°C for 1 hour. After the reaction, the aptamer is washed three times by centrifugation at 4000g using a 100 kDa ultrafiltration tube, with each wash using PBS buffer to remove unhybridized free aptamers. The EGFR-targeting DNA nanoprobe is obtained. The nucleotide sequence of the EGFR-targeting aptamer is shown in SEQ ID NO:5.

[0043] Example 2: Preparation of small-molecule TKI-modified DNA tetrahedral nanoprobes targeting cytoplasmic BCR-ABL fusion protein

[0044] 1. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA strands (named S1, S2, S3, and S4) were designed, each strand being 62-63 bases in length, with each edge containing approximately 20 base pairs. A carboxyl group was introduced at the 5' end of the S2 strand during solid-phase synthesis for subsequent amide reactions. The four single-stranded DNA strands (S1, S2 (carboxyl-modified), S3, and S4) were dissolved in an equimolar ratio (1 μM each) in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0), with a total volume of 100 μL. The mixture was heated to 95°C and held for 5 minutes, then slowly annealed to 4°C at a rate of 0.1°C / s to form a DNA tetrahedral nanostructure. Successful assembly was verified by 8% non-denaturing PAGE.

[0045] 2. Specific Site Functionalization: Amino-modified imatinib (BCR-ABL-targeting small molecule TKI) was diluted to 1.2 μM with MES buffer (pH 5.5). The assembled DNA tetrahedron (containing the 5' carboxyl group of the S2 chain) was diluted to 1 μM with MES buffer (pH 5.5), and EDC (final concentration 5 mM) and NHS (final concentration 10 mM) were added. The carboxyl group was activated at room temperature for 30 minutes. Then, a mixture of amino-modified imatinib and Cy3 fluorescent dye was added, and the reaction was carried out at room temperature for 2 hours, simultaneously achieving TKI small molecule conjugation and fluorescent labeling via an amide reaction. After the reaction, the sample was washed three times by centrifugation at 4000g using a 100 kDa ultrafiltration tube to remove unreacted small molecules and activating reagents. This yielded a small molecule TKI-modified DNA nanoprobe targeting the cytoplasmic BCR-ABL fusion protein.

[0046] Example 3: Preparation of antibody-modified DNA tetrahedral nanoprobes targeting the endoplasmic reticulum tumor stress protein GRP78

[0047] 1. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA strands (named S1, S2, S3, and S4) were designed, each strand being 62-63 bases in length, with each edge containing approximately 20 base pairs. An azide group (N3) was introduced at the 5' end of the S3 strand during solid-phase synthesis. The four single-stranded DNA strands (S1, S2, S3 (azon-modified), and S4) were dissolved in an equimolar ratio (1 μM each) in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0), with a total volume of 100 μL. The mixture was heated to 95°C and held for 5 minutes, then rapidly cooled in an ice-water bath for 1 minute, and finally annealed to 4°C at a rate of 0.1°C / s to form a DNA tetrahedral nanostructure. Successful assembly was verified by 8% non-denaturing PAGE.

[0048] 2. Site-Specific Functionalization: DBCO-modified anti-GRP78 antibodies were prepared and pre-labeled with Cy5 fluorescent dye. Assembled DNA tetrahedra (containing azide groups) and DBCO-modified Cy5-labeled anti-GRP78 nanobodies were dissolved in PBS buffer (pH 7.4) at a 1:1.2 molar ratio, with a total volume of 200 μL. The mixture was stirred at room temperature in the dark for 12 hours for copper-free click chemistry (SPAAC). After the reaction, the nanoparticles were washed three times by centrifugation at 4000g using a 100 kDa ultrafiltration tube to remove unreacted antibody. This yielded an antibody-modified DNA nanoprobe targeting the endoplasmic reticulum tumor stress protein GRP78.

[0049] Example 4: Preparation of DNA tetrahedral nanoprobes with EGFR targeting, fluorescent labeling, and TAT membrane-penetrating peptide functions

[0050] 1. Take a pre-synthesized S4 chain with a primary amino group at the 5' end (62 bases in length, prepared by standard phosphoramide solid-phase synthesis and purified before use), and dissolve it in PBS buffer (pH 7.4) to a concentration of 100 μM. Separately, dissolve the heterobifunctional crosslinking agent Sulfo-SMCC (sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester) in deionized water to a concentration of 20 mM. Add 10 molar excess of Sulfo-SMCC to the S4 chain solution and stir at room temperature for 2 hours to allow the 5' amino group of the S4 chain to react with the NHS ester of Sulfo-SMCC, introducing a maleimide group. After the reaction, purify using a desalting column to remove excess crosslinking agent, obtaining a maleimide-activated S4 chain. Simultaneously, a TAT-modified membrane peptide (sequence YGRKKRRQRRR, with a cysteine ​​residue introduced at the C-terminus to provide a thiol group, i.e., YGRKKRRQRRRC, or commercially available Cys-TAT) was synthesized and dissolved in PBS (pH 7.4) at a concentration of 200 μM. The maleimide-activated S4 chain was mixed with the Cys-TAT peptide at a molar ratio of 1:10 and reacted at room temperature in the dark for 4 hours to allow a specific addition reaction between the thiol group of the TAT peptide and the maleimide group on the S4 chain. After the reaction, the product was purified by reversed-phase HPLC to obtain the TAT-modified S4 chain (S4-TAT), which was then lyophilized for later use.

[0051] 2. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA molecules (S1, S2, S3, and S4-TAT) were dissolved in equimolar amounts (1 μM each) in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0), with a total volume of 100 μL. The mixture was heated to 95°C and held for 5 minutes, then annealed to 4°C at a rate of 0.1°C / s to form DNA tetrahedra. Assembly was verified by PAGE.

[0052] 3. Targeted Modification: An extension sequence (5'-AAAAA-3') was reserved at the 5' end of the S1 strand. The FITC-labeled EGFR aptamer (with the complementary sequence TTTTT at the 3' end) was mixed with the assembled tetrahedron at a 1:1 molar ratio and hybridized at 37°C for 1 hour. The mixture was then purified by ultrafiltration (100 kDa, 4000 g, 3 times). This yielded a DNA nanoprobe possessing EGFR targeting, fluorescent labeling, and TAT membrane-penetrating peptide functions. This probe can efficiently enter cells and promote lysosomal escape.

[0053] Example 5: Preparation of a small-molecule TKI-modified DNA tetrahedral nanoprobe targeting the cytoplasmic BCR-ABL fusion protein and possessing lysosomal escape function

[0054] 1. Synthesis of polyimide group molecules: Polylysine short peptide (K4, molecular weight about 450 Da) was reacted with imidazole-4-acetic acid under EDC / NHS, so that the amino group of lysine side chain formed an amide bond with the carboxyl group of imidazole acetic acid, and the product (K(Im)4) was obtained with one imidazole group modified on each lysine residue.

[0055] 2. Assembly of the DNA tetrahedral backbone: Four strands, S1, S2, S3, and S4, were designed, with an azide group introduced at the 5' end of the S4 strand during solid-phase synthesis. The four strands were dissolved in 1 μM each in equimolar ratio in TM buffer, annealed at 95°C for 5 minutes, and then assembled into a tetrahedron. Verification was performed using PAGE.

[0056] 3. Targeted modification at specific sites: Following the method in Example 2, amino-modified dasatinib and Cy3 fluorescent dye were coupled to the 5' carboxyl group of the S2 chain via an amide reaction to obtain an intermediate.

[0057] 4. Click coupling of imidazole groups: The above intermediate (containing the S4 chain 5' azide) and DBCO-PEG2-K(Im)4 were dissolved in PBS at a molar ratio of 1:10 and reacted at room temperature in the dark for 12 hours. Unreacted DBCO-imidazole molecules were removed by ultrafiltration. A small TKI-modified DNA nanoprobe targeting the cytoplasmic BCR-ABL fusion protein and possessing lysosomal escape function was obtained. In the acidic environment of the lysosome, the imidazole group of this probe is protonated, generating a "proton sponge effect," which promotes the probe's escape from the lysosome and entry into the cytoplasm.

[0058] Example 6: Preparation of probe Tetra-GRP78-Cy5-PS

[0059] 1. Synthesis of Phosphothiophosphate-Modified Single-Stranded DNA: Four single-stranded DNA strands were designed, named S1, S2, S3, and S4, each 62-63 bases in length, with each strand containing approximately 20 base pairs. Phosphothiophosphate (PS) modification was performed at 4 bases each at the 3' and 5' ends of each strand, replacing a non-bridging oxygen atom on the phosphodiester bond with a sulfur atom. The synthesis employed a standard phosphorous amide solid-phase synthesis method, introducing PS bonds at the appropriate positions using a thioreagent (such as Beaucage reagent or PADS). After synthesis, the DNA was purified by 8% denaturing polyacrylamide gel electrophoresis (PAGE). The target band was excised, eluted with sterile, nuclease-free ultrapure water, precipitated with ethanol, and vacuum dried. The purified PS-modified single-stranded DNA was dissolved in TM buffer (10 mM). Tris-HCl, 50mM MgCl2, pH In 8.0), the concentration was 10 μM, and the samples were aliquoted and stored at -20℃ for later use. During solid-phase synthesis, an additional azido group (a phosphorus amide monomer modified with azido) was introduced at the 5' end of the S3 chain for subsequent click chemical coupling.

[0060] 2. Assembly of the DNA tetrahedral backbone: Take the four PS-modified single-stranded DNA strands S1, S2, S3 (azide-modified), and S4, and mix them in an equimolar ratio (1 μM each) in TM buffer (total volume 100 μL). Heat the mixture to 95°C and hold for 5 minutes, then slowly anneal to 4°C at a rate of 0.1°C / second (or rapidly cool in an ice-water bath for 1 minute and then transfer to 4°C) to form a DNA tetrahedral nanostructure. Take 10 μL of the assembly product for 8% non-denaturing PAGE electrophoresis (90V, 2 hours) and observe the electrophoretic bands using a gel imaging system. Although PS modification may slightly affect the electrophoretic migration, a single master band is still visible, proving that the tetrahedral assembly was successful.

[0061] 3. Preparation of the GRP78-targeting aptamer: A DNA aptamer targeting the endoplasmic reticulum protein GRP78 was synthesized. Cy5 fluorescent dye was introduced at the 5' end of the aptamer via solid-phase synthesis, and a DBCO group was introduced at the 3' end of the aptamer via a hexaethylene glycol spacer arm. After synthesis, the aptamer was purified by HPLC and lyophilized for later use. The nucleotide sequence of the GRP78-targeting DNA aptamer is shown in SEQ ID NO:6.

[0062] The complete DNA aptamer sequence (5'→3') targeting GRP78 is: Cy5-AGCAGCACAGAGGTCAGATG-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-CCTATGCGTGCTACCGTGAA-(Spacer 18)-DBCO. The 5' end is labeled with Cy5 fluorescence, the middle 40 consecutive N's form a 40-base core recognition sequence specifically recognizing the GRP78 protein, obtained through SELEX screening, and the 3' end is connected to the DBCO group via a hexaethylene glycol spacer arm.

[0063] 4. Click chemical coupling (copper-free click reaction): The PS-modified DNA tetrahedron (containing the S3 chain 5' azide) assembled in step 2 is mixed with the Cy5-GRP78 aptamer-DBCO prepared in step 3 at a molar ratio of 1:1.2 in PBS buffer (pH 10). In step 7.4), the total volume was 200 μL, and the reaction was carried out at room temperature in the dark with stirring for 12 hours. During the reaction, the azide group and the DBCO group underwent strain-promoted azido-yne cycloaddition (SPAAC) to form a stable triazole bond, thereby covalently attaching the aptamer to the S3 vertex of the tetrahedron.

[0064] 5. Probe Purification and Characterization: After the reaction, the reaction solution was transferred to a 100 kDa ultrafiltration tube and washed three times by centrifugation at 4000 g, each time with PBS buffer (pH 7.4) to remove unreacted free aptamers and small molecule byproducts. The remaining solution in the ultrafiltration tube was collected as the purified probe, named Tetra-GRP78-Cy5-PS. A small sample was taken, and the absorbance at 260 nm (DNA) and 650 nm (Cy5) was measured using UV-Vis absorption spectroscopy. The molar ratio of Cy5 to DNA tetrahedra was calculated (usually approximately 0.8-1.2:1). Another sample was taken for dynamic light scattering detection, and the hydrated particle size (expected to be approximately 12-15 nm) and Zeta potential were recorded. The probe was aliquoted and stored at -20℃ in the dark for later use. Due to its phosphate thioester backbone modification, this probe can resist intracellular nuclease degradation and significantly prolong the live-cell imaging window.

[0065] Example 7: Preparation of DNA tetrahedral nanoprobes targeting Golgi matrix protein GM130

[0066] 1. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA strands (named S1, S2, S3, and S4) were designed, each strand being 62-63 bases in length, with each edge containing approximately 20 base pairs. The S2 strand had an azide group (N3) introduced at its 5' end during solid-phase synthesis for subsequent click chemilinking. The four single-stranded DNA strands (S1, S2 (azide-modified), S3, and S4) were dissolved in an equimolar ratio (1 μM each) in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0), with a total volume of 100 μL. The mixture was heated to 95 °C and held for 5 minutes, then slowly annealed to 4 °C at a rate of 0.1 °C / s to form a DNA tetrahedral nanostructure. Successful assembly was verified by 8% non-denaturing PAGE.

[0067] 2. Preparation of affinity molecules targeting Golgi GM130: A DNA aptamer targeting the Golgi matrix protein GM130 was synthesized. Cy5.5 fluorescent dye was introduced at the 5' end of the aptamer via solid-phase synthesis, and a DBCO group was introduced at the 3' end of the aptamer via a hexaethylene glycol spacer arm. After synthesis, the aptamer was purified by HPLC and lyophilized for later use. The nucleotide sequence of the DNA aptamer targeting GM130 is shown in SEQ ID NO:7. The complete sequence (5'→3') of the DNA aptamer targeting GM130 is as follows: Cy5.5-AGCAGCACAGAGGTCAGATG-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-CCTATGCGTGCTACCGTGAA-(Spacer 18)-DBCO. The 5' end is labeled with Cy5.5 fluorescently, the middle 40 consecutive N's are the 40-base core recognition sequence obtained by SELEX screening, which specifically recognizes the GM130 protein, and the 3' end is connected to the DBCO group via a hexaethylene glycol spacer arm.

[0068] 3. Click chemical coupling: The DNA tetrahedron (containing the S2 chain 5' azide) assembled in step 1 was mixed with the Cy5.5 labeled GM130 aptamer-DBCO prepared in step 2 at a molar ratio of 1:1.2 in PBS buffer (pH 7.4), with a total volume of 200 μL. The mixture was stirred at room temperature in the dark for 12 hours to carry out copper-free click chemical reaction (SPAAC).

[0069] 4. Probe Purification and Characterization: After the reaction, the probe was washed three times by centrifugation at 4000g using a 100kDa ultrafiltration tube, with each wash involving replacement with PBS buffer to remove unreacted free aptamers. The absorbance at 260nm and 650nm (Cy5.5) was measured using UV-Vis absorption spectroscopy to calculate the labeling efficiency. Dynamic light scattering was used to detect the hydrated particle size. The probe was stored at -20℃ in the dark. A DNA nanoprobe targeting the Golgi matrix protein GM130 was obtained.

[0070] Example 8: Preparation of small-molecule modified DNA tetrahedral nanoprobes targeting cytoplasmic KRAS mutant proteins

[0071] 1. Assembly of the DNA tetrahedral backbone: Four single-stranded DNA strands (named S1, S2, S3, and S4) were designed, each 62-63 bases in length, with each edge containing approximately 20 base pairs. The S4 strand had a primary amino group introduced at its 3' end during solid-phase synthesis (using an amino-modified CPG column) for subsequent amide reactions. The four single-stranded DNA strands (S1, S2, S3, and S4, amino-modified) were dissolved in 1 μM of TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0) in an equimolar ratio (1 μM each), for a total volume of 100 μL. The mixture was heated to 95°C and held for 5 minutes, then rapidly cooled in an ice-water bath for 1 minute, and finally annealed to 4°C at a rate of 0.1°C / second to form a DNA tetrahedral nanostructure. Successful assembly was verified by 8% non-denaturing PAGE.

[0072] 2. Preparation of small molecules targeting cytoplasmic KRAS mutant proteins: Carboxyl-modified sotorazib (KRASG12C targeting small molecule inhibitor) was prepared, and a carboxyl group was introduced into the small molecule structure through an alkyl linker for subsequent coupling; at the same time, NHS-activated Alexa Fluor 647 fluorescent dye was prepared.

[0073] 3. Amide Reaction Coupling: The DNA tetrahedra assembled in step 1 (containing the 3' amino group of the S4 chain) were dissolved in MES buffer (pH 5.5) to a final concentration of 1 μM. EDC (final concentration 5 mM) and NHS (final concentration 10 mM) were added, and the mixture was activated at room temperature for 30 minutes. Then, carboxyl-modified sotorasiib (final concentration 1 μM) and Alexa Fluor 647 fluorescent dye were added, and the mixture was reacted at room temperature for 2 hours.

[0074] 4. Probe Purification and Characterization: After the reaction, the probe was washed three times by centrifugation at 4000g using a 100kDa ultrafiltration tube to remove unreacted small molecules, dyes, and activating reagents. Successful labeling was confirmed by UV-Vis absorption spectroscopy and fluorescence spectroscopy, and the number of small molecules and fluorescent molecules coupled to each tetrahedron was calculated. Dynamic light scattering was used to detect hydration particle size and zeta potential. The probe was stored at -20℃ in the dark. A small-molecule modified DNA nanoprobe targeting the cytoplasmic KRAS mutant protein was obtained.

[0075] Example 9: Targeted and specific detection of subcellular target proteins by DNA tetrahedral fluorescent nanoprobes

[0076] 1. HCC827 cells (EGFR mutant) were induced at a rate of 2 × 10⁻⁶. 5 Cells were seeded in confocal culture dishes and cultured overnight in the appropriate medium containing 10% fetal bovine serum. When the cells adhered to the wall and reached approximately 60% confluence, the old medium was removed. The experimental group was treated with 1.5 mL of EGFR-targeting DNA nanoprobe prepared in Example 1 (100 nM). The competitive inhibition control group was pre-incubated at 37°C for 2 hours with 1 mL of EGFR-targeting TKI molecules (prepared in serum-free medium) to allow the free targeting TKI molecules to pre-bind to the cell surface and intracellular target sites; after removing the supernatant, the same concentration of probe solution was added.

[0077] 2. Both groups were incubated at 37℃ for another 4 hours. The culture medium containing the probe was discarded, and the cells were gently washed twice with pre-warmed PBS. 1 mL of 4% paraformaldehyde solution was added to each dish, and the cells were fixed at room temperature for 20 minutes, followed by washing three times with PBS for 5 minutes each time. Then, the cells were permeabilized with 0.1% Triton X-100 / PBS solution at room temperature for 10 minutes, followed by washing three times with PBS. A DAPI-containing anti-fluorescence quenching mounting medium was added, and the cells were carefully covered with a coverslip. The samples were placed under a laser confocal microscope, and images were acquired by exciting each fluorescence channel with the corresponding laser.

[0078] Result: From Figure 2 It can be observed that the fluorescence intensity of each target channel in the competitive inhibition group is significantly lower than that in the experimental group, confirming the high specificity of the DNA nanoprobe of the present invention in binding to the corresponding subcellular target protein.

[0079] Example 10: Quantitative analysis of subcellular target proteins using DNA tetrahedral fluorescent nanoprobes

[0080] 1. Culture different types of target cells separately (e.g., normal epithelial HUVEC cells and HepG2 liver cancer cells activated by abnormal endoplasmic reticulum proteins). Seed each type of cell into six-well plates, 1 × 10⁶ cells per well. 6 Cells were cultured overnight at 37°C in a 5% CO2 incubator. The old medium was removed, and 1.5 mL of fresh medium containing a 100 nM endoplasmic reticulum targeting probe mixture was added to each well. The cells were then incubated at 37°C for 4 hours. The medium was then replaced with fresh, probe-free medium, and the cells were cultured for another 2 hours.

[0081] 2. Aspirate the culture medium, wash once with PBS buffer, add 500 μL of 0.25% trypsin-EDTA digestion solution, and digest at 37°C for 2-3 minutes. After cell detachment, add 1 mL of serum-containing culture medium to terminate digestion. Collect the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 500 g for 5 minutes, and discard the supernatant. Resuspend the cells in PBS buffer, centrifuge again at 500 g for 5 minutes, and wash three times. Finally, resuspend the cells in 200 μL of PBS buffer and place the sample tube on the flow cytometer rack. Set the flow cytometer detection parameters to correspond to the channels of each fluorescent dye. Collect 20,000 cells for each sample and record the fluorescence intensity data for each channel. By using scatter plot gating analysis, the expression levels of target proteins between different cell lines can be compared, achieving protein quantification and cell subpopulation differentiation at the single-cell level.

[0082] Example 11: Verification of the subcellular localization accuracy of DNA tetrahedral fluorescent nanoprobes for cytoplasmic target proteins

[0083] 1. Take K562 cells in the logarithmic growth phase and adjust the cell concentration to 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL in confocal culture dishes and incubated overnight at 37°C with 5% CO2. The old culture medium was removed, and 1.5 mL of serum-free medium containing 100 nM BCR-ABL targeted DNA nanoprobes was added. The cells were incubated at 37°C in the dark for 4 hours. The medium was discarded, and the cells were gently washed twice with pre-warmed PBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by three washes with PBS. The cells were permeabilized with 0.1% Triton X-100 / PBS solution at room temperature for 10 minutes, and then blocked with 5% bovine serum albumin at room temperature for 30 minutes.

[0084] 2. Add Alexa Fluor 488-labeled anti-BCR-ABL monoclonal antibody working solution and incubate overnight at 4°C in the dark. The next day, wash the slides three times with PBS and mount them with anti-fluorescence quenching mounting medium containing DAPI. Observe under a laser confocal microscope, and acquire images of the probe fluorescence channel and the commercial antibody fluorescence channel, respectively. Calculate the co-localization coefficient of the two using image analysis software.

[0085] result: Figure 3 The fluorescence of the probe of this invention highly overlaps with that of commercial antibodies, confirming that the probe can accurately target the BCR-ABL fusion protein in the cytoplasm and has good subcellular localization accuracy.

Claims

1. A method for preparing a DNA tetrahedral fluorescent nanoprobe with modular subcellular targeting function, characterized in that, Includes the following steps: Step 1: Four DNA single strands S1, S2, S3 and S4, each containing three complementary regions, are self-assembled into a rigid tetrahedral backbone by the base pairing principle, thus obtaining a DNA tetrahedral nanostructure; at least one of the four single strands of DNA is provided with a functional modification site; the nucleotide sequences of DNA single strands S1, S2, S3 and S4 are shown in SEQ ID NO:1-4, respectively. Step 2: Couple the target molecule to the functional modification site via an active group reaction; the active group reaction is an amide reaction or click chemistry; the target molecule is an aptamer or affinity ligand for tumor-related proteins of the cell membrane, cytoplasm, or endoplasmic reticulum, and the target molecule can be replaced according to the detection target to adapt to the protein detection needs of different subcellular regions; Fluorescent signal molecules are covalently linked to the same functional modification site or another functional modification site on a different chain via reactive groups, or loaded with fluorescent signal molecules via hydrophobic or electrostatic interactions, to obtain crude probe products. Step 3: Purify the crude probe product obtained in Step 2 by polyacrylamide gel electrophoresis or size exclusion chromatography to remove unassembled single chains, assembly intermediates and unreacted modifications to obtain the target probe.

2. The preparation method according to claim 1, wherein in step one, the terminal regions of the four single-stranded DNA strands are modified with phosphate thioester or 2'-O-methyl.

3. The preparation method according to claim 1, wherein in step one, four DNA single strands in an equimolar ratio are dissolved in TM buffer, heated to 95°C and held for 5 minutes, and then rapidly cooled to 4°C to form a DNA tetrahedral nanostructure.

4. The preparation method according to claim 1, wherein in step two, the functionalization modification site is a reserved extension sequence or active group at the 5' end or 3' end of at least one selected single strand of DNA, and the active group is a carboxyl group, an amino group, or an azide group.

5. The preparation method according to claim 1, wherein in step two, when the fluorescent signal molecule is an organic fluorescent dye or a quantum dot, it is covalently linked by a reaction of similar active groups; when the fluorescent signal molecule is a small molecule fluorescent dye, it is loaded into the internal cavity of the tetrahedral framework through hydrophobic or electrostatic interactions.

6. The preparation method according to claim 1, wherein in step two, at least one single chain of the tetrahedral backbone is further connected with a cell-penetrating peptide and / or a pH-responsive polymer.

7. A single-cell detection method for abnormal activation of subcellular proteins, characterized in that, Includes the following steps: S1. The DNA tetrahedral fluorescent nanoprobe prepared by any one of claims 1 to 6 is mixed with the cells to be tested and incubated. S2. Detect the fluorescence signal of the cells to be tested using a laser confocal microscope and / or flow cytometry. S3. Based on the location distribution and / or fluorescence intensity of the fluorescence signal, determine the in situ spatial location and / or single-cell quantitative analysis of the abnormal activation of each subcellular protein in the test cell.

8. The detection method according to claim 7, wherein in step S1, the cell to be tested is a tumor cell line, a normal cell control, a solid tumor cell, or a disease model cell.

9. The detection method as described in claim 7, wherein in step S3, a laser confocal microscope is used to detect fluorescence signals, different channels are used to acquire fluorescence images of different probes, and cell nuclei are labeled with DAPI or Hoechst; the colocalization coefficient and relative intensity of the fluorescence signals are calculated using image analysis software to determine the expression and spatial distribution characteristics of abnormal activation of various subcellular proteins.

10. The detection method as described in claim 7, wherein in step S3, a flow cytometer is used to detect fluorescence signals, the fluorescence intensity of the corresponding channel is detected, at least 10,000 cells are collected for each sample, and the proportion of different protein expression subpopulations is calculated by scatter plot gating analysis.

Images