A probe combination for detecting mitochondrial respiratory chain supercomplex based on proximity ligation technology and hybridization chain reaction and its application

Abstract

The present invention discloses a probe combination and application for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology and hybridization chain reaction, belonging to the field of probe detection. By identifying two adjacent protein subunits that reflect the structural integrity of the supercomplex as pedestals, and utilizing the principle of antigen-antibody binding, the antigen-antibody that recognizes and binds to the pedestals triggers a proximity ligation reaction (PLA) due to nucleic acid labeling. Based on the principle of hybridization chain reaction (HCR), cascade amplification of the detection signal is achieved, and the activity of the oxidative phosphorylation system is determined by the structural integrity of the supercomplex. This overcomes the shortcomings and limitations of traditional detection methods and enables rapid, highly sensitive, and accurate detection of supercomplex structure. Furthermore, by fixing cells on a 96-well fluorescence plate and using a multifunctional microplate reader to detect the structural integrity of the mitochondrial supercomplex, the system ultimately enables parallel testing of up to 96 samples, achieving digital and high-throughput detection.

Description

Technical Field

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

[0002] Mitochondria, the center of eukaryotic energy metabolism, collaborate to transform energy through the electron transport chain (respiratory complexes I, II, III, and IV) and ATP synthase (complex V), ultimately generating large amounts of ATP to fuel cellular activity. Complexes I, III, and IV can assemble to form higher-level supramolecular structures called supercomplexes (SCs). SCs not only enhance the stability of the electron transport chain but also significantly improve energy conversion efficiency by optimizing electron transfer pathways, enabling cells to achieve more efficient ATP synthesis with lower energy consumption.

[0003] Proximity ligation assay (PLA) is a biological method for detecting molecular interactions based on the spatial proximity effect. Its core principle is to add proximity probes that specifically recognize and bind to the target protein, thereby reducing the spatial distance between the pair of proximity probes. When the spatial distance between the proximity probes is less than 40 nm, a proximity effect occurs. Fluorescent probes are then added to bind to the proximity probes, and the structural integrity of the target protein is verified by detecting the fluorescent signal. The hybridization chain reaction (HCR) is a signal amplification technology based on DNA self-assembly reactions. This system typically contains two or more DNA hairpins. In the presence of a target molecule, the DNA hairpins are triggered to alternately open and assemble, forming a double-stranded DNA structure containing a large number of repeating units, thereby amplifying the signal of the target molecule.

[0004] Defects in the mitochondrial respiratory chain supercomplex function essentially represent a disruption of the complex's structure. By assessing the structural integrity of the respiratory chain supercomplex, preliminary analysis of mitochondrial supercomplex function can be achieved. Mitochondrial disease (MD) is a metabolic disorder primarily characterized by defects in oxidative phosphorylation (OXPHOS) function. With an incidence of 1 in 5,000, MD is considered a rare disease. Due to their genetic heterogeneity and diverse clinical symptoms, their diagnosis is particularly complex and challenging. For a long time, the diagnosis of MD relied on measuring the activity of oxidative phosphorylation enzymes in affected tissues (such as muscle), a method considered the "gold standard" for confirming MD. In recent years, with the advancement of sequencing technology, the diagnostic criteria for mitochondrial diseases have evolved to include initial clinical screening of suspected patients, biochemical confirmation of MD, and combined genetic testing to identify pathogenic genes and mutations. However, the current diagnosis rate for mitochondrial diseases is low, primarily due to the following reasons: First, the number of pathogenic genes and mutation sites in mitochondrial diseases is numerous and continues to increase, making it difficult to confirm mitochondrial diseases through genetic screening. Second, testing for oxidative phosphorylation system enzyme activity requires highly invasive tissue biopsies, resulting in low patient and family compliance. Furthermore, the complex procedures and diverse compounds involved in tissue enzyme activity testing lead to significant deviations and instability in normal reference intervals measured by the same laboratory at different times. Therefore, establishing a non-invasive, simple, rapid, and reliable mitochondrial oxidative phosphorylation complex detection system would facilitate rapid and accurate diagnosis of mitochondrial diseases. However, there are currently no reports on the detection of mitochondrial supercomplex structure using PLA combined with HCR technology. Summary of the Invention

[0005] The present invention aims to provide a probe combination and application for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology and hybridization chain reaction to address the problems of the prior art. By identifying two adjacent protein subunits that reflect the structural integrity of the supercomplex as pedestals, and utilizing the principle of antigen-antibody binding, the antigen-antibody that recognizes and binds to the pedestals triggers a proximity ligation reaction (PLA) due to nucleic acid labeling. Based on the principle of hybridization chain reaction (HCR), cascade amplification of the detection signal is achieved, and oxidative phosphorylation system activity is determined by supercomplex structural integrity. This overcomes the shortcomings and limitations of traditional detection methods and enables rapid, highly sensitive, and accurate detection of supercomplex structure. Furthermore, by fixing cells on a 96-well fluorescence plate and using a multifunctional microplate reader to detect the structural integrity of the mitochondrial supercomplex, the present invention ultimately enables parallel detection of up to 96 samples, achieving digital and high-throughput 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 comprises a proximity probe and a detection probe, wherein the proximity probe comprises an antibody nucleic acid conjugate I and an antibody nucleic acid conjugate II. The antibody nucleic acid conjugate I is obtained by coupling a DNA1 probe having a nucleotide sequence as shown in SEQ ID NO.1 with a mitochondrial respiratory chain supercomplex antibody, and the antibody nucleic acid conjugate II is obtained by coupling a DNA2 probe having a nucleotide sequence as shown in SEQ ID NO.2 with a mitochondrial respiratory chain supercomplex antibody.

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

[0009] Preferably, one of the DNA3 probe and the DNA4 probe carries a fluorescent group, and the other probe carries a quenching group.

[0010] Preferably, the mitochondrial respiratory chain supercomplex antibody coupled to the DNA1 probe is the mitochondrial complex I subunit NDUFB8 antibody; and the mitochondrial respiratory chain supercomplex antibody coupled to the DNA2 probe is the complex IV subunit MT-CO1 antibody.

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

[0012] Preferably, the two ends of the H1 probe carry a fluorescent group and a quenching group respectively.

[0013] The present invention also provides a kit for detecting mitochondrial respiratory chain supercomplexes, wherein the kit comprises the PLA probe or the probe combination.

[0014] The present invention also provides use of the PLA probe or the probe combination in preparing 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, comprising the following steps:

[0016] The proximity probe is brought into contact with the sample to be tested, so that the proximity probe binds to the target protein in the sample to be tested, and then the detection probe or the HCR nucleic acid probe is added to react, and the expression level of the mitochondrial respiratory chain supercomplex is determined based on the fluorescent signal generated by the reaction system.

[0017] Preferably, 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 hours;

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

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

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

[0021] This invention, based on the PLA detection principle combined with HCR technology, analyzes the structure of the mitochondrial respiratory chain supercomplex, thereby enabling analysis of mitochondrial functional integrity. The two core components of PLA are a pair of proximity probes and a pair of detection probes. By designing these two pairs of probes and validating them using PAGE electrophoresis and fluorescence detection, the PLA detection platform was established, ultimately determining the optimal experimental conditions (proximity probe:fluorescence probe = 1:2, PLA incubation for 2 hours). Subsequently, the specificity and sensitivity of the PLA detection platform were verified. Results showed that PLA technology can specifically bind to mitochondrial supercomplexes and exhibits superior sensitivity to BN-PAGE electrophoresis in kinetic experiments of mitochondrial assembly and disassembly. The total experimental time for PLA detection is 15-19 hours, significantly shorter than that of BN-PAGE (42-46 hours). Furthermore, BN-PAGE has stringent requirements for protein content and sample preparation. A mild protein lysis agent (such as DDM) is required during protein extraction, which may affect complex stability. Furthermore, BN-PAGE gel preparation differs from conventional SDS-PAGE and requires a specific buffer system. PLA technology uses an antibody-protein in situ binding method to directly bind to the supercomplex subunits in the cell. PLA has low requirements for sample preparation and can detect low-abundance or weakly stable complexes.

[0022] In order to further improve the sensitivity of detection, the present invention constructs the detection platform based on the PLA principle combined with HCR technology, HCR has the remarkable advantages such as isothermal, enzyme-free, simple to operate and low cost, can be self-assembled by alternating open loops of DNA hairpins, form long-chain DNA, and realize signal amplification of target molecules. The results show that the detection sensitivity of HCR is better than PLA. In the assembly kinetics experiment of super complex, mitochondrial super complex can be detected after 2 h of recovery assembly. In addition, although the total experimental duration of HCR is 21 h -24 h, higher than PLA, the duration of HCR experiment is still much smaller than the experimental duration of BN-PAGE. The present invention provides a new method for the detection of mitochondrial super complex.

[0023] The present invention also constructs a digital detection platform, which uses HCR technology to detect the supercomplexes of cells fixed on the fluorescent plate, and outputs the fluorescence signal intensity through a multifunctional microplate reader. It is ultimately expected that 96 samples can be detected at a time to achieve high-throughput detection. In the future, it is expected to be widely used in the precise detection of mitochondrial supercomplexes in clinical serum samples and the diagnosis of mitochondrial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

[0026] Figure 2 Selection of supercomplex protein subunits; (A) Structural simulation of the supercomplex CI1CIII2CIV1; (B) List of candidate subunits, including 9 CI subunits and 4 CIV subunits; (C) Spatial distance between CI and CIV subunits;

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

[0028] Figure 4 Calculation results of respiratory chain supercomplex copy number; (A) Mitochondrial complex I protein copy number; (B) The proportion of supercomplexes assembled by complex CI1CIII2CIV1 in complex I;

[0029] Figure 5Figure 3. PLA probe copy number and Cy5 fluorescence spectra under different reaction conditions. (A) Cy5 fluorescence spectra of PLA detection probe. (B) Cy5 fluorescence spectra at different detection probe concentrations and reaction times. The ratio is the concentration of adjacent probes (DNA1+DNA2+refDNA): the concentration of detection probes (DNA3-Cy5+DNA4-BHQ2).

[0030] Figure 6 Results of proximity probe synthesis verification; (A) PAGE gel verification of PLA proximity probe coupling; (B) UV-visible spectrum of PLA proximity probe;

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

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

[0033] Figure 9 Kinetics of mitochondrial complex and supercomplex assembly; (A-B) BN-PAGE and quantitative analysis of supercomplex protein content; (B-D) Fluorescence analysis and quantitative analysis of supercomplex protein content; TOM70 is an internal control protein; ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0034] Figure 10 Kinetics of mitochondrial complex and supercomplex assembly (shortened time); (A-B) BN-PAGE and quantitative analysis of supercomplex protein content; (B-D) Fluorescence analysis and quantitative analysis of supercomplex protein content; TOM70 is an internal control protein; ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0035] Figure 11The results of the kinetic experiment of disassembly of mitochondrial complexes and supercomplexes; (AB) BN-PAGE and quantitative analysis of supercomplex protein content, respectively; (CD) fluorescence analysis and quantitative analysis of supercomplex protein content, respectively; TOM70 is an internal reference protein; ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0036] Figure 12 This is the HCR detection principle diagram;

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

[0038] Figure 14 is the fluorescence spectrum of Cy5 at different reaction times;

[0039] Figure 15 Optimization of intracellular detection and experimental conditions for HCR assay; (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 of mitochondrial supercomplex assembly after CAP removal; (B) Fluorescence analysis of supercomplexes in 143B ρ0 cells; (C) Fluorescence analysis of supercomplexes in Hela COX7a2l KO cells;

[0041] Figure 17 Kinetics of mitochondrial complex and supercomplex assembly; (A) Fluorescence analysis of supercomplex; (B) Quantitative fluorescence analysis of supercomplex; ns = no significant difference; ***P < 0.001; ****P < 0.0001;

[0042] Figure 18 Kinetics of disassembly of mitochondrial complexes and supercomplexes; (A) Fluorescence analysis of supercomplexes; B) Quantitative fluorescence analysis of supercomplexes; ***P<0.001; ****P<0.0001;

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

[0044] Figure 20 Kinetics of mitochondrial complex and supercomplex assembly; (A) Fluorescence analysis of supercomplex; (B) Quantitative fluorescence analysis of supercomplex; ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0045] Figure 21 Kinetic experiments on the disassembly of mitochondrial complexes and supercomplexes; (A) Fluorescence analysis of supercomplexes; (B) Quantitative fluorescence analysis of supercomplexes; ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are 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 were kindly provided by Professor Gu Haihua's laboratory, School of Laboratory Medicine (School of Life Sciences), Wenzhou Medical University.

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

[0055] The nucleic acid sequences involved in this example (as shown in Table 1) were all synthesized by Shanghai Sangon Biotechnology 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 connecting dT-Cy5 to the 5' end of the sequence shown in SEQ ID NO.3; DNA4-BHQ2 was obtained by connecting dT-BHQ2 to the 3' end of the sequence shown in SEQ ID NO.4; DNA1-SH was obtained by connecting a thiol group to the 3' end of the sequence shown in SEQ ID NO.1; DNA2-SH was obtained by connecting a thiol group to the 5' end of the sequence shown in SEQ ID NO.2; H1-Cy5 was obtained by inserting dT-BHQ2 between positions 12 and 13 and dT-Cy5 between positions 43 and 44 of the sequence shown in SEQ ID NO.6.

[0059] 2. Establishment of PLA method

[0060] 2.1 DNA sequence incubation

[0061] First, five single-stranded DNAs, DNA1, DNA2, DNA3, DNA4, and ref DNA, were dissolved in 1× PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM. The single-stranded stock solution was then denatured in a water bath at 95°C for 5 minutes, removed, and incubated at room temperature for 30 minutes. Subsequently, equimolar amounts of the five single-stranded stock solutions were mixed in a 1:1 ratio in a 1.5 mL EP tube according to the different strand binding conditions. The mixture was mixed using a vortex shaker and incubated in a water bath at 37°C for 2 hours. During this incubation, the DNA single strands hybridized with each other through the proximity ligation principle and completed the strand displacement reaction.

[0062] 2.2 PAGE gel verification

[0063] Prepare a 12% polyacrylamide gel (PAGE) and mix 5 µL of each sample with 1 µL of 6× loading buffer. Load the gel into the sample wells. Use 1× TBE buffer as the electrophoresis buffer. Set the electrophoresis instrument to a constant voltage of 110 V and run for 70 minutes at room temperature. After electrophoresis, slowly remove the PAGE gel, cut it, and stain it. Immerse the gel in nucleic acid stain and react on a shaker for 30 minutes. Remove the gel, photograph it, and analyze the electrophoretic bands using a gel imaging system.

[0064] Before the electrophoresis experiment, 10 samples were prepared, and the samples in each lane were set as follows: the left and right lanes were 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 DNA fluorescent sequence incubation

[0067] The ratios of DNA3-Cy5 and DNA4-BHQ2, as well as the incubation time, were carefully adjusted, resulting in the design of different ratio and time gradient combinations, including 1 µM, 2 µM, and 3 µM DNA3-Cy5 and DNA4-BHQ2, and incubation times of 2, 4, and 8 hours. First, the three single-stranded DNAs 1, DNA2, and ref DNA were dissolved in 1× PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM. Separately, the two single-stranded detection probes, DNA3-Cy5 and DNA4-BHQ2, were dissolved in 1× PBS buffer to prepare single-stranded stock solutions with final concentrations of 1 µM, 2 µM, and 3 µM. Because DNA3-Cy5 and DNA4-BHQ2 carry fluorescent moieties, they were placed in a 1.5 mL brown, light-proof EP tube. Subsequently, the single-stranded stock solution was denatured at 95°C in a water bath for 5 minutes and then incubated at room temperature for 30 minutes. Subsequently, five single-chain storage solutions of equal concentration and equimolar amounts were prepared in 1.5 mL brown light-proof EP tubes according to different chain binding conditions, mixed with a vortex oscillator, and incubated in a 37°C water bath for 2 h, 4 h, and 8 h, respectively, and the fluorescence intensity was detected using a fluorimeter.

[0068] 3.2 Fluorometer Verification

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

[0070] 4. Preparation and Validation of PLA Probes

[0071] 4.1 Preparation of PLA probes

[0072] First, dilute Ab1 solution (anti-NDUFB8 (0.65 mg / mL)) to 0.1 mg / mL using PBS1 buffer. Add 50 µL of sulfo-SMCC solution to 450 µL of the Ab1 solution (0.1 mg / mL) prepared above to make a 500 µL mixture, which was then shaken and incubated at room temperature for 2 h. The activated Ab1 solution was purified by ultrafiltration through a 10 KD cutoff membrane (Millipore, 10,000 rpm, 10 min) and redissolved in 500 µL of PBS2 buffer. Next, reduce 3 µL of 100 µM DNA1-SH in 500 µL of PBS1 buffer with 4 µL of 100 mM DTT at 37°C for 1 h. The reduced DNA1 solution was purified by ultrafiltration through a 10 KD cutoff membrane (Millipore, 10,000 rpm, 10 min) and redissolved in 500 µL of PBS2. Third, mix 500 µL of the activated Ab1 solution with 500 µL of the reduced DNA1-SH prepared above and incubate overnight at 4°C. Then, wash at least three times with PBS1 buffer using a 10 kDa cutoff membrane (Millipore, 10,000 rpm, 10 min). The remaining DNA1Ab1 conjugate after ultrafiltration was redissolved in 1 mL of PBS2 buffer to serve as the storage solution for the recognition probe DNA1Ab1. The recognition probe DNA2Ab2 (anti-MT-CO1 and DNA2-SH) was prepared in the same manner as above.

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

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

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

[0076] 4.2 PLA probe coupling verification

[0077] 4.2.1 PAGE gel verification

[0078] Prepare a 12% polyacrylamide gel (PAGE) and run the gel using the same steps and procedures as in 2.2. Use ultrafiltered DNA1, DNA2, DNA1Ab1, and DNA2Ab2 solutions as lanes and samples, respectively. After staining, analyze the PAGE gel using a gel imaging system.

[0079] 4.2.2 UV detection

[0080] 200 μL of the final reaction product was placed in a quartz cuvette and scanned using a UV spectrophotometer at room temperature to measure the UV-visible absorbance of Ab-DNA at 260 nm and 280 nm.

[0081] 5. Cell culture

[0082] 5.1 Cell culture and passaging

[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 culture medium and add an appropriate amount of fresh culture medium. Change the culture medium every 2 days or when the culture medium liquid color turns yellow. Subculture when the growth density reaches 80%. Discard the culture medium, rinse once with PBS, and add trypsin cell digestion solution. Observe cell shrinkage under an inverted microscope, then add complete culture medium to stop digestion, blow the cells, and subculture at a ratio of 1:3.

[0087] (2) CAP treatment of HeLa cells:

[0088] Culture medium: DMEM plus complete culture 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 replacement and subculturing: Aspirate the culture medium and add an appropriate amount of fresh culture medium. Change the medium every day. When the growth density reaches 80%, subculturing is performed, following the same steps as (1).

[0091] (3) Removal of CAP after CAP treatment of Hela cells:

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

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

[0094] Medium replacement and subculture: Aspirate the culture medium and add an appropriate amount of fresh 12CS+CAP+U culture medium. Change the medium every day. When the growth density reaches 80%, subculture the cells. The steps are the same as (1). After 7 days of CAP treatment, the translation of most mitochondrial proteins in the cells is inhibited. This is Day 0 of CAP treatment. According to different experimental groups, the culture medium is replaced with 12CS+U after different treatment times.

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

[0096] Culture medium: 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 replacement and subculturing: Aspirate the culture medium and add an appropriate amount of fresh culture medium. Change the medium every day. When the growth density reaches 80%, subculturing is performed, following the same steps as (1).

[0099] 5.2 Cell Thaw and Cryopreservation

[0100] Cell recovery: Remove cells from liquid nitrogen and quickly place them in a 37°C water bath to thaw frozen cells within 1 minute. Centrifuge at 500 × g for 3 minutes at room temperature. Discard the supernatant, resuspend in complete culture medium, and add to a culture dish for subsequent culture.

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

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

[0103] 6.1 Cell plating

[0104] (1) Place 9 groups of cell slides in a 24-well plate, 3 in each group, soak them in 1 mL of alcohol for 30 minutes, then rinse with 1 mL of PBS. Replace the PBS after 5 minutes, and rinse three times in total;

[0105] (2) Plant 1×10 4 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 imaging

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

[0108] (2) Remove ice 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 with PBS three times, 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 diluent (Anti-TOM70), 50 μL of coupled probe DNA1Ab1 and 50 μL of coupled probe DNA2Ab2, and incubate overnight on a shaker at 4°C. The antibody diluent is 30 μL of TritonX-100 added to 10 mL of 1×PBS, mixed thoroughly, and then 0.1 g of BSA is added and mixed. The antibody diluent used in subsequent experiments is the same.

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

[0114] (8) Start avoiding light;

[0115] (9) Add 150 μL of secondary antibody diluent (Anti-Rabbit Alexa Fluor® 488) and 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 a shaker at room temperature in the dark according to the above conditions;

[0119] (11) Remove the mixed solution and rinse with PBS on a shaker three times, 5 min each time;

[0120] (12) Add 4 μL of antifade mounting medium (containing DAPI, Beyotime) to the slide, hook out the cell slide, and place the cell side in contact with the antifade mounting medium. After it dries slightly, add nail polish around the sides to fix it. Place it in a dark box and store it in a refrigerator at 4°C until it is ready for filming.

[0121] (13) Use a confocal microscope to capture the last three channels (DAPI: 405 nm, TOM70: 488 nm, CY5: 638 nm) and the images after MERGE and save them (take pictures in time and store them at 4°C for up to 7 days);

[0122] (14) After the images were taken, they were analyzed using Image J software. At least 15 cells were circled and their fluorescence intensity was analyzed. Fluorescence intensity was used to represent the content of supercomplexes in the cells. The mean fluorescence intensity of the control cells was used as a calibration, and the results were statistically analyzed.

[0123] 7. Intracellular detection of probes

[0124] 7.1 Cell Plating

[0125] The plating steps are the same as 6.1. Divide the HeLa cells into 7 groups, perform three independent replicates in each group, and place a total of 21 slides.

[0126] 7.2 Probe Incubation and Confocal Imaging

[0127] The experimental steps are the same as 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 consisted of 150 μL of secondary antibody dilution and 100 μL of PBS. The remaining groups were treated with 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 the cell pellet and add 40 μL of lysis buffer A (1 mL of lysis buffer A plus 10 μL of PMSF protease inhibitor (Sigma-Aldrich)) and 20 μL of 10% digitonin (strong irritant non-ionic detergent, Sigma-Aldrich) to every 10 mg of cell pellet. Mix well by pipetting with a pipette tip 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, dissolved in deionized water, pH 7.4, stored at 4°C;

[0135] (2) Centrifugation: 20,000 × g, 20 min, 4°C;

[0136] (3) Transfer the supernatant and determine 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 on ice until ready to use;

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

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

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

[0141] The samples prepared above were subjected to non-denaturing gradient polyacrylamide gel electrophoresis. Electrophoresis procedure 1: 45V constant voltage electrophoresis for 50 min; procedure 2: 180V constant voltage electrophoresis for 150 min. The inner tank solution was cathode liquid B and the outer tank solution was 1× imidazole. The electrophoresis was performed on ice throughout.

[0142] 9. Western blot (WB)

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

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

[0145] (3) Arrange the layers from bottom to top in the following order: sponge - 2 layers of transfer filter paper - glue - PVDF membrane - 2 layers of transfer filter paper - sponge, arrange them tightly and remove any bubbles;

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

[0147] (5) Transfer procedure: 100V, 120 min (different transfer times are set according to the molecular weight of the protein, generally 1 min for 1 kD), and the entire transfer process is performed on ice;

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

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

[0150] (8) Incubation with primary antibody: Cut out the entire lane of different groups and place it in the primary antibody, then block it on a shaker at 4°C for 24 h;

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

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

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

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

[0155] The antibody list is shown in Table 4:

[0156] Table 4 Antibody list

[0157]

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

[0159] Sample preparation steps are as follows:

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

[0161] (2) Centrifugation: 14,000 × g, 10 min, 4°C;

[0162] (3) Transfer the supernatant and do not aspirate the lower sediment;

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

[0164] The samples prepared above were subjected to SDS-PAGE electrophoresis.

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

[0166] Plasmid transfection and verification steps are as follows:

[0167] (1) When the Hela cells grow to a density of 60% in a 6-well plate, transfect according to the lipofectamine 3000 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) Limiting dilution: dilute to 1 cell / well in a 96-well cell culture plate;

[0170] (4) Selecting fluorescent monoclonal cells: On the second day, select the wells expressing green fluorescence under a fluorescence microscope and check under an ordinary optical microscope to see if there is only one cell in the well, which will be used as the cell to be verified;

[0171] (5) When the culture was expanded to a 6-well plate, the cell pellet was collected to verify the protein knockout level. After three verifications, the Hela-COX7A2L knockout cell model was obtained.

[0172] 12. Analysis of assembly and disassembly of mitochondrial complexes and supercomplexes

[0173] 12.1 Analysis of Mitochondrial Complexes and Supercomplexes

[0174] (1) According to the dynamic detection time point setting, 6 dishes of Hela cells were planted in a small dish. The number of cells planted can be fully grown in about 6-8 days according to the cell doubling time, and cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% triple antibody;

[0175] (2) After 24 hours, the cells were well attached to the wall. CAP was added to the culture medium of 5 dishes of cells (10 μL of CAP was added to every 10 mL of culture medium) and cultured for another 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 once a day, otherwise the efficacy cannot be guaranteed;

[0177] (4) On the 7th day, the culture medium containing CAP was discarded, the cells were washed three times with preheated PBS, and then replaced with regular culture medium for culture to allow the mitochondrial complex to resynthesize and begin to reassemble the supercomplex;

[0178] (5) After culturing in a 37°C incubator for 0 h, 12 h, 24 h, 48 h, and 72 h, the cell pellets were collected, washed once with PBS, and frozen in a -80°C refrigerator;

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

[0180] 12.2 Assembly Analysis of Mitochondrial Complexes and Supercomplexes (Shortening Assembly Time)

[0181] (1) In the subsequent period, shorten the assembly time to further analyze the assembly of the mitochondrial complex. At this time, the cell culture steps are the same as 12.1;

[0182] (2) After 7 days of CAP treatment, the CAP-containing medium was replaced with normal medium. Cell pellets were collected after culturing in a 37°C incubator for 0 h, 4 h, 8 h, 12 h, and 24 h. The cell pellets were washed once with PBS and frozen in a -80°C refrigerator.

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

[0184] 12.3 Analysis of Disassembly of Mitochondrial Complexes and Supercomplexes

[0185] (1) According to the dynamic detection time point setting, 5 plates of Hela cells were planted in a small dish. The number of cells planted can be fully grown in about 4-6 days according to the cell doubling time. They were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% triple antibody;

[0186] (2) When the cell density reaches about 40%, the medium is changed and CAP is added to the culture medium at 6, 4, 2, and 1 days (10 μL of CAP is added to every 10 mL of culture medium) and culture is continued to remove the mitochondrial complexes and assembled super complexes originally synthesized by the cells;

[0187] (3) The culture medium containing CAP needs to be changed once a day, otherwise the efficacy cannot be guaranteed;

[0188] (4) After culturing in a 37°C incubator for 6 days, the cell pellets were collected and rinsed once with PBS. After collection, the proteins were extracted with digitonin and subjected to non-denaturing gradient polyacrylamide gel electrophoresis.

[0189] 13 Establishment of HCR detection platform

[0190] 13.1 Reagent Preparation

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

[0192] 13.2 DNA Sequence Incubation

[0193] First, five single-stranded DNA strands, DNA1, DNA2, H1, H2, and refDNA, were dissolved in 1× PBS buffer to prepare a single-stranded stock solution with a final concentration of 1 µM. The single-stranded stock solution was then denatured in a water bath at 95°C for 5 minutes, removed, and incubated at room temperature for 30 minutes. Subsequently, equimolar amounts of the five single-stranded stock solutions were mixed in a 1:1 ratio in a 1.5 mL EP tube according to the different strand binding conditions. The mixture was mixed using a vortex shaker and incubated in a water bath at 37°C for 8 hours. During this incubation period, the DNA strands hybridized with each other according to the proximity ligation principle, completing the hybridization chain amplification reaction.

[0194] 13.3 PAGE gel verification

[0195] Prepare a 10% polyacrylamide gel (PAGE) and mix 5 µL of each sample with 1 µL of 6× loading buffer. Load the gel into the sample wells. Use 1× TBE buffer as the electrophoresis buffer. Set the electrophoresis instrument to a constant voltage of 110 V and run for 90 minutes at room temperature. After electrophoresis, slowly remove the PAGE gel, cut it, and stain it. Immerse the gel in nucleic acid stain and react on a shaker for 30 minutes. Remove the gel, photograph it, and analyze the electrophoretic bands using a gel imaging system.

[0196] Before the electrophoresis experiment, 7 samples were prepared, and the corresponding sample information of the lane settings was: the left and right lanes were 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, and lane 7 was DNA1+DNA2+H1+H2+refDNA.

[0197] 14. In vitro detection of fluorescence signals using the HCR detection platform

[0198] 14.1 DNA Fluorescent Sequence Incubation

[0199] To obtain the best experimental results, a series of detailed experimental optimization steps were carried out. Since the optimal reaction conditions were determined in the early stage when testing the in vitro fluorescence of the PLA platform, the concentration ratio of the proximity probe: detection probe was 1:2, and incubation was carried out at 37°C for 2 hours, the basic conditions established when testing the HCR platform were the same as above. The fluorescence intensity was subsequently tested by changing the reaction time. First, the five single-stranded DNAs 1, DNA2, H1-Cy5, H2, and refDNA were dissolved in 1× PBS buffer to prepare a single-stranded storage solution with a final concentration of 1 µM. H1-Cy5 should be placed in the dark. Subsequently, the single-stranded storage solution was placed in a water bath for denaturation at 95°C for 5 minutes, removed and placed at room temperature for 30 minutes. Subsequently, five single-chain storage solutions of equal concentration and equimolar amounts were prepared in 1.5 mL brown light-proof EP tubes according to different chain binding conditions, 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. The fluorescence intensity was detected by a fluorimeter.

[0200] 14.2 Fluorometer Verification

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

[0202] 15. Optimization of HCR probe fluorescence intracellular detection conditions

[0203] 15.1 Cell Plating

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

[0205] 15.2 Probe Incubation and Confocal Imaging

[0206] During the first incubation, 150 μL of secondary antibody dilution (Anti-Rabbit Alexa Fluor® 488) and 100 μL of detection probe were added. The experimental conditions are shown in the table below:

[0207] Table 5 Experimental groups and conditions

[0208]

[0209] The rest of the experimental steps are the same as 6.2.

[0210] 16. Establishment and verification of digital testing platform

[0211] 16.1 Cell Treatment

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

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

[0214] (3) Digest the cells, count 5000 cells / well using flow cytometry, and seed them into a 96-well plate;

[0215] (4) After 48 h of culture, when the cells are in good adhesion state, remove them and prepare for subsequent experiments.

[0216] 16.2 Probe Incubation and Multi-function Microplate Reader Detection

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

[0218] (2) Remove the ice methanol and rinse with PBS for 5 minutes gently.

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

[0220] (4) Remove the permeabilization solution and rinse 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 avoiding light;

[0223] (7) Add 50 μL of proximity probe and 50 μL of 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 fluorescent plate. Set two wavelengths: the first for DAPI fluorescence, with an excitation light of 340 nm and an emission light of 405 nm; the second for Cy5 fluorescence, with an excitation light of 649 nm and an emission light of 679 nm. After the detection is completed, the relative fluorescence intensity is obtained by taking the mean of Cy5 fluorescence / DAPI fluorescence.

[0228] 17. Statistical methods

[0229] Statistical analysis was performed using Prism 8.0 (GraphPad). All statistical analyses were performed based on three independent replicates. For comparisons between two groups, t-tests were used for pairwise comparisons. Data were considered significant below the P < 0.05 level (*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 are as follows Figure 1 As shown. The PLA detection platform has two key components: (1) a pair of proximity 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 antibody coupled to the proximity probe recognizes the corresponding protein on the mitochondrial respiratory chain supercomplex, the spatial distance between the proximity probes is shortened, so that the complementary sequences of the single-stranded DNA bases 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 a fluorescent group Cy5. Because it partially binds to the DNA4-BHQ2 with the quenching group BHQ2, the fluorescence is quenched and cannot be detected. The exposed DNA single strand of the proximity probe can completely bind to the recognition probe DNA3, so DNA3-Cy5 is displaced from DNA4-BHQ2 and binds to the proximity probe, 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, primarily composed of complexes I, III, and IV, is also known as the respiratory body. The 5XTH (Cryo-EM structure of human respiratory supercomplex) file, including the fasta sequence file (rcsb_pdb_5XTH.fasta) and the 3D structure PDB file (5xth-pdb-bundle), was downloaded from the PDB database. The PDB file was viewed and distances calculated using UCSF Chimera software. For a group of atoms, the center of mass is a point in space that represents the center of mass distribution for that group of atoms. In molecular structure analysis, calculating the center of mass of a subunit or the entire molecule helps understand its mass distribution. For example, when studying protein-protein interactions, calculating the center of mass of two interacting proteins allows analysis of their relative positional changes during binding. The center of mass is often used to assess molecular stability and determine the center of interaction between molecules. For example, in molecular dynamics simulations, tracking the trajectory of the center of mass can provide insights into the overall translational motion of the molecule.

[0235] Based on the HPA and PDB databases, nine subunits of complex I (CI) (NDUFV1, NDUFA6, NDUFA5, NDUFB9, NDUFV3, NDUFS4, NDUFS6, NDUFA2, and NDUFB8) and four subunits of complex IV (CIV) (MT-CO1, COX4, COX5A, and COX5B) were screened and labeled. The spatial distance between the two proteins was calculated by calculating their center of mass. NDUFB8 and MT-CO1 were found to be the closest, and this met the requirements of the PLA detection principle. Therefore, NDUFB8 and MT-CO1 were selected for the construction of PLA proximity probes in this study (see [1]). Figure 2 .

[0236] 18.3 Establishment and Validation of PLA Detection Platform

[0237] 18.3.1 Establishment of PLA Detection Platform and PAGE Validation

[0238] The DNA sequence was designed and its binding was predicted using the Nupack website, and the construction of the PLA platform was subsequently 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 serves as an extracellular surrogate for the mitochondrial supercomplex, closing the spatial distance between the proximity probes. Lanes 4 and 10 demonstrate that when only a pair of proximity probes are present, they are unable to bind due to the large spatial distance. However, when refDNA is present, the proximity probes and refDNA can combine to produce new ligation products. Lane 7 demonstrates that DNA3 and DNA4 can bind. Lane 11 demonstrates that when both proximity probes and detection probes are present, DNA3 can be displaced by the ligation action of refDNA to bind to the proximity probes. These results demonstrate the successful establishment of the PLA platform.

[0239] 18.3.2 Calculation of Respiratory Chain Supercomplex Copy Numbers

[0240] The mitochondrial bottleneck effect refers to the "bottleneck effect" that occurs when certain mitochondrial genes or mitochondrial mutations are genetically transmitted during cell division, especially during egg cell development. 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 offspring individuals. Studies have shown that almost all complex I proteins participate in the assembly of the mitochondrial supercomplex. Therefore, the minimum copy number of mitochondrial complex I proteins determines the copy number of the mitochondrial supercomplex. Figure 4 As shown in Figure A, the copy numbers of all proteins in complex I were statistically analyzed in a single cell, 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 4. 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 approximately 1.7×10 3 ~6.9×10 5 .

[0241] 18.3.3 Fluorescence Spectrometer Verification

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

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

[0244]

[0245] 18.3.4 In-cell Validation and Experimental Condition Optimization

[0246] 18.3.4.1 Antibody-Nucleic Acid Conjugation

[0247] In order to verify the feasibility of the PLA method in cells, first, a pair of proximity probes was synthesized by coupling DNA single strands and antibodies, and then verified by using PAGE electrophoresis and spectrophotometer to scan the absorption peak at a wavelength of 200-450 nm. Figure 6As shown in Figure A, the molecular weight of a single-stranded DNA is 54 bp. When an antibody is coupled to a single-stranded DNA, a substance with a larger molecular weight of approximately 160 bp is produced. Single-stranded DNA is a nucleic acid, which has a maximum absorption peak around 260nm. Antibodies are proteins, which have a maximum absorption peak around 280nm. When antibodies are coupled to nucleic acids, the maximum absorption peak relative to nucleic acids tends to shift to the right, and similarly, the maximum absorption peak relative to proteins tends to shift to the left. Therefore, the conjugate will have a maximum absorption peak between 260-280nm. Figure 6 As shown in Figure B, the maximum absorption peak of the proximity probe DNA-Ab was detected at 267 nm. These results demonstrate the successful construction of the PLA proximity probe.

[0248] 18.3.4.2 In-cell Detection and Condition Optimization

[0249] In order to verify the optimal experimental conditions for the detection reaction in cells, different detection probe concentrations and reaction times were divided into 9 groups according to the results of in vitro fluorescence detection. The fluorescence was photographed and qualitatively analyzed using a confocal microscope. Figure 7 As shown in Figure A, the fluorescence signal increases with increasing probe concentration and prolonged reaction time, and Cy5 fluorescence colocalizes with the mitochondrial marker protein TOM70, indicating that the probe binds to the mitochondria. However, at 2 μM DNA3-Cy5 and a 4 h reaction time, nonspecific fluorescence was detected in the nucleus, suggesting that some unbound fluorescent probe may have entered the nucleus. In addition, the specificity of the probe was also tested, as shown in Figure 5. Figure 7 As shown in Figure B, fluorescence signals were detected in cells only when a proximity probe and detection probe were present and fully coupled. Confocal MERGE images showed that the probes colocalized with mitochondrial membrane-specific proteins. Considering both sensitivity and specificity, a 2 μM detection probe, a 1 μM proximity probe, and a 2-h reaction time were selected as the experimental conditions for subsequent studies. These results demonstrate the feasibility of this assay.

[0250] 18.4 Specificity Analysis of PLA Detection Platform

[0251] Currently, the conventional method for laboratory detection of respiratory chain supercomplexes is non-denaturing gradient polyacrylamide gel electrophoresis (BN-PAGE), such as Figure 8As shown in Figure A, the location of the supercomplex in Hela cells was located by BN-PAGE electrophoresis, and this was used as a standard for subsequent experiments. To verify the specificity of the assay, three different treatment groups were set up: (1) Hela cell group treated with CAP for 7 days and the control group; (2) 143B group and 143B ρ0 cell group; (3) COX7A2L KO Hela cell group and the control group. Chloramphenicol (CAP) is a broad-spectrum antibiotic that has inhibitory effects on a variety of bacteria. The effect of CAP on mitochondrial proteins is mainly reflected in its inhibition of mitochondrial translation. CAP reduces mitochondrial protein expression by inhibiting mitochondrial translation, but does not affect mitochondrial copy number. As the CAP treatment time increases, the degree of inhibition of mitochondrial protein translation 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 was basically inhibited. Rho 0 cells, also known as ρ0 cells, are a cell line in which mitochondrial mtDNA is completely eliminated, unable to encode the corresponding subunits of the mitochondrial respiratory chain enzyme complexes. Consequently, they lose their oxidative phosphorylation function and survive primarily through glycolysis. 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 is encoded by nuclear DNA. It participates in the assembly of the respiratory chain supercomplex.

[0252] like Figure 8 As shown in B and C, the levels of mitochondrial supercomplex proteins were detected by BN-PAGE experiments. Compared with the control group, the expression of mitochondrial supercomplex proteins was almost undetectable in the CAP-treated group and the 143B ρ0 cell group. Since COX7A2L can directly inhibit the assembly of supercomplexes CI1CIII2CIV1, its effect on the assembly of other supercomplexes is still unclear, such as Figure 8 As shown in Figure D, the content of mitochondrial supercomplex proteins in the Hela COX7A2L KO cell group decreased. Subsequently, confocal microscopy was used to detect the specificity of the probe. Figure 8As shown in Figures E, F, and G, Cy5 fluorescence signal expression was almost undetectable in the CAP-treated and 143B ρ0 cell groups compared to the control group. However, fluorescence signal expression decreased in the Hela COX7A2L KO cell group compared to the control group, but the signal was still detectable, which is consistent with the results of the BN-PAGE experiment. These results demonstrate that the PLA method can specifically detect the expression of the mitochondrial supercomplex CI1CIII2CIV1 in cells.

[0253] 18.5 Sensitivity Analysis of PLA Detection Platform

[0254] 18.5.1 Kinetics of Mitochondrial Complex and Supercomplex Assembly

[0255] In order to verify the sensitivity of the PLA method, the CAP removal experiment was conducted, and the PLA and BN-PAGE methods were used for quantitative detection and comparison. The sensitivity of the PLA method was explored based on the dynamic detection of the assembly of mitochondrial complexes and supercomplexes. 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 culture medium at 72, 48, 24, 12, and 0 hours, and the cells continued to be cultured. After the treatment time was reached, the CAP-treated group and Ctrl (Hela cell group not treated with CAP) were divided into 6 groups, and the mitochondrial supercomplex was detected by BN-PAGE experiment. Three independent repeated experiments were performed, and quantitative analysis was performed using Image j software. Figure 9 As shown in A and B, the expression of mitochondrial supercomplex proteins in the 0h group of the BN-PAGE method was completely inhibited, and the expression of proteins was basically undetectable. After 12 hours of CAP removal, the mitochondrial supercomplex began to assemble. Compared with the 0h group, the expression of mitochondrial supercomplex proteins in the 24h group recovered by 16%, and the difference was significant (P=0.0034). Because the grayscale of the gel image background is high during quantification by the BN-PAGE method, it has a greater impact on quantification, resulting in inaccurate quantification and a large target value. Compared with BN-PAGE, the PLA method shows better sensitivity, as shown in Figure 2. Figure 9 As shown in Figures C and D, the expression of mitochondrial supercomplex proteins recovered to 24% in the 12-hour group; after 48 hours of assembly, the protein recovered to over 50%, and by the 72-hour group, the protein recovered to 78%. Because the PLA method quantifies punctate fluorescence signals, it is relatively simple and accurate, and the differences between the groups were significant and statistically significant.

[0256] 18.5.2 Kinetics of Disassembly of Mitochondrial Complexes and Supercomplexes (Shortening Assembly Time)

[0257] In order to further verify the sensitivity of the PLA method, the CAP removal experiment was performed to reduce the time gradient between the groups, and the PLA and BN-PAGE methods were compared. 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 culture medium at 24, 12, 8, 4, and 0 hours, and the cells continued to be cultured. After the treatment time was reached, the CAP-treated group and Ctrl (Hela cell group not treated with CAP) were divided into 6 groups, and the mitochondrial supercomplex was detected by BN-PAGE experiment. Three independent repeated experiments were performed, and quantitative analysis was performed using Image j software. Figure 10 As shown in Figures A and B, the BN-PAGE method detected almost no protein expression at 4 h compared to the 0 h group. There was no difference between the two groups. Protein differences were only detected 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, the PLA method was used to detect and quantify the protein. Figure 10 As 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 of Disassembly of Mitochondrial Complexes and Supercomplexes

[0259] As the CAP treatment time increases, the synthesis of mitochondrial supercomplex proteins is gradually inhibited. Therefore, the sensitivity of the CAP method was verified by using both PLA and BN-PAGE methods and quantitative detection of Hela cells treated with CAP for 0, 1, 2, 4, and 6 days. First, the same number of Hela cells were inoculated into the culture medium on the same day by flow cytometry counting. Subsequently, the culture medium was changed from 12CS to 12CS+CAP+U in the order of 6, 4, 2, 1, and 0 days, and the medium was changed every day. After the treatment time was reached, the cells were divided into 5 groups, of which the group treated for 0 day was the Ctrl group, and the mitochondrial supercomplex was detected by BN-PAGE experiment. Three independent repeated experiments were performed, and quantitative analysis was performed using Image j software. Figure 11As shown in A and B, after 1 day of CAP treatment, the assembly of mitochondrial supercomplex proteins detected by BN-PAGE method decreased to 56%, and the difference was significant (P=0.0314); after 6 days of CAP treatment, the assembly of mitochondrial supercomplex proteins decreased to 8%, and the difference was significant (P=0.0024); however, the difference between the two comparisons of CAP treatment for 1 day and treatment for 2 days and CAP treatment for 4 days and 6 days was not statistically significant. Figure 11 As shown in Figures C and D, PLA analysis of the CAP-treated group revealed that 62% of mitochondrial supercomplex protein assembly was still detectable after one day of CAP treatment, whereas only 3% of protein assembly was detectable after six days of CAP treatment. Pairwise comparisons of the five cell groups revealed significant differences across all groups (P < 0.001).

[0260] These results demonstrate that PLA demonstrates greater sensitivity for detecting the respiratory chain supercomplex CI1III2IV1 compared to BN-PAGE. Specifically, PLA can detect mitochondrial protein recovery in a shorter timeframe and, within the same timeframe, can detect a greater degree of supercomplex protein recovery. Furthermore, PLA is more sensitive in CAP removal experiments than in CAP treatment experiments, with more significant differences.

[0261] 18.6 HCR Detection Principle

[0262] The construction and operation principle of the HCR detection platform are as follows Figure 12 As 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. Based on the PLA detection platform, the hairpin structure and H1 and H2 detection probes are introduced. Due to the presence of the fluorescent group -Cy5 and the quenching group -BHQ2 on the H1 hairpin, the fluorescence signal is quenched. After PLA occurs, the detection probes H1 and H2 are added. The long single-stranded DNA exposed after the proximity probes bind can open the H1 hairpin, so the H1 hairpin combines with the proximity probes and emits a fluorescent signal. At this time, the H2 hairpin, which is 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 the hybridization chain reaction (HCR). HCR can cause a cascade reaction in the detection signal and enhance the fluorescence signal.

[0263] 18.7 Establishment and Validation of HCR Reaction System

[0264] 18.7.1 Establishment of the HCR Detection Platform Reaction System and PAGE Verification

[0265] The DNA sequence was designed and its binding was predicted using the Nupack website, and the construction of the HCR platform was subsequently verified by 10% denaturing polyacrylamide gel electrophoresis (PAGE). Figure 13 ).like Figure 13 As shown in Figure A, DNA1 and DNA2 represent a pair of proximity probes, H1 and H1 are detection probes of hairpin structure, and refDNA is used as an in vitro substitute for the mitochondrial respiratory chain supercomplex to shorten the spatial distance between the proximity probes. Figure 13 As shown in B, lanes 4 and 5 show that when there is only a pair of proximity probes, they cannot bind due to the large spatial distance, but when refDNA is present, the proximity probe and refDNA can combine to produce a ligation product. Lane 6 shows that the H1 and H2 structures are stable and cannot bind in the absence of a priming chain. Lane 8 shows that when the proximity probe and the detection probe are present at the same time, under the ligation effect of refDNA, the H1 hairpin can be opened and combined with the proximity probe, and the binding of H1 to DNA1 and DNA2 can trigger H2 to be opened, triggering a hybridization chain reaction. The above results can prove the successful establishment of the HCR platform.

[0266] 18.7.2 Fluorescence Spectrometer Verification

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

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

[0269] In order to verify the optimal experimental conditions for in-cell detection, the cells were divided into 7 groups according to the results of in vitro fluorescence detection and different reaction times, of which the PLA reaction group for 2 h served as the control group. Confocal microscopy was used to capture and qualitatively analyze the fluorescence. Figure 15As shown in Figures A and B, compared with the PLA group, the fluorescence signal detected increased with increasing HCR reaction time. At 8 hours, the fluorescence detected in the HCR group was approximately three times that of the PLA group. However, nonspecific fluorescence was detected in the nucleus at 16 hours, and the fluorescence was relatively strong, presumably due to the partial entry of unbound fluorescent probe into the nucleus. Confocal MERGE images showed that the probe colocalized with mitochondrial membrane-specific proteins. Considering both sensitivity and specificity, 2 μM H1-Cy5 and 2 μM H2, along with an 8-hour reaction time, were selected as the experimental conditions for subsequent studies.

[0270] 18.9 Analysis of HCR Detection Specificity

[0271] In order to verify the specificity of the HCR method for the detection of respiratory chain supercomplexes, three cell models were also used: (1) Hela cell group treated with CAP for 7 days and the control group; (2) 143B group and 143B ρ0 cell group; (3) COX7a2l KO Hela cell group and the control group.

[0272] like Figure 16 As shown in Figures A to C, HCR assays were performed on three cell groups, and the results showed similar trends to those observed with PLA. Compared to the control group, Cy5 fluorescence signal was almost undetectable in the CAP-treated and 143B rho0 cell groups, indicating that assembly of the mitochondrial supercomplex was inhibited. While fluorescence signal expression decreased in the COX7a2l KO cell group compared to the control group, it was still detectable. These results demonstrate that the HCR assay can specifically detect mitochondrial supercomplex expression in cells.

[0273] 18.10 Sensitivity Analysis of HCR Detection Platform

[0274] 18.10.1 Kinetics of Mitochondrial Complex and Supercomplex Assembly

[0275] In the early stage of the test, it was found that the BN-PAGE method can detect the difference as early as 12 hours after CAP removal, while the PLA method can detect the difference as early as 4 hours after CAP removal. In addition, the in vitro fluorescence experiment can prove that HCR can release more fluorescent signals than the PLA method under the same detection conditions. Therefore, in order to verify the sensitivity of the HCR method, the present invention set up a total of 7 groups when setting the groups, namely the 0, 1, 2, 4, 8, and 12h removal groups after 7 days of CAP treatment and the Ctrl group (Hela cell group not treated with CAP), and used the HCR method to detect the mitochondrial supercomplex, and used Image j software for quantitative analysis. Figure 17As shown in Figures A and B, the HCR method could detect differences as early as 2 hours after CAP removal (P=0.0314), and the expression of mitochondrial supercomplex proteins was significantly restored to 30% by 12 hours (P<0.0001). This demonstrates improved sensitivity compared to the PLA method.

[0276] 18.10.2 Kinetics of Disassembly of Mitochondrial Complexes and Supercomplexes

[0277] Similarly, the sensitivity of the HCR method was tested using the CAP treatment experiment, and the sensitivity of the CAP method was further verified by quantitative analysis of Hela cells treated with CAP for 0, 1, 2, 4, and 6 days. First, the same number of Hela cells were inoculated into the culture medium on the same day by flow cytometry counting. Subsequently, the supercomplex fluorescence was detected using the HCR detection platform. Figure 18 As shown in Figures A and B, only 9% of mitochondrial supercomplex protein assembly was detected after 6 days of CAP treatment. Pairwise comparisons of the five cell groups revealed significant differences among all groups (P < 0.001). These results demonstrate that HCR has greater sensitivity than BN-PAGE, but no significant difference compared to PLA.

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

[0279] 18.11 Establishment and Verification of Digital Testing Platform

[0280] 18.11.1 Testing Principles 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 constructs a digital detection platform. Figure 19 As shown, the cells were fixed on a 96-well fluorescent plate (sample capture), and an antigen-antibody in situ reaction (proximity point reaction) was performed by adding a pair of proximity probes (DNA1Ab1 and DNA2Ab2). Subsequently, HCR detection probes (H1 and H2) were added for amplification, and finally, proximity point fluorescence quantitative detection was performed using a multifunctional microplate reader.

[0282] 18.11.2 Digital Testing Platform Verification

[0283] 18.11.2.1 Kinetics of Mitochondrial Complex and Supercomplex Assembly

[0284] To validate the detection platform, cells were treated with CAP for 7 days and then divided into CAP removal 0, 1, 2, 4, 8, 12, and 24 h groups and the Ctrl group (Hela cells not treated with CAP). The cells were amplified using the HCR method, and the fluorescence intensity was quantitatively detected using a multifunctional microplate reader. The experiment was repeated three times independently. Figure 20 As shown in middle A, the fluorescence intensity detected increases with the extension of CAP removal time, which also indicates the increase of supercomplex protein assembly; Figure 20 Figure B shows the fluorescence intensity of the 0-hour CAP removal group as a negative control, with the fluorescence intensity of the 0-hour group subtracted from the fluorescence intensity of the remaining groups. The results show that the assembly of mitochondrial supercomplex proteins increases with increasing CAP removal time. However, due to the large target values ​​between the test results, the differences are not statistically significant.

[0285] 18.11.2.2 Kinetics of Disassembly of Mitochondrial Complexes and Supercomplexes

[0286] To further validate the digital detection platform, Hela cells were treated with CAP for 0, 1, 2, 4, 6, and 7 days, and amplified using the HCR method. The fluorescence intensity was then quantitatively detected using a multifunctional microplate reader, and the experiment was repeated three times independently. Figure 21 As shown in middle A, the fluorescence intensity detected weakened with the extension of CAP treatment time; Figure 21 Panel B shows the fluorescence intensity of the 7-day CAP removal group as a negative control, with the fluorescence intensity of the remaining groups minus that of the 7-day group. The results show that the assembly of mitochondrial supercomplex proteins decreases with prolonged CAP treatment.

[0287] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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 mitochondrial respiratory chain supercomplex is the mitochondrial respiratory chain supercomplex CI1CIII2CIV1; the PLA probe includes a proximity probe and a detection probe, wherein the proximity probe includes an antibody nucleic acid conjugate I and an antibody nucleic acid conjugate II, the antibody nucleic acid conjugate I is obtained by coupling a DNA1 probe having a nucleotide sequence as shown in SEQ ID NO.1 and a mitochondrial respiratory chain supercomplex antibody, and the antibody nucleic acid conjugate II is obtained by coupling a DNA2 probe having a nucleotide sequence as shown in SEQ ID NO.2 and a mitochondrial respiratory chain supercomplex antibody; The detection probes include 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; One of the DNA3 probe and the DNA4 probe carries a fluorescent group, and the other carries a quenching group; The mitochondrial respiratory chain supercomplex antibody coupled to the DNA1 probe is the mitochondrial complex I subunit NDUFB8 antibody; the mitochondrial respiratory chain supercomplex antibody coupled to the DNA2 probe is the complex IV subunit MT-CO1 antibody.

2. A probe combination for detecting mitochondrial respiratory chain supercomplexes based on proximity ligation technology and hybridization chain reaction, characterized in that: The method comprises the proximity probe and the HCR nucleic acid probe according to claim 1, wherein the HCR nucleic acid probe comprises an H1 probe with a nucleotide sequence as shown in SEQ ID NO.10 and an H2 probe with a nucleotide sequence as shown in SEQ ID NO.11; The two ends of the H1 probe are respectively provided with a fluorescent group and a quenching group; The mitochondrial respiratory chain super complex is the mitochondrial respiratory chain super complex CI1CIII2CIV1.

3. A kit for detecting mitochondrial respiratory chain supercomplex, characterized in that: The kit comprises the PLA probe according to claim 1 or the probe combination according to claim 2, and the mitochondrial respiratory chain supercomplex is the mitochondrial respiratory chain supercomplex CI1CIII2CIV1.

4. Use of the PLA probe according to claim 1 or the probe combination according to claim 2 in preparing a kit for detecting mitochondrial respiratory chain supercomplexes, characterized in that: The mitochondrial respiratory chain super complex is the mitochondrial respiratory chain super complex CI1CIII2CIV1.

5. A method for detecting mitochondrial respiratory chain supercomplexes for non-diagnostic purposes, characterized in that: The following steps are involved: contacting the proximity probe of claim 1 with a sample to be tested, allowing the proximity probe to bind to the target protein in the sample to be tested, then adding the detection probe of claim 1 or the HCR nucleic acid probe of claim 2 to react, and determining the expression level of the mitochondrial respiratory chain supercomplex based on the fluorescent signal generated by the reaction system; The mitochondrial respiratory chain super complex is the mitochondrial respiratory chain super complex CI1CIII2CIV1.

6. The method according to claim 5, 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 hours; 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 hours.

7. Use of the PLA probe according to claim 1 or the probe combination according to claim 2 in preparing a digital detection platform for detecting mitochondrial respiratory chain supercomplexes, characterized in that: The mitochondrial respiratory chain super complex is the mitochondrial respiratory chain super complex CI1CIII2CIV1.

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