Probe combination for detecting mitochondrial respiratory chain super complex based on proximity ligation assay and hybridization chain reaction and application

Abstract

The invention discloses a probe combination for detecting a mitochondrial respiratory chain super complex based on a proximity ligation assay and a hybridization chain reaction and application, and belongs to the field of probe detection. Two adjacent protein subunits capable of reflecting the structural integrity of the hypercomplex are determined as bases, the antigen-antibody combination principle is utilized to promote the antigen-antibody which recognizes and combines the bases to initiate proximity ligation reaction (PLA) due to nucleic acid labeling, cascade amplification of detection signals is realized according to the hybridization chain reaction (HCR) principle, and the detection sensitivity of the hypercomplex is improved. The activity of the oxidative phosphorylation system is judged through the structural integrity of the super-complex, the defects and limitation of a traditional detection method are overcome, and rapid, high-sensitivity and accurate detection on the structure of the super-complex is achieved. Meanwhile, the cells are fixed on a 96-hole fluorescent plate, and the structural integrity of the mitochondrial hypercomplex is detected by using a multifunctional microplate reader, so that parallel detection on 96 samples at the same time is finally realized, and digitization and high throughput of detection are realized.

Description

Technical Field

[0001] The present invention relates to the field of probe detection, and particularly to a probe combination and application for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation assay and hybridization chain reaction. Background Art

[0002] As the center of energy metabolism in eukaryotes, mitochondria complete energy conversion through the cooperation of the electron transport chain (respiratory chain complexes I, II, III, IV) and ATP synthase (complex V), and finally generate a large amount of ATP to supply energy for cell activities. Respiratory chain complexes I, III, and IV can assemble to form a higher-level supramolecular structure - supercomplexes (SCs). SCs not only enhance the stability of the electron transport chain but also significantly improve the energy conversion efficiency by optimizing the electron transport path, enabling the cell to achieve more efficient ATP synthesis with lower energy consumption.

[0003] Proximity ligation assay (PLA) is a biological detection method for molecular interaction based on the proximity effect. Its core principle is to add proximity probes to specifically recognize and bind to the target protein, thereby reducing the spatial distance between this pair of proximity probes. When the spatial distance between the proximity probes is <40 nm, a proximity effect is generated. Subsequently, a fluorescence probe is added to bind to the proximity probes, and the structural integrity of the target protein is verified by detecting the fluorescence signal. Hybridization chain reaction (HCR) is a signal amplification technology based on DNA self-assembly reaction. This system usually contains two or more DNA hairpins. In the presence of the target molecule, it will trigger the alternating opening and assembly of the DNA hairpins to form a double-stranded DNA structure containing a large number of repeating units, thereby achieving signal amplification of the target molecule.

[0004] The functional defect of mitochondrial respiratory chain supercomplex is essentially the destruction of the complex structure. By detecting the integrity of the respiratory chain supercomplex structure, a preliminary analysis of the function of mitochondrial supercomplex can be achieved. Mitochondrial Disease (MD) is a metabolic disease mainly characterized by the functional defect of oxidative phosphorylation (OXPHOS). The incidence of mitochondrial disease is 1 / 5000, belonging to rare diseases. Due to the genetic heterogeneity and diverse clinical symptoms of mitochondrial diseases, the diagnosis of mitochondrial diseases is particularly complex and difficult. For a long time, the diagnosis of mitochondrial diseases has relied on the detection of the enzyme activity of the oxidative phosphorylation system in affected tissues (such as muscles), and this method is considered the "gold standard" for diagnosing mitochondrial diseases. In recent years, with the development of sequencing technology, the diagnosis standard of mitochondrial diseases has gradually evolved into: clinically screening suspected patients initially, diagnosing mitochondrial diseases through biochemical tests, and combining gene detection to identify pathogenic genes and mutations. However, the current diagnosis rate of mitochondrial diseases is relatively low, and the main reasons are as follows: on the one hand, there are numerous and continuously increasing pathogenic genes and mutation sites for mitochondrial diseases, and it is impossible to diagnose mitochondrial diseases through gene screening; on the other hand, the detection of the enzyme activity of the oxidative phosphorylation system requires highly invasive tissue biopsies, resulting in a low cooperation rate of patients and their families; in addition, due to the complex operation steps of enzyme activity detection in tissues and the variety of compounds involved, the normal reference intervals measured by the same laboratory at different times vary greatly, with poor stability. Therefore, establishing a non-invasive, simple, rapid, and stable mitochondrial oxidative phosphorylation complex detection system will contribute to the rapid and accurate diagnosis of mitochondrial diseases. Currently, there is no relevant report on detecting the structure of mitochondrial supercomplex based on the combination of PLA technology and HCR technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a probe combination and its application for detecting mitochondrial respiratory chain supercomplex based on proximity ligation assay (PLA) and hybridization chain reaction (HCR), so as to solve the problems existing in the above-mentioned prior art. By determining two adjacent protein subunits that can reflect the integrity of the supercomplex structure as the base, and using the principle of antigen-antibody binding, the antigen-antibody that recognizes and binds to the base is induced to undergo proximity ligation reaction (PLA) due to nucleic acid labeling. According to the principle of hybridization chain reaction (HCR), the detection signal is amplified cascadingly, and the activity of the oxidative phosphorylation system is determined by the integrity of the supercomplex structure, overcoming the deficiencies and limitations of traditional detection methods, and achieving rapid, highly sensitive, and accurate detection of the supercomplex structure. At the same time, by fixing cells on a 96-well fluorescence plate and using a multifunctional microplate reader to detect the integrity of the mitochondrial supercomplex structure, finally, parallel detection of up to 96 samples can be achieved simultaneously, realizing the digitization and high-throughput of detection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a PLA probe for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology. The PLA probe includes a proximity probe and a detection probe. The proximity probe includes antibody-nucleic acid conjugate I and antibody-nucleic acid conjugate II. Antibody-nucleic acid conjugate I is obtained by conjugating a DNA1 probe with a nucleotide sequence as shown in SEQ ID NO.1 and an antibody against mitochondrial respiratory chain supercomplexes. Antibody-nucleic acid conjugate II is obtained by conjugating a DNA2 probe with a nucleotide sequence as shown in SEQ ID NO.2 and an antibody against mitochondrial respiratory chain supercomplexes;

[0008] The detection probe includes a DNA3 probe with a nucleotide sequence as shown in SEQ ID NO.3 and a DNA4 probe with a nucleotide sequence as shown in SEQ ID NO.4.

[0009] Preferably, one of the DNA3 probe and the DNA4 probe is labeled with a fluorophore, and the other probe is labeled with a quencher.

[0010] Preferably, the antibody against mitochondrial respiratory chain supercomplexes conjugated to the DNA1 probe is an antibody against subunit NDUFB8 of mitochondrial complex I; the antibody against mitochondrial respiratory chain supercomplexes conjugated to the DNA2 probe is an antibody against subunit MT-CO1 of complex IV.

[0011] The present invention also provides a probe combination for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology and hybridization chain reaction, including the proximity probe and HCR nucleic acid probes. The HCR nucleic acid probes include an H1 probe with a nucleotide sequence as shown in SEQ ID NO.6 and an H2 probe with a nucleotide sequence as shown in SEQ ID NO.7.

[0012] Preferably, both ends of the H1 probe are labeled with a fluorophore and a quencher respectively.

[0013] The present invention also provides a kit for detecting mitochondrial respiratory chain supercomplexes, which includes the above-mentioned PLA probe or the above-mentioned probe combination.

[0014] The present invention also provides the use of the above-mentioned PLA probe or the above-mentioned probe combination in the preparation of a kit for detecting mitochondrial respiratory chain supercomplexes.

[0015] The present invention also provides a method for detecting mitochondrial respiratory chain supercomplexes for non-diagnostic purposes, including the following steps:

[0016] Contact the adjacent probe with the sample to be tested, so that the adjacent probe binds to the target protein in the sample to be tested, and then add the detection probe or the HCR nucleic acid probe to react, and judge the expression level of the mitochondrial respiratory chain supercomplex according to the fluorescence signal generated by the reaction system.

[0017] Preferably, when the detection probe is added to the reaction system, the molar concentration ratio of the adjacent probe to the detection probe is 1:2, and the reaction time is 2 h;

[0018] When the HCR nucleic acid probe is added to the reaction system, the molar concentration ratio of the adjacent probe to the HCR detection probe is 1:2, and the reaction time is 8 h.

[0019] The present invention also provides the application of the PLA probe or the probe combination in the preparation of a digital detection platform for detecting mitochondrial respiratory chain supercomplexes.

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

[0021] Based on the PLA detection principle combined with the HCR technology, the present invention realizes the analysis of the mitochondrial function integrity by detecting the structure of the mitochondrial respiratory chain supercomplex. The two core components of PLA include a pair of adjacent probes and a pair of detection probes. By designing two pairs of probes and using PAGE electrophoresis and fluorescence spectrometer detection to verify the establishment of the PLA detection platform, the optimal experimental conditions were finally determined (adjacent probe: fluorescence probe = 1:2, PLA incubation for 2 h). Subsequently, the specificity and sensitivity of the PLA detection platform were verified. The results showed that the PLA technology could specifically bind to the mitochondrial supercomplex, and in the kinetic experiments of mitochondrial assembly and disassembly, its detection sensitivity was better than that of BN-PAGE electrophoresis. The total experimental duration of PLA detection was 15 h - 19 h, which was much shorter than the experimental duration of BN-PAGE (42 h - 46 h). In addition, BN-PAGE has high requirements for protein content and sample preparation. Mild protein lysing agents (such as DDM) need to be used during protein extraction, which may affect the stability of the complex. Moreover, the gel preparation of BN-PAGE is different from conventional SDS-PAGE and requires a specific buffer system. The PLA technology, on the other hand, adopts the in-situ antibody-protein binding method to directly bind to the supercomplex subunits in cells. PLA has lower requirements for sample preparation and can detect low-abundance or weakly stable complexes.

[0022] To further improve the detection sensitivity, the present invention constructs a detection platform based on the combination of the PLA principle and the HCR technology. HCR has significant advantages such as isothermal, enzyme-free, simple operation, and low cost. It can form long-chain DNA through the alternating ring-opening self-assembly of DNA hairpins, realizing signal amplification for target molecules. The results show that the detection sensitivity of HCR is superior to that of PLA. In the assembly kinetics experiment of the supercomplex, mitochondrial supercomplexes can be detected 2 hours after the restoration of assembly. In addition, although the total experimental duration of HCR is 21 h - 24 h, which is longer than that of PLA, the experimental duration of HCR is still much shorter than that of BN-PAGE. The present invention provides a new method for the detection of mitochondrial supercomplexes.

[0023] The present invention also constructs a digital detection platform. The supercomplexes of cells fixed on a fluorescence plate are detected by the HCR technology, and the fluorescence signal intensity is output through a multifunctional microplate reader. Finally, it is expected to detect 96 samples simultaneously at one time, realizing high-throughput detection. In the future, it is expected to be widely used in the accurate detection of mitochondrial supercomplexes in clinical serum samples and the diagnosis of mitochondrial diseases. Brief Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is the schematic diagram of PLA detection;

[0026] Figure 2 It is the selection of supercomplex protein subunits; (A) Structural simulation diagram of the supercomplex CI1CIII2CIV1; (B) List of candidate subunits, screening out 9 subunits of CI and 4 subunits of CIV; (C) Spatial distance between CI subunits and CIV subunits;

[0027] Figure 3 It is the establishment and verification of the PLA system; (A) Schematic diagram of in vitro binding of sequences; (B) 12% PAGE gel analysis of the assembly of DNA components;

[0028] Figure 4 It is the calculation result of the copy number of respiratory chain supercomplexes; (A) Copy number of mitochondrial complex I protein; (B) Proportion of the complex CI1CIII2CIV1 in the supercomplexes assembled with the participation of complex I;

[0029] Figure 5Cy5 fluorescence spectra of PLA probes at different reaction conditions and copy numbers; (A) Cy5 fluorescence spectra of PLA detection probes; (B) Cy5 fluorescence spectra at different detection probe concentrations and different reaction times, with the ratio being the concentration of proximity probes (DNA1 + DNA2 + refDNA): concentration of detection probes (DNA3-Cy5 + DNA4-BHQ2).

[0030] Figure 6 Results of proximity probe synthesis verification; (A) Verification of PLA proximity probe coupling by PAGE gel; (B) UV-visible spectra of PLA proximity probes.

[0031] Figure 7 Results of intracellular detection by the PLA method and optimization of experimental conditions; (A) Fluorescence analysis under different experimental conditions; (B) Specificity verification of PLA detection probes, with CY5 being the detection probe pair (DNA3-Cy5, DNA4-BHQ2).

[0032] Figure 8 Results of specificity analysis of the PLA detection platform; (A) Localization of supercomplexes on the BN-PAGE membrane; (B) Protein content analysis after CAP removal of mitochondrial supercomplex assembly; (C) Protein content analysis of supercomplexes in 143B ρ0 cells; (D) Protein content analysis of supercomplexes in Hela COX7a2l KO cells; (E) Fluorescence analysis after CAP removal of mitochondrial supercomplex assembly; (F) Fluorescence analysis of supercomplexes in 143B ρ0 cells; (G) Fluorescence analysis of supercomplexes in Hela COX7a2l KO cells.

[0033] Figure 9 Kinetic experiments on the assembly of mitochondrial complexes and supercomplexes; (A - B) BN-PAGE and quantitative analysis of supercomplex protein content respectively; (C - D) Fluorescence analysis and quantitative analysis of supercomplexes respectively; TOM70 is the internal reference protein; n.s = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0034] Figure 10 Kinetic experiments on the assembly of mitochondrial complexes and supercomplexes (shortened time); (A - B) BN-PAGE and quantitative analysis of supercomplex protein content respectively; (C - D) Fluorescence analysis and quantitative analysis of supercomplexes respectively; TOM70 is the internal reference protein; n.s = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0035] Figure 11Results of the kinetic experiments on the disassembly of mitochondrial complexes and supercomplexes; (A-B) BN-PAGE and quantitative analysis of the protein content of supercomplexes, respectively; (C-D) Fluorescence analysis and quantitative analysis of supercomplexes, respectively; TOM70 is the reference protein; n.s = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0036] Figure 12 Schematic diagram of the HCR detection principle;

[0037] Figure 13 Establishment and validation of the HCR detection platform; (A) Schematic diagram of in vitro binding of sequences; (B) 10% PAGE gel analysis of the assembly of DNA components;

[0038] Figure 14 Fluorescence spectra of Cy5 at different reaction times;

[0039] Figure 15 Intracellular detection and experimental condition optimization by the HCR method; (A) Fluorescence detection analysis under different experimental conditions; (B) Fluorescence quantitative analysis under different experimental conditions;

[0040] Figure 16 Specificity analysis of the HCR detection platform; (A) Fluorescence analysis after removing mitochondrial supercomplex assembly by CAP; (B) Fluorescence analysis of supercomplexes in 143B ρ0 cells; (C) Fluorescence analysis of supercomplexes in Hela COX7a2l KO cells;

[0041] Figure 17 Kinetic experiments on the assembly of mitochondrial complexes and supercomplexes; (A) Fluorescence analysis of supercomplexes; (B) Fluorescence quantitative analysis of supercomplexes; n.s = no significant difference; ***P < 0.001; ****P < 0.0001;

[0042] Figure 18 Kinetic experiments on the disassembly of mitochondrial complexes and supercomplexes; (A) Fluorescence analysis of supercomplexes; (B) Fluorescence quantitative analysis of supercomplexes; ***P < 0.001; ****P < 0.0001;

[0043] Figure 19 Principle and flow chart of the mathematical detection platform;

[0044] Figure 20 Kinetic experiments on the assembly of mitochondrial complexes and supercomplexes; (A) Fluorescence analysis of supercomplexes; (B) Fluorescence quantitative analysis of supercomplexes; n.s = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0045] Figure 21 For the kinetic experiment of mitochondrial complex and supercomplex disassembly; (A) Fluorescence analysis of supercomplex; (B) Fluorescence quantitative analysis of supercomplex; n.s = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Detailed implementation manners

[0046] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0050] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0051] Example 1

[0052] 1. Experimental materials

[0053] Cell lines: Hela cells, 143B cells, and 143B ρ0 cells, which were kindly provided by the laboratory of Professor Gu Haihua of the School of Laboratory Medicine (School of Life Sciences), Wenzhou Medical University.

[0054] Plasmid: COX7A2L KO plasmid, purchased from Tsingke Biotechnology Co., Ltd.

[0055] The nucleic acid sequences involved in this example (as shown in Table 1) were all synthesized by Shanghai Sangon Biotech Co., Ltd. and purified by high performance liquid chromatography (HPLC).

[0056] Table 1 Nucleic Acid Sequences and Their Modifications

[0057]

[0058] Note: In Table 1, DNA3-Cy5 was obtained by ligating dT-Cy5 to the 5' end of the sequence shown in SEQ ID NO.3; DNA4-BHQ2 was obtained by ligating dT-BHQ2 to the 3' end of the sequence shown in SEQ ID NO.4; DNA1-SH was obtained by ligating a mercapto group to the 3' end of the sequence shown in SEQ ID NO.1; DNA2-SH was obtained by ligating a mercapto group to the 5' end of the sequence shown in SEQ ID NO.2; H1-Cy5 was obtained by inserting dT-BHQ2 between the 12th and 13th positions and dT-Cy5 between the 43rd and 44th positions of the sequence shown in SEQ ID NO.6.

[0059] 2. Establishment of the PLA method

[0060] 2.1 Incubation of DNA sequences

[0061] First, dissolve 5 single-stranded DNAs, DNA1, DNA2, DNA3, DNA4, and ref DNA, in 1×PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM. Subsequently, place the single-stranded stock solution in a water bath at 95°C for 5 min for denaturation, then take it out and place it at room temperature for 30 min. Subsequently, configure equimolar amounts of the 5 single-stranded stock solutions in a 1.5 mL EP tube according to different strand-binding situations 1:1, mix them with a vortex oscillator, and incubate them in a water bath at 37°C for 2 h. During this incubation process, the DNA single strands hybridize with each other through the principle of proximity ligation and complete the strand displacement reaction.

[0062] 2.2 Verification by PAGE gel

[0063] Prepare a 12% PAGE gel. Subsequently, mix 5 µL of different samples to be tested with 1 µL of 6× loading buffer and load the mixture into the wells. Use 1× TBE buffer as the electrophoresis buffer, set the constant voltage of the electrophoresis apparatus to 110 V at room temperature, and run for 70 minutes. After electrophoresis is completed, slowly take out the PAGE gel for cutting and staining. Immerse the gel in the nucleic acid staining solution and react on a shaker for 30 min. Then take it out, take pictures using a gel imaging analysis system, and perform electrophoresis band analysis.

[0064] Before the electrophoresis experiment, 10 samples were prepared. The samples in each lane were set as follows: the left and right lanes were both markers, lane 1 was DNA1, lane 2 was DNA2, lane 3 was DNA1 + DNA2, lane 4 was DNA3, lane 5 was DNA4, lane 6 was DNA3 + DNA4, lane 7 was DNA1 + DNA2 + DNA3 + DNA4, lane 8 was ref DNA, lane 9 was DNA1 + DNA2 + refDNA, and lane 10 was DNA1 + DNA2 + DNA3 + DNA4 + ref DNA.

[0065] 3. In vitro detection of DNA sequence binding fluorescence signal

[0066] 3.1 Incubation of DNA fluorescent sequences

[0067] The ratio of DNA3-Cy5 to DNA4-BHQ2 and the incubation time were carefully adjusted. Different combinations of ratios and time gradients were designed, including 1 µM, 2 µM, 3 µM DNA3-Cy5:DNA4-BHQ2 and incubation times of 2, 4, 8 h. First, dissolve 3 single-stranded DNAs, DNA1, DNA2, and ref DNA, in 1× PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM. In addition, dissolve 2 detection probe single-stranded DNAs, DNA3-Cy5 and DNA4-BHQ2, in 1× PBS buffer to prepare single-stranded stock solutions with final concentrations of 1 µM, 2 µM, 3 µM. Since DNA3-Cy5 and DNA4-BHQ2 are strands with fluorescent modification groups, they should be placed in 1.5 mL brown light-proof EP tubes. Subsequently, place the single-stranded stock solutions in a water bath at 95 °C for 5 min of denaturation, take them out and place them at room temperature for 30 min. Then, prepare 5 single-stranded stock solutions with equal concentrations and equal molar amounts in 1.5 mL brown light-proof EP tubes according to different strand binding situations, mix them with a vortex oscillator, and incubate them in a water bath at 37 °C for 2 h, 4 h, 8 h respectively, and detect the fluorescence intensity using a fluorometer.

[0068] 3.2 Verification by fluorometer

[0069] Place 200 µL of the final reaction product in a quartz cuvette and record the fluorescence spectrum from 650 nm to 800 nm at an excitation wavelength of 646 nm, an emission wavelength of 664 nm, and a slit size of 5 nm using an F-7000 fluorescence spectrophotometer at room temperature; and measure the fluorescence intensity at 650 nm.

[0070] 4. Preparation and verification of the PLA probe

[0071] 4.1 Preparation of the PLA probe

[0072] First, dilute the Ab1 solution (anti-NDUFB8 (0.65 mg / mL)) to 0.1 mg / mL using PBS1 buffer. Add 50 µL of the sulfo-SMCC solution to 450 µL of the above-prepared Ab1 solution (0.1 mg / mL) to prepare a 500-µL mixture, and incubate it with shaking at room temperature for 2 h. The activated Ab1 solution is ultrafiltered and purified through a 10 KD cut-off membrane (Millipore, 10000 rpm, 10 min), and then dissolved in 500 µL of PBS2 buffer. Second, reduce 3 µL of 100 µM DNA1-SH with 4 µL of 100 mM DTT in 500 µL of PBS1 buffer at 37 °C for 1 h. The reduced DNA1 solution is ultrafiltered and purified through a 10 KD cut-off membrane (Millipore, 10000 rpm, 10 min), and then dissolved in 500 µL of PBS2. Third, mix 500 µL of the activated Ab1 solution with 500 µL of the above-prepared reduced DNA1-SH and incubate overnight at 4 °C. Then, wash it with PBS1 buffer at least 3 times using a 10KD cut-off membrane (Millipore, 10 000 rpm, 10 min). The remaining DNA1Ab1 conjugate after ultrafiltration is redissolved in 1 mL of PBS2 buffer as the stock solution of the recognition probe DNA1Ab1. The operation of the recognition probe DNA2Ab2 (anti-MT-CO1 and DNA2-SH) is the same as above.

[0073] PBS1 buffer: 55 mM Na2HPO4, 150 mM NaCl and 20 mM EDTA, adjusted to pH 7.40 with HCl, stored at room temperature.

[0074] PBS2 buffer: 55 mM NaH2PO4, 150 mM NaCl and 5 mM EDTA, adjusted to pH 7.40 with HCl, stored at room temperature.

[0075] Sulfo-SMCC solution: Accurately weigh 4.95 mg of sulfo-SMCC powder, dissolve it in 1 mL of DMSO, and store it in the refrigerator at -30°C in the dark.

[0076] 4.2 Verification of PLA probe coupling

[0077] 4.2.1 Verification by PAGE gel

[0078] Prepare a 12% PAGE gel, and the steps and procedures for running the gel are the same as in 2.2. The lanes and samples are the ultrafiltered DNA1, DNA2, DNA1Ab1, and DNA2Ab2 solutions respectively. After the PAGE gel is stained by soaking, use a gel imaging system for analysis.

[0079] 4.2.2 Ultraviolet detection

[0080] Place 200 μL of the final reaction product in a quartz cuvette and scan it at room temperature using an ultraviolet spectrophotometer to measure the ultraviolet-visible absorbance of Ab-DNA at 260 nm and 280 nm.

[0081] 5. Cell culture

[0082] 5.1 Cell culture and subculture

[0083] (1) Hela cells:

[0084] Culture medium: DMEM complete medium (containing 12% calf serum (CS)), labeled as 12CS;

[0085] Culture environment: 37°C, 5% CO2 incubator;

[0086] Medium change and subculture: Discard the medium, add an appropriate amount of fresh medium, and change the medium every 2 days or when the medium turns yellow. Subculture when the growth density reaches 80%. Discard the medium, rinse once with PBS, add trypsin cell digestive solution, observe the cell shrinkage under an inverted microscope, then add complete medium to stop digestion, pipette the cells, and subculture at a ratio of 1:3.

[0087] (2) Hela cells treated with CAP:

[0088] Culture medium: DMEM plus complete medium (containing 12% CS, 5 mg / mL uridine, and 4 mg / mL chloramphenicol (CAP)), labeled as: 12CS+CAP+U;

[0089] Culture environment: 37°C, 5% CO2 incubator;

[0090] Medium change and subculture: Aspirate and discard the medium, add an appropriate amount of fresh medium, and change the medium every day. Subculture when the growth density reaches 80%, and the steps are the same as in (1).

[0091] (3) After treating Hela cells with CAP, remove CAP:

[0092] Culture media: 12CS + CAP + U, 12CS + U;

[0093] Culture environment: 37°C, 5% CO2 incubator;

[0094] Medium change and subculture: Aspirate the culture medium, add an appropriate amount of fresh 12CS + CAP + U medium, change the medium every day, and subculture when the growth density reaches 80%. The steps are the same as in (1); After treating cells with CAP for 7 days, the translation of most mitochondrial proteins in cells is inhibited. At this time, it is Day0 of CAP treatment. According to different experimental groups, change the medium to 12CS + U after different treatment times.

[0095] (4) 143B cells and 143B ρ0 cells:

[0096] Culture media: DMEM complete medium (containing 10% FBS and 5 mg / mL uridine), labeled as 10% FBS + U;

[0097] Culture environment: 37°C, 5% CO2 incubator;

[0098] Medium change and subculture: Aspirate the culture medium, add an appropriate amount of fresh medium, change the medium every day, and subculture when the growth density reaches 80%. The steps are the same as in (1).

[0099] 5.2 Cell resuscitation and cryopreservation

[0100] Cell resuscitation: Take out the cells from liquid nitrogen, quickly put them into a 37°C water bath, and melt the frozen cells within 1 min; Centrifugation: 500×g, 3 min, centrifuge at room temperature; Aspirate the supernatant, resuspend with complete medium and add it to a culture dish for subsequent culture;

[0101] Cell cryopreservation: Aspirate the culture medium, rinse once with PBS, then add an appropriate amount of trypsin cell digestive solution to digest the cells. Aspirate the digestive solution, stop digestion with complete medium, pipette and collect the cells into a centrifuge tube; Centrifugation: 500×g, 3 min, at room temperature; Discard the supernatant, resuspend the precipitate with 1 mL cell cryopreservation solution, transfer to a cell cryopreservation tube, put it into a gradient freezing box, overnight; Transfer to liquid nitrogen for storage on the second day.

[0102] 6. Optimization of intracellular detection conditions for PLA probes

[0103] 6.1 Cell seeding

[0104] (1)Place 9 sets of cell slides in a 24-well plate, with 3 slides in each set. Immerse them in 1 mL of alcohol for 30 min, then rinse with 1 mL of PBS. Replace the PBS after 5 min and repeat the rinsing three times in total;

[0105] (2)Seed 1×10 4 cells on each slide. After two days, when the cell density reaches 60%-70%, take out the 24-well plate and prepare for probe detection.

[0106] 6.2 Probe Incubation and Confocal Microscopy

[0107] (1)Remove the culture medium, rinse twice with PBS, and add 500 μL of ice-cold methanol that has been pre-cooled in a -30°C refrigerator for 20 min, and fix at room temperature for 20 min;

[0108] (2)Remove the ice-cold methanol and rinse twice with PBS, 5 min each time;

[0109] (3)Add 500 μL of 1% TritonX-100 and permeabilize for 10 min;

[0110] (4)Remove the permeabilization solution and rinse 3 times with PBS, 5 min each time;

[0111] (5)Add 500 μL of 5% BSA blocking solution and block for 10 min;

[0112] (6)Add 150 μL of primary antibody dilution (Anti-TOM70), 50 μL of conjugated probe DNA1Ab1, and 50 μL of conjugated probe DNA2Ab2, and incubate overnight on a shaker in a 4°C refrigerator; The antibody dilution is prepared by adding 30 μL of TritonX-100 to 10 mL of 1×PBS, mixing well, then adding 0.1 g of BSA and mixing well. The antibody dilution used in subsequent experiments is the same;

[0113] (7)Recover the primary antibody mixture (it can be reused 3-5 times), and rinse 3 times with PBS on a shaker, 5 min each time;

[0114] (8)Start avoiding light;

[0115] (9)Add 150 μL of secondary antibody dilution (Anti-Rabbit Alexa Fluor® 488), 50 μL of fluorescent probe DNA3-CY5, and 50 μL of fluorescent probe DNA4-BHQ2. The experimental conditions for the 9 groups are shown in the following table:

[0116] Table 2 Experimental Groups and Conditions

[0117]

[0118] (10) Incubate in the dark at room temperature on a shaker according to the above conditions;

[0119] (11) Remove the mixture and rinse 3 times with PBS on a shaker for 5 min each time;

[0120] (12) Add 4 μL of anti-quenching mounting medium (containing DAPI, Beyotime) on the glass slide, outline the cell culture insert, and make the side with cells contact the anti-quenching mounting medium. After it dries slightly, add nail polish around to fix it. Place it in a dark box and store it in a refrigerator at 4 °C for later imaging;

[0121] (13) After confocal microscopy imaging, take pictures of the last three channels (DAPI: 405 nm, TOM70: 488 nm, CY5: 638 nm) and the merged image and save them (image in time, store at 4 °C for at most 7 days);

[0122] (14) After imaging, analyze the pictures with Image J software. Outline at least 15 cells to analyze their fluorescence intensity. Use the fluorescence intensity to represent the content of supercomplexes in the cells, calibrate with the average fluorescence intensity of the control cells as 1, and perform statistical analysis on the results.

[0123] 7. Intracellular detection of probes

[0124] 7.1 Cell seeding

[0125] The seeding steps are the same as in 6.1. Divide the Hela cells into 7 groups, with three independent replicates in each group, and a total of 21 cell culture inserts are placed.

[0126] 7.2 Probe incubation and confocal imaging

[0127] The experimental steps are the same as in 6.2. The groups in step (6) are shown in the following table:

[0128] Table 3 Experimental groups and conditions

[0129]

[0130] In step (9), the mixture added to group 6 is 150 μL of secondary antibody dilution and 100 μL of PBS. The other groups are 150 μL of secondary antibody dilution, 50 μL of 2 μM DNA3-CY5, and 50 μL of 2 μM DNA4-BHQ2.

[0131] 8. Native gradient polyacrylamide gel electrophoresis (BN-PAGE)

[0132] Sample preparation steps are as follows:

[0133] (1)Collect cell pellet. Add 40 μL of lysis buffer A (10 μL of PMSF protease inhibitor (Sigma - Aldrich) is added to 1 mL of lysis buffer A) and 20 μL of 10% digitonin (a strong - irritant non - ionic detergent, Sigma - Aldrich) to every 10 mg of cell pellet. Pipette to mix well and lyse on ice for 20 min;

[0134] Lysis buffer A: 50 mM sodium chloride, 50 mM Tris - Base, 2 mM 6 - aminohexanoic acid, 1 mM EDTA. After dissolving in deionized water, adjust the pH to 7.4 and store at 4 °C;

[0135] (2)Centrifuge: 20000×g, 20 min, 4 °C;

[0136] (3)Transfer the supernatant and measure the protein concentration according to the instructions of the BCA protein concentration assay kit (Thermo Fisher Scientific);

[0137] (4)Dilute the supernatant to 1× with 6× loading buffer and keep it on ice for standby;

[0138] 6× loading buffer: Mix 80% glycerol and cathode buffer B in a ratio of 1:1 and store at 4 °C;

[0139] (5)Take 20 μg of total protein and add 5× loading buffer according to the volume ratio, denature at 95 °C for 5 min, and use it for WB as the loading amount correction;

[0140] 5× loading buffer: 2.5 mL of 20% (w / v) SDS, 0.5 mL of mercaptoethanol, 4.25 mL of glycerol, 5 mg of bromophenol blue. Make up the volume to 0.5 L with ddH2O and store at room temperature;

[0141] Perform native gradient polyacrylamide gel electrophoresis on the above - prepared samples. Electrophoresis program 1: Constant voltage electrophoresis at 45 V for 50 min; Program 2: Constant voltage electrophoresis at 180 V for 150 min. The inner tank solution is cathode buffer B, and the outer tank solution is 1× imidazole. The whole electrophoresis process is carried out on ice.

[0142] 9. Western blot (WB)

[0143] (1)After the electrophoresis program is completed, take out the gel, cut off the stacking gel, and soak the gel in the transfer buffer;

[0144] (2)Soak the PVDF membrane in methanol for 1 min, then transfer it to the transfer buffer and wash it;

[0145] (3) Arrange them in order from bottom to top as follows: sponge - 2 layers of transfer filter paper - glue - PVDF membrane - 2 layers of transfer filter paper - sponge, arrange them closely and expel air bubbles;

[0146] (4) Place the "sandwich" model for membrane transfer into the membrane transfer apparatus according to the corresponding electrodes, immerse the membrane transfer clamp with the electrotransfer buffer, and place an ice box in the empty space;

[0147] (5) Membrane transfer procedure: 100 V, 120 min (set different membrane transfer times according to the molecular weight of the protein, generally 1 min for 1 KD), and the whole process of membrane transfer is carried out on ice;

[0148] (6) Blocking: Immerse the PVDF membrane in 5% skim milk on a shaker at room temperature for 2 h;

[0149] (7) Wash the membrane: Wash the membrane on a shaker with 1×TBST, 5 min each time, for a total of 3 times;

[0150] (8) Incubate with the primary antibody: Cut the whole lanes of different groups and place them in the primary antibody, and block them on a shaker at 4°C for 24 h;

[0151] (9) Wash the membrane: Wash the membrane on a shaker with TBST, 5 min each time, for a total of 3 times;

[0152] (10) Incubate with the secondary antibody: Select the correct secondary antibody according to the property of the primary antibody, and block it on a shaker at 4°C for 24 h;

[0153] (11) Wash the membrane: Wash the membrane on a shaker with TBST, 5 min each time, for a total of 3 times;

[0154] (12) Band development: Prepare the developing solution, cover the PVDF membrane with the developing solution, incubate it on a shaker at room temperature, stop it by rinsing with running water after development, mark the bands and the date, and save the electronic image after scanning with a scanner.

[0155] The list of antibodies is shown in Table 4:

[0156] Table 4 List of antibodies

[0157]

[0158] 10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS - PAGE)

[0159] Sample preparation, the steps are as follows:

[0160] (1) Add 50 μL of 1×RIPA lysis buffer (add 10 μL of PMSF to each mL of lysis buffer) to every 10 mg of protein precipitate, pipette the precipitate, and lyse it on ice for 10 min;

[0161] (2) Centrifugation: 14000×g, 10 min, 4°C;

[0162] (3) Transfer the supernatant without aspirating the lower precipitate;

[0163] (4) Determine the concentration using a BCA protein assay kit. Take 20 - 30 μg of the protein sample and add 5× loading buffer, then react in a metal bath at 95 °C for 5 min;

[0164] Detect the prepared samples by SDS - PAGE electrophoresis.

[0165] 11. Construction of the COX7A2L knockout cell model (Hela - COX7A2L - KO)

[0166] Plasmid transfection and verification are as follows:

[0167] (1) When the growth density of Hela cells in a 6 - well plate reaches 60%, perform transfection according to the operating steps of the lipofectamine3000 transfection kit to transfer the COX7A2L plasmid into the cells;

[0168] (2) After 48 h, check the transfection efficiency under a fluorescence microscope;

[0169] (3) Limited dilution: Dilute to 1 cell / well in a 96 - well cell culture plate;

[0170] (4) Select monoclonal cells with fluorescence: The next day, select the wells expressing green fluorescence under a fluorescence microscope and check that there is only one cell in the well under an ordinary optical microscope as the cells to be verified;

[0171] (5) When expanding the culture to a 6 - well plate, collect the cell precipitate for verification of the protein knockout level. After 3 verifications, obtain the Hela - COX7A2L knockout cell model.

[0172] 12. Assembly and disassembly analysis of mitochondrial complexes and supercomplexes

[0173] 12.1 Assembly analysis of mitochondrial complexes and supercomplexes

[0174] (1) According to the setting of dynamic detection time points, plant 6 dishes of Hela cells in small dishes. The number of cells planted can grow to confluence in about 6 - 8 days according to the cell doubling time, and culture them with DMEM high - glucose medium containing 10% fetal bovine serum and 1% triple antibody;

[0175] (2) After 24 h, when the cells are well - adhered, add CAP to the culture medium of 5 of the dishes (add 10 μL of CAP to every 10 mL of culture medium) and continue culturing for 6 days to remove the mitochondrial complexes and assembled supercomplexes originally synthesized by the cells;

[0176] (3)The culture medium containing CAP needs to be changed daily; otherwise, the drug efficacy cannot be guaranteed.

[0177] (4)On the 7th day, discard the culture medium containing CAP, wash the cells 3 times with pre-warmed PBS, and replace it with a conventional culture medium for cultivation to allow the mitochondrial complexes to re-synthesize and start re-assembling into supercomplexes.

[0178] (5)After culturing in a 37°C incubator for 0 h, 12 h, 24 h, 48 h, and 72 h, collect the cell pellet, wash the cell pellet once more with PBS, and freeze the pellet in an -80°C refrigerator.

[0179] (6)After collecting all the cell pellets at all time points, extract the proteins with digitonin and perform non-denaturing gradient polyacrylamide gel electrophoresis.

[0180] 12.2 Assembly analysis of mitochondrial complexes and supercomplexes (shortening the assembly time)

[0181] (1)In subsequent experiments, further analyze the assembly of mitochondrial complexes by shortening the assembly time. The cell culture procedure is the same as in 12.1 at this time.

[0182] (2)After 7 days of CAP treatment, change the culture medium containing CAP to a normal culture medium. After culturing in a 37°C incubator for 0 h, 4 h, 8 h, 12 h, and 24 h, collect the cell pellet, wash the cell pellet once more with PBS, and freeze the pellet in an -80°C refrigerator.

[0183] (3)After collecting all the cell pellets at different time points, extract the proteins with digitonin and perform non-denaturing gradient polyacrylamide gel electrophoresis.

[0184] 12.3 Disassembly analysis of mitochondrial complexes and supercomplexes

[0185] (1)According to the settings of the dynamic detection time points, seed 5 dishes of Hela cells in small dishes. The number of seeded cells can cover the dish in about 4 - 6 days according to the cell doubling time, and culture them with DMEM high-glucose medium containing 10% fetal bovine serum and 1% triple antibody.

[0186] (2)When the cell density reaches about 40%, change the medium, and add CAP to the medium according to the time of 6, 4, 2, and 1 days (add 10 μL of CAP to every 10 mL of culture medium) and continue culturing to remove the originally synthesized mitochondrial complexes and assembled supercomplexes in the cells.

[0187] (3)The culture medium containing CAP needs to be changed daily; otherwise, the drug efficacy cannot be guaranteed.

[0188] (4) After culturing in a 37 °C incubator for 6 days, collect the cell pellet, rinse it once with PBS, and after collection, extract the protein with digitonin and perform non-denaturing gradient polyacrylamide gel electrophoresis.

[0189] Establishment of the 13 HCR Detection Platform

[0190] 13.1 Reagent Preparation

[0191] 10% PAGE Gel: 3.3 mL of 30% acrylamide mixture, 2.5 mL of 5× TBE buffer, 4 mL of ddH2O, 100 μL of 10% ammonium persulfate (AP), 10 μL of TEMED.

[0192] 13.2 Incubation of DNA Sequences

[0193] First, dissolve 5 single-stranded DNAs, DNA1, DNA2, H1, H2, and refDNA, in 1× PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 μM. Subsequently, place the single-stranded stock solution in a water bath at 95 °C for 5 min of denaturation, then take it out and place it at room temperature for 30 min. Subsequently, configure equal molar amounts of the 5 single-stranded stock solutions in a 1.5 mL EP tube according to different strand-binding situations at a ratio of 1:1, mix with a vortex oscillator, and incubate in a water bath at 37 °C for 8 h. During this incubation process, the DNA single strands hybridize with each other according to the principle of proximity ligation and complete the hybridization chain reaction.

[0194] 13.3 Verification of PAGE Gel

[0195] Prepare a 10% PAGE gel, then mix 5 μL of different samples to be tested with 1 μL of 6× loading buffer and load the sample into the loading well. Use 1× TBE buffer as the electrophoresis buffer, set the constant voltage of the electrophoresis apparatus to 110 V at room temperature and run for 90 minutes. After electrophoresis is completed, slowly take out the PAGE gel for cutting and staining. Immerse the gel in the nucleic acid staining solution and react on a shaker for 30 min, then take it out, take pictures with a gel imaging analysis system and perform electrophoresis band analysis.

[0196] Before the electrophoresis experiment, 7 samples were prepared, and the corresponding sample information for the lane settings was: the left and right lanes were both markers, lane 1 was DNA1, lane 2 was DNA2, lane 3 was DNA1 + DNA2, lane 4 was DNA1 + DNA2 + refDNA, lane 5 was H1 + H2, lane 6 was DNA1 + DNA2 + H1 + H2, lane 7 was DNA1 + DNA2 + H1 + H2 + refDNA.

[0197] 14. In Vitro Detection of Fluorescent Signals of the HCR Detection Platform

[0198] 14.1 Incubation of DNA Fluorescent Sequences

[0199] To obtain the best experimental results, a series of detailed experimental optimization steps were carried out. Since the optimal reaction conditions for detecting the in vitro fluorescence of the PLA platform were determined in the early stage, with the concentration ratio of the proximity probe to the detection probe being 1:2 and incubating at 37 °C for 2 h, the same basic conditions were set when detecting the HCR platform. Subsequently, the fluorescence intensity was detected by changing the reaction time. First, five single-stranded DNAs, DNA1, DNA2, H1-Cy5, H2, and refDNA, were dissolved in 1×PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM, and H1-Cy5 should be placed in the dark. Subsequently, the single-stranded stock solution was placed in a water bath at 95 °C for 5 min, taken out and placed at room temperature for 30 min. Then, equal-concentration and equal-molar amounts of the five single-stranded stock solutions were configured in 1.5 mL brown light-shielded EP tubes according to different chain-binding situations, mixed with a vortex oscillator, and incubated in a water bath at 37 °C for 2 h, 4 h, 8 h, 16 h, 24 h, and 32 h respectively, and the fluorescence intensity was detected by a fluorometer.

[0200] 14.2 Verification of the Fluorometer

[0201] The detection steps are the same as those in 3.2.

[0202] 15. Optimization of the Intracellular Detection Conditions of HCR Probe Fluorescence

[0203] 15.1 Cell Seeding

[0204] The experimental steps are the same as those in 6.1.

[0205] 15.2 Probe Incubation and Confocal Microscopy Imaging

[0206] Add 150 μL of secondary antibody dilution (Anti-Rabbit Alexa Fluor® 488) and 100 μL of detection probe during the first incubation, and the experimental conditions are shown in the following table:

[0207] Table 5 Experimental Groups and Conditions

[0208]

[0209] The remaining experimental steps are the same as those in 6.2.

[0210] 16. Establishment and Verification of the Digital Detection Platform

[0211] 16.1 Cell Treatment

[0212] (1)After treating Hela cells with CAP for 7 days, change the culture medium from 12CS + CAP + U to 12CS + U at 0, 1, 2, 4, 8, 12, and 24 h groups;

[0213] (2)Seed 6 dishes of Hela cells and change the culture medium from 12CS + U to 12CS + CAP + U at 0, 1, 2, 4, 6, and 7 day groups;

[0214] (3)Detach the cells, count 5000 cells / well using a flow cytometer, and seed them into 96-well plates;

[0215] (4)After culturing for 48 h, when the cells are in a good adherent state, take them out for subsequent experiments.

[0216] 16.2 Probe Incubation and Detection with a Multifunctional Microplate Reader

[0217] (1)Remove the culture medium, rinse twice with PBS, add 100 μL of ice-cold methanol pre-cooled in a -30°C refrigerator for 20 min, and fix at room temperature for 20 min;

[0218] (2)Remove the ice-cold methanol, rinse once with PBS for 5 min, gently, the same below;

[0219] (3)Add 100 μL of 1% TritonX-100 to each well for permeabilization for 10 min;

[0220] (4)Remove the permeabilization solution, rinse once with PBS for 5 min;

[0221] (5)Add 100 μL of 5% BSA blocking solution to each well and block for 10 min;

[0222] (6)Start shielding from light;

[0223] (7)Add 50 μL of the proximity probe and 50 μL of the detection probe to each well and incubate on a shaker at room temperature for 8 h;

[0224] (8)Rinse once with PBS for 5 min;

[0225] (9)Add 50 μL of DAPI detection solution diluted 1:100 with PBS to each well and incubate at room temperature for 10 min;

[0226] (10)Rinse once with PBS for 5 min;

[0227] (11) Use a multifunctional microplate reader to detect the excitation light of a 96-well fluorescence plate, setting two wavelengths. The first is DAPI fluorescence, with an excitation light of 340 nm and an emission light of 405 nm; the second is Cy5 fluorescence, with an excitation light of 649 nm and an emission light of 679 nm. After the detection is completed, use the mean value of Cy5 fluorescence / DAPI fluorescence to obtain the relative fluorescence intensity.

[0228] 17. Statistical methods

[0229] Statistical analysis was performed using Prism 8.0 (GraphPad) to analyze the data. All data for statistical analysis were obtained from three independent repeated experiments. For the part of two groups of research objects, a t-test was used to compare pairwise, and the data were statistically analyzed. For those with a significance level below (P<0.05), it was considered to be different, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0230] 18. Results and analysis

[0231] 18.1 PLA detection principle

[0232] The construction and operation principle of the PLA detection platform is as Figure 1 shown. The PLA detection platform has two key components: (1) a pair of proximity ligation probes DNA1Ab1 and DNA2Ab2; (2) a pair of detection probes including a DNA3-Cy5 with a fluorescent group and a DNA4-BHQ2 with a quenching group. When the antibodies conjugated on the proximity probes recognize the corresponding proteins on the mitochondrial respiratory chain supercomplex, the spatial distance between the proximity probes is shortened, so that the single-stranded DNA base complementary sequences on the probes are combined, and a longer single-stranded DNA is exposed. At this time, the recognition probes DNA3-Cy5 and DNA4-BHQ2 are added. DNA3-Cy5 is modified with the fluorescent group Cy5, and because it binds to the DNA4-BHQ2 part with the quenching group BHQ2, the fluorescence is quenched and cannot be detected. The single-stranded DNA exposed by the proximity probes can completely bind to the recognition probe DNA3. Therefore, DNA3-Cy5 is displaced from DNA4-BHQ2 and binds to the proximity probes, emitting a fluorescent signal, thereby verifying the integrity of the mitochondrial supercomplex structure.

[0233] 18.2 Selection of supercomplex protein subunits

[0234] The mitochondrial respiratory chain supercomplex is mainly assembled from Complex I + III2 + IV, and it is also known as the respirasome. The 5XTH (Cryo-EM structure of human respiratory supercomplex) file was downloaded from the PDB database, including the fasta sequence file (rcsb_pdb_5XTH.fasta) and the three-dimensional structure PDB file (5xth-pdb-bundle). The UCSF Chimera software was used to view the PDB file and calculate distances. For a set of atoms, the centroid is a point in space that represents the center of the mass distribution of this set of atoms. In molecular structure analysis, calculating the centroid of a subunit or the entire molecule helps to understand its mass distribution characteristics. For example, in the study of protein-protein interactions, calculating the centroids of the two interacting proteins respectively can analyze the relative position changes during their binding process. The centroid is often used to evaluate the stability of molecules, determine the central position of intermolecular interactions, etc. For instance, in molecular dynamics simulations, tracking the movement trajectory of the centroid can understand the overall translational motion of the molecule.

[0235] Nine subunits of Complex I (CI) (NDUFV1, NDUFA6, NDUFA5, NDUFB9, NDUFV3, NDUFS4, NDUFS6, NDUFA2, NDUFB8) and four subunits of Complex IV (CIV) (MT-CO1, COX4, COX5A, COX5B) were screened according to the HPA database and the PDB database, and their relative positions on the PDB model were compared and marked. The spatial distance between the two was obtained by calculating the centroid between the proteins. Among them, the spatial distance between NDUFB8 and MT-CO1 was the closest and met the requirements of the PLA detection principle. Therefore, the two protein subunits NDUFB8 and MT-CO1 were selected in this invention to construct the PLA proximity probe, as shown in Figure 2 .

[0236] 18.3 Establishment and verification of the PLA detection platform

[0237] 18.3.1 Establishment and PAGE verification of the PLA detection platform

[0238] The DNA sequences were designed using the Nupack website and their binding was predicted, and then the construction of the PLA platform was verified by 12% denaturing polyacrylamide gel electrophoresis (PAGE) ( Figure 3). As shown in the figure, DNA1 and DNA2 represent a pair of proximity probes, DNA3 and DNA4 represent a pair of detection probes, and refDNA is used as an extracellular substitute for the mitochondrial supercomplex to shorten the spatial distance between the proximity probes. Lanes 4 and 10 show that when there is only a pair of proximity probes, they cannot bind due to the large spatial distance, and when refDNA exists, the proximity probes and refDNA can be combined to produce new connection products. Lane 7 shows that DNA3 and DNA4 can be combined. Lane 11 shows that when the proximity probe and the detection probe are present at the same time, DNA3 can be displaced and combined with the proximity probe under the connection effect of refDNA. The above results can prove the successful establishment of the PLA platform.

[0239] 18.3.2 Calculation of respiratory chain supercomplex copy number

[0240] The mitochondrial bottleneck effect refers to the "bottleneck effect" of certain mitochondrial genes or mitochondrial mutations during the cell division of mitochondrial DNA, especially during the development of egg cells and the process of genetic transmission. This effect causes the number of mitochondrial DNA copies to decrease during the genetic transmission process, ultimately affecting the diversity of the mitochondrial gene pool in future generations. Studies have shown that almost all complex I proteins are involved in the assembly of mitochondrial supercomplexes. Therefore, the minimum number of copies of mitochondrial complex I proteins determines the number of copies of mitochondrial supercomplexes. Figure 4 As shown in Figure A, the copy numbers of all proteins in complex I in a single cell were statistically analyzed, and it was found that the low copy number of CI protein in a single cell ranged from 4967 to 8222. The proportion of complex CI1CIII2CIV1 in the super complex assembled by complex I was then quantitatively analyzed, as shown in Figure 2. Figure 4 As shown in B, the proportion of supercomplex CI1CIII2CIV1 is 34.14%, so the copy number of mitochondrial supercomplex CI1CIII2CIV1 in a single cell is about 1.7×10 3 ~6.9×10 5 .

[0241] 18.3.3 Fluorescence Spectrometer Verification

[0242] The concentration and incubation time of the fluorescent probe DNA3-Cy5 as a detection probe have a direct impact on the fluorescence detection efficiency. By setting different concentration gradients and action times, the fluorescence spectrum is analyzed in detail. When the copy number of the detection probe concentration is lower than the mitochondrial copy number, the probe cannot be completely combined with the mitochondrial complex. Therefore, the present invention obtains the copy number of the detection probe by calculation. According to the DNA copy number calculation formula: (6.02×10 23copies / mol) × (concentration g / mL) / (MW g / mol) = copies / mL, it can be obtained that the copy number of the detection probe at 100 μM in each microliter of the reaction solution is 1.83×10 10 . Since the maximum copy number of the mitochondrial supercomplex is 1.7×10 3 , usually 20,000 cells are seeded in a 24-well plate, so the maximum copy number of the total mitochondrial supercomplex is 3.39×10 7 . In a 24-well plate, a total of 100 μL of the detection probe is added. In order to make the probe copy number slightly greater than the mitochondrial complex copy number and meet the added volume, the concentration of the detection probe needs to be diluted to 1 μM. The copy number of the probe in the 96-well plate was calculated according to the same method, and the results are shown in Table 6. The copy number of the PLA probe is much larger than that of the mitochondrial supercomplex. Therefore, the probe concentration of 1 μM and the reaction time of 2 h are used as the basic conditions. Subsequently, by changing the ratio of the detection probe to the proximity probe and the reaction time of PLA, different experimental control groups are set. As Figure 5 shown in A, when DNA3-Cy5 binds to DNA4-BHQ2, its fluorescence signal only shows extremely weak fluorescence due to quenching. And only when refDNA exists, DNA1 and DNA2 can undergo a proximity ligation reaction, causing DNA3-Cy5 to be displaced from DNA4-BHQ2 and combine with DNA1 and DNA2, emitting a fluorescence signal. The results show that the detected fluorescence signal increases with the increase of the ratio of the detection probe to the proximity probe and the extension of the total reaction time, and the ratio has a more significant effect on the fluorescence intensity, as shown in Figure 5 B.

[0243] Table 6 PLA probe copy numbers in 24-well plates and 96-well plates Mitochondrial complex I protein copy numbers

[0244]

[0245] 18.3.4 Intracellular verification and experimental condition optimization

[0246] 18.3.4.1 Antibody-nucleic acid conjugation

[0247] To verify the feasibility of the PLA method in cells, first, a pair of proximity probes was synthesized by conjugating single-stranded DNA and antibodies, and it was verified by PAGE electrophoresis and spectrophotometer scanning of the absorption peak at a wavelength of 200 - 450 nm. As Figure 6As shown in A, the molecular weight of the single-stranded DNA is 54 bp. When the antibody is conjugated to the single-stranded DNA, a substance with a larger molecular weight will be produced, approximately 160 bp. The single-stranded DNA is nucleic acid, which has a maximum absorption peak at around 260 nm; the essence of the antibody is protein, which has a maximum absorption peak at around 280 nm. When the antibody-nucleic acid is conjugated, there is a tendency for the maximum absorption peak to shift to the right relative to that of the nucleic acid, and similarly, there is a tendency for the maximum absorption peak to shift to the left relative to that of the protein. Therefore, the conjugate will have a maximum absorption peak between 260 - 280 nm. As Figure 6 shown in B, the maximum absorption peak of the proximity probe DNA-Ab was detected at 267 nm. The above results can prove that the PLA proximity probe was successfully constructed.

[0248] 18.3.4.2 Intracellular Detection and Condition Optimization

[0249] To verify the optimal experimental conditions for the intracellular detection reaction, according to the results of in vitro fluorescence detection, different detection probe concentrations and reaction times were divided into 9 groups, and confocal microscopy was used for imaging and qualitative analysis of the fluorescence. As Figure 7 shown in A, as the concentration of the detection probe increased and the reaction time extended, the detected fluorescence signal also increased, and the Cy5 fluorescence co-localized with the mitochondrial marker protein TOM70, indicating that the probe bound to the mitochondria. However, from 2 μM DNA3-Cy5 and 4 h reaction time, non-specific fluorescence was detected in the nucleus, presumably because some unbound fluorescence probes entered the nucleus. In addition, the specificity of the probe was also detected. As Figure 7 shown in B, fluorescence signals could only be detected intracellularly when a pair of proximity probes and detection probes were present simultaneously and the probes were completely conjugated. According to the confocal MERGE image, the probe could co-localize with the specific protein on the mitochondrial membrane. Considering the sensitivity and specificity of the detection comprehensively, 2 μM detection probe, 1 μM proximity probe, and 2 h reaction time were finally selected as the subsequent experimental conditions. The above results all demonstrated the feasibility of the detection in this study.

[0250] 18.4 Specificity Analysis of the PLA Detection Platform

[0251] Currently, the conventional method for laboratory detection of respiratory chain supercomplexes is native gradient polyacrylamide gel electrophoresis (BN-PAGE). As Figure 8As shown in A, the positions of supercomplexes in Hela cells were located by BN-PAGE electrophoresis, and subsequent experiments were carried out based on this standard. To verify the specificity of the detection, three different treatment methods were set: (1) the Hela cell group treated with CAP for 7 days and the control group; (2) the 143B group and the 143B ρ0 cell group; (3) the Hela cell group with COX7A2L KO and the control group. Chloramphenicol (CAP) is a broad-spectrum antibiotic that inhibits a variety of bacteria. The effect of CAP on mitochondrial proteins is mainly reflected in its inhibition of the mitochondrial translation process. CAP reduces the expression of mitochondrial proteins by inhibiting mitochondrial translation, but does not affect mitochondrial copy number. As the CAP treatment time prolongs, the degree of inhibition of mitochondrial protein translation and synthesis gradually increases, which is also reflected in the assembly of mitochondrial complexes and supercomplexes. After 7 days of CAP treatment, the expression of mitochondrial complex proteins is basically inhibited. Rho 0 cells, also known as ρ0 cells, are a type of cell line in which mtDNA in the mitochondria is completely cleared, unable to encode the corresponding subunits of the mitochondrial respiratory chain enzyme complex, losing the function of oxidative phosphorylation, and mainly surviving by the glycolysis pathway. Cytochrome C oxidase subunit VIIa 2-like protein (COX7A2L), also known as Supercomplex assembly factor 1 (SCAF1), is located in the inner mitochondrial membrane and encoded by nuclear DNA, and can participate in the assembly of respiratory chain supercomplexes.

[0252] As Figure 8 shown in B and C, the protein levels of mitochondrial supercomplexes were detected by BN-PAGE experiments. Compared with the control group, almost no expression of mitochondrial supercomplex proteins was detected in the CAP treatment group and the 143B ρ0 cell group; since COX7A2L can directly inhibit the assembly of supercomplex CI1CIII2CIV1, its effect on the assembly of other supercomplexes is not yet clear. As Figure 8 shown in D, the content of mitochondrial supercomplex proteins in the Hela COX7A2L KO cell group decreased. Subsequently, confocal microscopy was used to detect the probe specificity. As Figure 8As shown in E, F, and G, in the CAP treatment group and the 143B ρ0 cell group, almost no Cy5 fluorescence signal expression was detected compared with the control group. In the Hela COX7A2L KO cell group, the fluorescence signal expression decreased compared with the control group, but the signal could still be detected. This was the same trend as the results shown in the BN-PAGE experiment. The above results proved that the PLA method could specifically detect the expression of mitochondrial supercomplex CI1CIII2CIV1 in cells.

[0253] 18.5 Sensitivity analysis of the PLA detection platform

[0254] 18.5.1 Kinetics experiment on the assembly of mitochondrial complexes and supercomplexes

[0255] To verify the sensitivity of the PLA method, through the CAP removal experiment, and use both PLA and BN-PAGE methods for quantitative detection and comparison. According to the dynamic detection of the assembly of mitochondrial complexes and supercomplexes, the sensitivity of the PLA method was explored. First, Hela cells were treated with CAP for 7 days, and the culture medium was changed every day. At this time, the expression of mitochondrial supercomplex proteins was basically inhibited. Subsequently, according to the time of 72, 48, 24, 12, 0 h, CAP was removed from the culture medium and the cells were continuously cultured. After reaching the treatment time, the CAP treatment group and Ctrl (the Hela cell group not treated with CAP) were divided into 6 groups in total, and the mitochondrial supercomplex was detected by BN-PAGE experiment, and three independent repeated experiments were done. Image j software was used for quantitative analysis. As Figure 9 shown in A and B, the mitochondrial supercomplex proteins in the 0 h group of the BN-PAGE method were completely inhibited, and almost no protein expression was detected. After 12 h of CAP removal, the mitochondrial supercomplex began to assemble. Compared with the 0 h group, the expression of mitochondrial supercomplex proteins in the 24 h group recovered by 16%, and the difference was significant (P = 0.0034). Because the background gray value of the gel image was relatively high during quantification by the BN-PAGE method, it had a greater impact on quantification, resulting in inaccurate quantification and a larger target value. Compared with BN-PAGE, the PLA method showed better sensitivity. As Figure 9 shown in C and D, the expression of mitochondrial supercomplex proteins could be detected to recover to 24% in the 12 h group; after 48 h of assembly, the protein recovered to more than 50%, and the mitochondrial supercomplex proteins could be detected to recover to 78% in the 72 h group. Since the PLA method quantifies the punctate fluorescence signals, the quantification is relatively simple and accurate, and the differences between groups are obvious and all have statistical significance.

[0256] 18.5.2 Kinetics experiment on the disassembly of mitochondrial complexes and supercomplexes (shortening the assembly time)

[0257] To further verify the sensitivity of the PLA method, CAP removal experiments were carried out to reduce the time gradient between groups, and the PLA method was compared with the BN-PAGE method. First, Hela cells were treated with CAP for 7 days, and the medium was changed every day. At this time, the expression of mitochondrial supercomplex proteins was basically inhibited. Subsequently, CAP was removed from the medium at 24, 12, 8, 4, and 0 h, and the cells were continuously cultured. After the treatment time was reached, the CAP-treated group and Ctrl (the group of Hela cells not treated with CAP) were divided into 6 groups in total, and the mitochondrial supercomplex was detected by BN-PAGE experiments. Three independent replicates were performed, and quantitative analysis was carried out using Image j software. As Figure 10 shown in A and B, compared with the 0 h group, the expression of proteins could hardly be detected in the 4 h group by the BN-PAGE method, and there was no difference between the two groups. The difference in proteins was detected only at 12 h. Since the PLA method showed better sensitivity in the early stage, in addition to the above 6 groups, 1 and 2 h groups were added during grouping. After the treatment time was reached, it was detected and quantified by the PLA method. As Figure 10 shown in C and D, compared with the BN-PAGE method, the expression of mitochondrial supercomplex proteins could be detected in the 4 h group, and the difference was statistically significant (P = 0.0048).

[0258] 18.5.3 Kinetics experiments on the disassembly of mitochondrial complexes and supercomplexes

[0259] As the CAP treatment time increased, the synthesis of mitochondrial supercomplex proteins was gradually inhibited. Therefore, the PLA and BN-PAGE methods were used, and the sensitivity of the CAP method was verified by quantitatively detecting Hela cells treated with CAP for 0, 1, 2, 4, and 6 days. First, by flow cytometry counting, the same number of Hela cells were inoculated into the medium on the same day. Subsequently, in the order of 6, 4, 2, 1, and 0 days, the medium was changed from 12CS to 12CS + CAP + U, and the medium was changed every day. After the treatment time was reached, the cells were divided into 5 groups, and the group treated for 0 days was the Ctrl group. The mitochondrial supercomplex was detected by BN-PAGE experiments. Three independent replicates were performed, and quantitative analysis was carried out using Image j software. As Figure 11As shown in A and B, after treatment with CAP for 1 day, the assembly of mitochondrial supercomplex proteins detected by BN-PAGE decreased to 56%, with a significant difference (P = 0.0314); after treatment with CAP for 6 days, the assembly of mitochondrial supercomplex proteins decreased to 8%, with a significant difference (P = 0.0024); however, there was no significant difference between the 1-day and 2-day CAP treatments and between the 4-day and 6-day CAP treatments when compared pairwise. As Figure 11 As shown in C and D, the CAP treatment group was detected by the PLA method, and it was found that 62% of the assembly of mitochondrial supercomplex proteins could still be detected after 1 day of CAP treatment; while after 6 days of CAP treatment, only 3% of the protein assembly could be detected. By comparing the 5 groups of cells pairwise, there were differences in each group, and the differences were significant (P < 0.001).

[0260] The above results prove that compared with the BN-PAGE method, the PLA method shows higher sensitivity in detecting the respiratory chain supercomplex CI1III2IV1, specifically manifested in that the PLA method can detect the recovery of mitochondrial proteins in a shorter time; within the same time, the PLA method can detect a greater degree of recovery of supercomplex proteins. And compared with the CAP treatment experiment, the PLA method is more sensitive and the difference is more significant in the detection of the CAP removal experiment.

[0261] 18.6 Detection principle of HCR

[0262] The construction and operation principle of the HCR detection platform are as Figure 12 shown. The HCR detection platform has two key components: (1) a pair of proximity ligation probes DNA1Ab1 and DNA2Ab2; (2) a pair of hairpin detection probes H1 and H2. On the basis of the PLA detection platform, the hairpin structure and the detection probes H1 and H2 are introduced. Since there are a fluorescent group - Cy5 and a quenching group - BHQ2 on the H1 hairpin, the fluorescence signal is quenched. When PLA occurs and the detection probes H1 and H2 are added, the long single-stranded DNA exposed after the binding of the proximity probes can open the hairpin of H1, so the H1 hairpin binds to the proximity probes and emits a fluorescence signal. At this time, the H2 hairpin complementary to the H1 hairpin sequence is also opened. When there are enough detection probes H1 and H2, a long-chain nucleic acid structure will be formed, also known as hybridization chain reaction (HCR). HCR can cause a cascade reaction of the detection signal and enhance the fluorescence signal.

[0263] 18.7 Establishment and verification of the HCR reaction system

[0264] 18.7.1 Establishment of the HCR detection platform reaction system and PAGE verification

[0265] The DNA sequences were designed using the Nupack website and their binding was predicted. Subsequently, the construction of the HCR platform was verified by 10% denaturing polyacrylamide gel electrophoresis (PAGE). Figure 13 ). As Figure 13 shown in A of, DNA1 and DNA2 represent a pair of proximity probes, H1 and H1 are detection probes for hairpin structures, and refDNA serves as an in vitro substitute for mitochondrial respiratory chain supercomplexes to shorten the spatial distance between proximity probes. As Figure 13 shown in B of, lanes 4 and 5 indicate that when only a pair of proximity probes are present, they cannot bind due to the large spatial distance, while when refDNA is present, the proximity probes and refDNA can combine to produce ligation products. Lane 6 shows that the H1 and H2 structures are stable and cannot bind in the absence of the initiating strand. Lane 8 shows that when the proximity probes and detection probes are present simultaneously, under the ligation of refDNA, the H1 hairpin can be opened to bind to the proximity probes, and the binding of its H1 to DNA1 and DNA2 can trigger the opening of H2, initiating a hybridization chain reaction. The above results can prove the successful establishment of the HCR platform.

[0266] 18.7.2 Verification by Fluorescence Spectrometer

[0267] Previously, when using the PLA method, the set detection conditions were a detection probe concentration of 2 μM and a reaction time of 2 h. At this time, the probe copy number was much larger than the supercomplex protein copy number. Therefore, in this invention, by changing the reaction time, the fluorescence spectrum was analyzed to optimize the experimental conditions. As Figure 14 shown, when H1 and H2 probes were added, the fluorescence signal only showed extremely weak fluorescence due to quenching. In addition, this invention also set the probes for the PLA reaction under optimal conditions as the control group. When refDNA is present, DNA1 and DNA2 can undergo proximity ligation reaction and amplification reaction with the addition of H1 and H2, amplifying the fluorescence signal. Since the total experimental time of BN-PAGE is 42 h, plus the experimental time for pre-processing the probes, the maximum reaction time set for the HCR reaction is 32 h. The results show that the detected fluorescence signal increases with the increase of the reaction time.

[0268] 18.8 Optimization of Experimental Conditions for the HCR Detection Platform

[0269] To verify the optimal experimental conditions for in-cell detection reactions, according to the results of in vitro fluorescence detection, 7 groups were divided according to different reaction times, among which the 2 h group of the PLA reaction was used as the control group, and confocal microscopy was used for imaging and qualitative analysis of the fluorescence. As Figure 15As shown in A and B, compared with the PLA group, as the HCR reaction time extended, the detected fluorescence signal also increased. At 8 h, the fluorescence detected in the HCR group was approximately 3 times that in the PLA group. However, starting from 16 h of the reaction, non-specific fluorescence was detected in the nucleus, and the fluorescence was strong. It was speculated that some unbound fluorescent probes might have entered the nucleus. According to the confocal MERGE images, the probe could co-localize with the mitochondrial membrane specific protein. Considering the detection sensitivity and specificity comprehensively, 2 μM of H1-Cy5, 2 μM of H2, and an 8-h reaction time were finally selected as the subsequent experimental conditions.

[0270] 18.9 Specificity analysis of HCR method detection

[0271] To verify the specific detection of the respiratory chain supercomplex in the laboratory by the HCR method, three cell models were also used: (1) the Hela cell group treated with CAP for 7 days and the control group; (2) the 143B group and the 143B ρ0 cell group; (3) the Hela cell group with COX7a2l KO and the control group.

[0272] As Figure 16 As shown in A-C, the three groups of cells were detected using the HCR method, and the experimental results showed the same trend as the PLA method. Compared with the control group, almost no Cy5 fluorescence signal expression was detected in the CAP-treated group and the 143B ρ0 cell group, indicating that the assembly of mitochondrial supercomplexes was inhibited. The fluorescence signal expression in the COX7a2l KO cell group decreased compared with the control group, but the signal could still be detected. The above results demonstrated that the HCR method could specifically detect the expression of mitochondrial supercomplexes in cells.

[0273] 18.10 Sensitivity analysis of HCR detection platform

[0274] 18.10.1 Kinetics experiment on the assembly of mitochondrial complexes and supercomplexes

[0275] Since it was found in the previous detection that the BN-PAGE method could first detect differences 12 h after CAP removal, and the PLA method could first detect differences 4 h after CAP removal, and it could be proven from in vitro fluorescence experiments that HCR could release more fluorescence signals than the PLA method under the same detection conditions. Therefore, to verify the sensitivity of the HCR method, a total of 7 groups were set in this invention, namely the groups of 0, 1, 2, 4, 8, 12 h after removing CAP after being treated with CAP for 7 days and the Ctrl group (the Hela cell group not treated with CAP), and the mitochondrial supercomplex was detected using the HCR method, and quantitative analysis was performed using Image j software. As Figure 17As shown in A and B, the difference could be detected as early as 2 h after CAP removal by the HCR method (P = 0.0314), and the expression of mitochondrial supercomplex proteins could be detected to recover to 30% in the 12-h group, with a significant difference (P < 0.0001). This showed better detection sensitivity compared with the PLA method.

[0276] 18.10.2 Kinetics experiment on disassembly of mitochondrial complexes and supercomplexes

[0277] Similarly, the sensitivity of the HCR method was detected using the CAP treatment experiment, and quantitative analysis was performed on Hela cells treated with CAP for 0, 1, 2, 4, and 6 days to further verify the sensitivity of the CAP method. First, by flow cytometry counting, the same number of Hela cells were seeded into the culture medium on the same day. Subsequently, the fluorescence of the supercomplex was detected using the HCR detection platform. As Figure 18 shown in A and B, after 6 days of CAP treatment, only 9% of the mitochondrial supercomplex protein assembly could be detected. By pairwise comparison of the 5 groups of cells, there were differences in each group, and the differences were significant (P < 0.001). The above results showed that the HCR method had better detection sensitivity than the BN-PAGE method, but there was no significant difference compared with the PLA method.

[0278] The above results showed that compared with the BN-PAGE method, the PLA method could detect the assembly of supercomplexes in a shorter time and the assembly of mitochondrial supercomplexes after CAP treatment in a longer time range, which indicated that the PLA method had a lower detection limit (4 h after CAP removal) and a larger detection range (6 days of CAP treatment); while compared with the PLA method, the HCR method had a lower detection limit (2 h after CAP removal), but there was no obvious advantage in the detection range.

[0279] 18.11 Establishment and verification of the digital detection platform

[0280] 18.11.1 Detection principle and process

[0281] In order to establish an in vitro microplate integrated detection system that includes sample pretreatment, proximity ligation reaction, and signal detection, the present invention constructed a digital detection platform. As Figure 19 shown, the cells were fixed on a 96-well fluorescence plate (sample capture), an antigen-antibody in-situ reaction (proximity ligation reaction) was carried out by adding a pair of proximity probes (DNA1Ab1 and DNA2Ab2), then HCR detection probes (H1 and H2) were added for amplification, and finally, a multifunctional microplate reader was used for proximity ligation fluorescence quantitative detection.

[0282] 18.11.2 Verification of the digital detection platform

[0283] 18.11.2.1 Kinetics experiment on the assembly of mitochondrial complexes and supercomplexes

[0284] To verify the detection platform, cells were treated with CAP for 7 days and then divided into CAP removal 0, 1, 2, 4, 8, 12, 24 h groups and the Ctrl group (the group of Hela cells not treated with CAP). The amplification was carried out using the HCR method, and then the fluorescence intensity was quantitatively detected using a multifunctional microplate reader, and three independent repeated experiments were performed. As Figure 20 shown in A, the detected fluorescence intensity increased with the extension of CAP removal time, which also indicated an increase in the assembly of supercomplex proteins; Figure 20 In B, the group with CAP removal of 0 h was used as the negative control, and the fluorescence intensity of the remaining groups was subtracted from that of the 0 h group for plotting. The results showed that with the extension of CAP removal time, the assembly of mitochondrial supercomplex proteins showed an upward trend. However, due to the large target values among the detection results, the difference was not statistically significant.

[0285] 18.11.2.2 Kinetics experiment on the disassembly of mitochondrial complexes and supercomplexes

[0286] To further verify the digital detection platform, Hela cells were treated with CAP for 0, 1, 2, 4, 6, 7 days, and the amplification was carried out using the HCR method. Then the fluorescence intensity was quantitatively detected using a multifunctional microplate reader, and three independent repeated experiments were performed. As Figure 21 shown in A, the detected fluorescence intensity decreased with the extension of CAP treatment time; Figure 21 In B, the group with CAP treatment for 7 days was used as the negative control, and the fluorescence intensity of the remaining groups was subtracted from that of the 7-day group for plotting. The results showed that with the extension of CAP treatment time, the assembly of mitochondrial supercomplex proteins showed a downward trend.

[0287] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A PLA probe for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology, characterized in that, The PLA probe includes a proximity probe and a detection probe. The proximity probe includes antibody-nucleic acid conjugate I and antibody-nucleic acid conjugate II. The antibody-nucleic acid conjugate I is obtained by conjugating a DNA1 probe with a nucleotide sequence shown in SEQ ID NO.1 and a mitochondrial respiratory chain supercomplex antibody. The antibody-nucleic acid conjugate II is obtained by conjugating a DNA2 probe with a nucleotide sequence shown in SEQ ID NO.2 and a mitochondrial respiratory chain supercomplex antibody. The detection probe includes a DNA3 probe with a nucleotide sequence shown in SEQ ID NO.3 and a DNA4 probe with a nucleotide sequence shown in SEQ ID NO.

4.

2. The PLA probe according to claim 1, wherein One of the DNA3 probe and the DNA4 probe is labeled with a fluorophore, and the other is labeled with a quencher.

3. The PLA probe according to claim 1, characterized in that, The mitochondrial respiratory chain supercomplex antibody conjugated with the DNA1 probe is a mitochondrial complex I subunit NDUFB8 antibody; the mitochondrial respiratory chain supercomplex antibody conjugated with the DNA2 probe is a complex IV subunit MT-CO1 antibody.

4. A probe combination for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology and hybridization chain reaction, characterized in that, It includes the proximity probe described in claim 1 and an HCR nucleic acid probe. The HCR nucleic acid probe includes an H1 probe with a nucleotide sequence shown in SEQ ID NO.6 and an H2 probe with a nucleotide sequence shown in SEQ ID NO.

7.

5. The probe combination according to claim 4, wherein Both ends of the H1 probe are labeled with a fluorophore and a quencher, respectively.

6. A kit for detecting mitochondrial respiratory chain supercomplexes, characterized in that, The kit includes the PLA probe described in any one of claims 1-3, or the probe combination described in any one of claims 4-5.

7. Use of the PLA probe described in any one of claims 1-3 or the probe combination described in any one of claims 4-5 in the preparation of a kit for detecting mitochondrial respiratory chain supercomplex.

8. A method for detecting mitochondrial respiratory chain supercomplexes for non-diagnostic purposes, characterized in that, It includes the following steps: Contact the proximity probe described in claim 1 with a test sample to bind the proximity probe to the target protein in the test sample, and then add the detection probe described in claim 1 or the HCR nucleic acid probe described in claim 4 for reaction. Judge the expression level of the mitochondrial respiratory chain supercomplex according to the fluorescence signal generated by the reaction system.

9. The method according to claim 8, wherein When the detection probe is added to the reaction system, the molar concentration ratio of the proximity probe to the detection probe is 1:2, and the reaction time is 2 h. When the HCR nucleic acid probe is added to the reaction system, the molar concentration ratio of the proximity probe to the HCR nucleic acid probe is 1:1, and the reaction time is 8 h.

10. Use of the PLA probe described in any one of claims 1-3 or the probe combination described in any one of claims 4-5 in the preparation of a digital detection platform for detecting mitochondrial respiratory chain supercomplex.

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