Signal amplification system for detecting mitochondrial enzyme activity and application

Abstract

The invention discloses a signal amplification system for detecting mitochondrial enzyme activity and application. The signal amplification system comprises a depurination and depyrimidine endonuclease 1 response module, an rRNA activation module and a signal report and amplification module. The depurination and depyrimidine endonuclease 1 response module contains a DNA duplex AP-I of an AP site, wherein the DNA duplex AP-I can be specifically recognized and cut by the depurination and depyrimidine endonuclease 1; the rRNA activation module contains a hairpin probe R-H1 of a mitochondrial rRNA recognition sequence; and the signal reporting and amplifying module comprises a fuel hairpin H2 marked with a fluorescence reporting group and a quenching group. The system provided by the invention successfully realizes real-time and in-situ imaging of APE1 enzyme activity in living cell mitochondria, and provides a powerful tool for researching related life processes at a subcellular level.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and molecular diagnostics technology, and relates to a signal amplification system for detecting mitochondrial enzyme activity and its application. Background Technology

[0002] Precise regulation of cellular life activities relies on the orderly distribution and function of biomolecules in specific subcellular structures. Among these, the spatiotemporal regulation of enzyme activity directly determines core processes such as cellular metabolism, signal transduction, and fate selection. Therefore, achieving real-time, in-situ monitoring of specific enzyme activity at subcellular resolution is crucial for a deeper understanding of the molecular mechanisms of complex life processes.

[0003] Mitochondria, as the central hub of cellular energy metabolism and apoptosis regulation, are closely related to various pathological processes, including tumors and neurodegenerative diseases. Monitoring the dynamic activity of key enzymes within mitochondria is crucial for understanding their physiological functions and pathological roles. Among them, depurine-depyrimidine endonuclease 1 (APE1) is a typical bifunctional DNA repair enzyme. This enzyme is usually located in the cell nucleus and is responsible for DNA damage repair and transcriptional regulation. Studies have shown that under various pathological conditions, including tumors, APE1 can migrate to mitochondria and participate in maintaining mitochondrial genome stability and regulating redox balance. Its activity changes are closely related to disease progression and treatment response. Therefore, developing methods for real-time, in-situ monitoring of APE1 activity within mitochondria is of great significance for elucidating its functional mechanisms in physiological and pathological processes.

[0004] Currently, research methods targeting APE1 in cells mainly include traditional molecular biology methods such as Western blotting and real-time quantitative PCR (rt-qPCR). While these methods can detect the total expression level of APE1, they cannot provide information on its activity and spatial distribution in subcellular compartments, let alone achieve dynamic monitoring. DNA-based molecular probes have been developed for the analysis of intracellular enzyme activity. By designing DNA probes that can be cleaved or modified by specific enzymes, enzyme activity can be converted into a receptive optical signal. Based on this, researchers have achieved in situ imaging of cytoplasmic APE1. However, when applied to the imaging and analysis of enzymes in mitochondria, significant challenges are faced: on the one hand, DNA probes themselves lack organelle localization capabilities; on the other hand, the absolute abundance of APE1 in mitochondria is low, resulting in weak signals; furthermore, DNA probes are susceptible to background signals generated by non-specific activation of cytoplasmic APE1, making it difficult to achieve highly organelle-specific in situ monitoring.

[0005] To improve detection sensitivity, DNA signal amplification strategies such as catalytic hairpin assembly (CHA) have been used for highly sensitive detection of trace intracellular targets. However, uncontrolled amplification reactions can simultaneously amplify weak, nonspecific background signals, leading to serious false positives. Therefore, precisely limiting the efficient signal amplification process to the target organelle is a key technical challenge for achieving high-fidelity mitochondrial imaging. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a signal amplification system and its application for detecting mitochondrial enzyme activity. The system uses mitochondrial rRNA as an activation element and combines it with catalytic hairpin assembly (CHA) signal amplification technology to construct an rRNA-activated CHA system (R-CHA), achieving high spatial resolution and high sensitivity detection of APE1 in mitochondria.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a signal amplification system for detecting the activity of depurinylpyrimidine endonuclease 1 in mitochondria, the signal amplification system comprising: a depurinylpyrimidine endonuclease 1 response module, an rRNA activation module, and a signal reporting and amplification module; The depurine-depyrimidine endonuclease 1 response module contains a DNA duplex AP-I with an AP site that can be specifically recognized and cleaved by depurine-depyrimidine endonuclease 1, and the duplex releases initiation chain I after enzymatic cleavage. The rRNA activation module contains a hairpin probe R-H1 with a mitochondrial rRNA recognition sequence. After the hairpin probe R-H1 binds to the mitochondrial rRNA, it releases the hairpin H1 through a strand displacement reaction. The signal reporting and amplification module contains a fuel hairpin H2 labeled with fluorescent reporter groups and quencher groups; The initiation chain I and the hairpin H1 together trigger the catalytic hairpin assembly reaction between the fuel hairpin H2, thereby achieving cascaded amplification and output of the signal.

[0008] The design of this invention ensures that the signal amplification reaction can only be initiated after the probe enters the mitochondria and is activated by endogenous rRNA, fundamentally eliminating interference from non-target compartments and achieving extremely high spatial localization accuracy.

[0009] Preferably, the mitochondrial rRNA includes mitochondrial 12S rRNA.

[0010] Preferably, the nucleic acid sequence of the mitochondrial 12S rRNA includes the sequence shown in SEQ ID NO.6.

[0011] SEQ ID NO. 6: AAGCAUCCCCGUUCCAGUGAGU.

[0012] Preferably, the DNA duplex AP-I is formed by hybridization of the initiating strand I with a complementary strand carrying at least two AP sites, for example, two, three, or four AP sites.

[0013] Preferably, the hairpin probe R-H1 is formed by partial hybridization of hairpin H1 with a closed strand containing a toehold region that specifically binds to mitochondrial rRNA.

[0014] Preferably, the toehold region is 8-15 nucleotides in length, for example, 8 nt, 10 nt or 15 nt.

[0015] Preferably, the fluorescent reporter group includes any one or a combination of at least two of Cy5, FAM, HEX, VIC or ROX, and the quencher group includes any one or a combination of at least two of BHQ1, BHQ2 or MGB.

[0016] In a second aspect, the present invention provides a mitochondrial-targeted nanocarrier for delivering the signal amplification system described in the first aspect, wherein the mitochondrial-targeted nanocarrier has a layered structure.

[0017] Preferably, the mitochondrial-targeting nanocarrier uses lanthanide upconversion nanoparticles coated with poly-L-lysine as the core; the signal amplification system described in the first aspect is loaded onto the surface of the core by electrostatic adsorption to form an intermediate layer; and the outermost layer is a poly-L-lysine coating layer covalently linked with triphenylphosphine targeting molecules.

[0018] In this invention, the poly-L-lysine can be replaced with cationic polymers such as poly-D-lysine or polyethyleneimine.

[0019] Preferably, the lanthanide upconversion nanoparticles include NaGdF4:Yb / Tm nanoparticles.

[0020] Preferably, the poly-L-lysine coating layer is covalently linked to triphenylphosphine via an amide reaction to form a poly-L-lysine-triphenylphosphine complex.

[0021] Thirdly, the present invention provides the application of the signal amplification system described in the first aspect or the mitochondrial-targeted nanocarrier described in the second aspect in the preparation of products for the detection or imaging of depurinylpyrimidine endonuclease 1 activity in mitochondria of living cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The core advantage of this invention is that it constructs a signal amplification system gated by endogenous molecules in organelles. This system uses 12S rRNA, which is inherent and has a stable abundance in mitochondria, as an essential activating element, so that the signal amplification process is strictly restricted to start inside the mitochondria. This spatial site-specific design avoids false positive signals caused by non-specific cleavage of similar enzymes in the cytoplasm, and exhibits extremely high accuracy and reliability in complex living cell environments. (2) The system of the present invention achieves high-sensitivity imaging of low-abundance targets in mitochondria. By coupling CHA, the single target recognition event is converted into a large number of signal outputs, which effectively amplifies the weak initial signal generated by low-abundance APE1, thereby achieving a detection sensitivity far exceeding that of non-amplified probes. Compared with traditional molecular biology endpoint detection methods such as Western Blot and rt-qPCR, the present invention has achieved a technological leap. The operation is extremely simple and can be directly applied to live cell systems without complicated sample processing steps. (3) The mitochondrial-targeting nanocarrier constructed in this invention effectively solves the technical problem of delivering functional DNA probes to mitochondria. This nanocarrier can not only achieve efficient mitochondrial targeting through surface-modified triphenylphosphine, but its outer cationic polymer can also effectively load negatively charged DNA probes through electrostatic interaction and provide protection during delivery. This design successfully delivers the complete signal amplification system to the mitochondrial matrix, providing a key delivery guarantee for high-fidelity in situ mitochondrial analysis in living cells. (4) The design framework of the system of the present invention is clear, and each functional module (depurinylpyrimidine endonuclease 1 response module and rRNA activation module) is relatively independent. Researchers can extend this strategy to the detection of other organelles or target enzymes and RNA combinations by replacing the corresponding recognition sequences according to the established principles, showing broad application prospects. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the structure and working principle of the R-CHA signal amplification system described in this embodiment of the invention; Figure 2 The figure shows the results of verifying the basic sensing performance of the R-CHA signal amplification system in an in vitro test tube environment; Figure 3 The graph shows the results of evaluating the target detection sensitivity of the R-CHA signal amplification system. Figure 4 The result diagram shows the target detection specificity of the R-CHA signal amplification system. Figure 5 The diagram shows the dynamic results of the R-CHA signal amplification system for target detection. Figure 6Figure showing the results of verifying the sensing mechanism of the R-CHA signal amplification system in vitro; Figure 7 The results of validating the sensing performance of the R-CHA signal amplification system inside cells are shown in Figure a, which is a laser confocal microscopy image; Figure b is a flow cytometry quantitative analysis of the average fluorescence intensity of Cy5 in three groups of treated cells; and Figure c is a histogram of relative luminescence intensity distribution plotted based on flow cytometry data. Detailed Implementation

[0024] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0025] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0026] The sources of raw materials used in the following examples are as follows: The AP chain, I chain, H1 chain, closed chain, H2 chain, AP mutant chain, closed mutant chain, and PLL are all from Sangon Biotech Co., Ltd. 12S rRNA: from Huzhou Hippo Biotechnology Co., Ltd.; APE1 and other nucleases were purchased from New England Biotechnology Co., Ltd., USA. (4-Carboxybutyl)triphenylphosphine bromide (TPP) was purchased from Anaiji Chemical Co., Ltd., product number: E010098; Rare earth oxides, oleylamine (OM, 90%) and 1-octadecene (ODE, 95%) were purchased from Across Technologies, Belgium. Oleic acid (OA, 90%) and trifluoroacetic acid (99%) were purchased from Sigma-Aldrich Biotechnology Co., Ltd., USA. MCF-7 breast cancer cells were obtained from the American Type Culture Collection (ATCC). Hoechst33342 and Mito-Tracker Green: purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0027] Example 1 This embodiment provides an R-CHA rRNA-activated CHA signal amplification system capable of detecting mitochondrial APE1 enzyme.

[0028] The R-CHA comprises a depurinase-depyrimidine endonuclease 1 response module, an rRNA activation module, and a signal reporting and amplification module. The depurinase-depyrimidine endonuclease 1 response module contains a DNA duplex AP-I with an AP site that can be specifically recognized and cleaved by depurinase-depyrimidine endonuclease 1. After enzymatic cleavage, the duplex releases initiating chain I. The rRNA activation module contains a hairpin probe R-H1 with a mitochondrial rRNA recognition sequence. After binding to mitochondrial rRNA, the hairpin probe R-H1 releases hairpin H1 through a strand displacement reaction. The signal reporting and amplification module contains a fuel hairpin H2 labeled with a fluorescent reporter group Cy5 at the 5' end and a quencher group BHQ2 at the 3' end. The initiating chain I and the hairpin H1 jointly trigger a catalytic hairpin assembly reaction between the fuel hairpins H2, achieving cascaded amplification of the signal output. The DNA duplex AP-I is formed by hybridization of the initiating strand I with the complementary strand carrying two AP sites; the hairpin probe R-H1 is formed by hybridization of the hairpin H1 with a closed strand, the closed strand containing a toehold region that specifically binds to mitochondrial rRNA. Figure 1 This is a schematic diagram illustrating the structure and working principle of the R-CHA amplification system. The relevant nucleic acid sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are shown in Table 1. The RNA used was synthesized by Huzhou Hippo Biotechnology Co., Ltd. All DNA and RNA powders were first dissolved in sterile, enzyme-free water, and then the absorbance was measured using a UV spectrophotometer to calculate the accurate concentration. The DNA double-stranded probe AP-I was prepared by mixing the initiating strand I and the complementary strand in an equimolar ratio, heating in an annealing buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2) in a 95°C metal bath for 5 min, and then allowing it to cool naturally to obtain a DNA probe solution with a final concentration of 10 μM. Similarly, R-H1 was prepared by annealing the H1 strand and the blocking strand in an annealing buffer at a molar ratio of 1:1.1. The hairpin H2 probe solution was also prepared using a similar procedure to obtain a 10 μM probe solution. Subsequent reactions were all carried out in a reaction buffer containing 20 mM Tris-HCl, 100 mM NaCl, 50 mM KCl, 5 mM MgCl2, and 0.1 mg / mL BSA.

[0029] Table 1 Note: SEQ ID NO.2 contains two deoxyribose abase sites, denoted as idSp.

[0030] Example 2 This embodiment is used to verify the basic response performance of the R-CHA signal amplification system prepared in Example 1 in an in vitro test tube environment. The steps are as follows: Take 1 μL of 10 μM AP-I solution, 5 μL of 10 μM R-H1 solution, 5 μL of 10 μM H2 solution, 7 μL of 10 μM 12S rRNA solution, and 1 μL of 500 U / mL APE1 solution and add them to the buffer solution described in Example 1. Add enzyme-free sterile water to a final volume of 500 μL, so that the above system contains 20 nM AP-I, 100 nM R-H1, 100 nM H2, 120 nM 12S rRNA, and 0.5 U / mL APE1. Incubate the above reaction solution in a metal bath for 1 h. Controls were set up with no APE1 solution, no 12S rRNA solution, and neither. The results were measured using a fluorescence spectrophotometer. Figure 2 As shown.

[0031] Depend on Figure 2 It can be seen that the R-CHA system prepared in Example 1 can only generate a fluorescent signal when 12S rRNA and APE1 are present together. Neither of the two targets alone can generate a signal response, indicating that this system can amplify and detect APE1 in a 12S rRNA-triggered manner.

[0032] Example 3 This embodiment tests the detection sensitivity of the R-CHA signal amplification system prepared in Example 1 for the APE1 enzyme. The specific operating steps are as follows: (1) Detection performance test of signal amplification system at different APE1 concentrations The only difference from Example 1 is that the APE1 concentration is 0 U / mL, 0.025 U / mL, 0.05 U / mL, 0.075 U / mL, 0.1 U / mL, 0.15 U / mL, 0.2 U / mL, 0.3 U / mL, 0.4 U / mL, and 0.5 U / mL. All other raw materials and reaction conditions are the same as in Example 1.

[0033] (2) Performance testing of the amplification system at low APE1 concentration The only difference from Example 1 is that the APE1 concentrations are 0 U / mL, 0.005 U / mL, 0.01 U / mL, 0.015 U / mL, and 0.02 U / mL. All other components and reaction conditions are the same as in Example 1.

[0034] The test was performed using a fluorescence spectrophotometer, and the results are as follows: Figure 3 As shown, at low target concentrations, the fluorescence signal intensity of the amplification system exhibits a good linear relationship with the concentration of APE1 (R1). 2 >0.99), and the calculated detection limits for APE1 are 4.1 × 10⁻⁶.-5 U / mL indicates that the R-CHA amplification system can perform highly sensitive detection of APE1.

[0035] Example 4 This embodiment tests the specificity of the R-CHA signal amplification system prepared in Example 1 for APE1 enzyme detection. The specific operation steps are as follows: The difference from Example 1 is that the selected control group had APE1 enzyme replaced with equal amounts of EcoRI, BamH1, EcoR, Lambda Exo, and T7 Exo. All other components and reaction conditions were the same as in Example 1.

[0036] The test was performed using a fluorescence spectrophotometer, and the results are as follows: Figure 4 As shown.

[0037] Depend on Figure 4 It was observed that a significant increase in fluorescence signal was only observed in the experimental group where the APE1 enzyme was present; however, no significant increase in fluorescence signal was observed when APE1 was replaced with the corresponding enzyme in the control group. These results indicate that the signal amplification system R-CHA designed in Example 1 exhibits high specificity in recognizing APE1 and can effectively eliminate interference from other structurally or functionally similar biomolecules.

[0038] Example 5 This embodiment aims to evaluate the reaction kinetics of the R-CHA signal amplification system prepared in Example 1 with the APE1 enzyme. The specific operating steps are as follows: The difference from Example 1 is that the selected control group did not contain 12S rRNA, while the other components and reaction conditions were the same as in Example 1.

[0039] The test was performed using a fluorescence spectrophotometer, and the results are as follows: Figure 5 As shown.

[0040] Depend on Figure 5 The fluorescence intensity in the experimental group increased rapidly after the reaction was initiated and reached a plateau within approximately 60 minutes, indicating that the system has a rapid response kinetic to APE1. In contrast, the fluorescence intensity in the control group remained at baseline throughout the monitoring period without significant change. These results confirm that the activation and rapid response of the signal amplification system are strictly dependent on the presence of 12S rRNA, further validating its function as a mitochondrial-specific "switch".

[0041] Example 6 This embodiment verifies the response mechanism of the R-CHA signal amplification system prepared in Example 1 to APE1 sensing triggered by 12S rRNA by designing a control system. The specific operation steps are as follows: Two sets of mutation-type signal amplification systems, mR-CHA and R-nCHA, were designed. In mR-CHA, the 10 nucleotide bases in the closed strand sequence of the R-H1 module used to recognize 12S rRNA were randomly replaced, rendering it unable to bind to 12S rRNA. The remaining components were identical to those of R-CHA. In R-nCHA, the AP site in the AP-I module was replaced with a normal base that is completely complementary to the initiator I, thus preventing it from being cleaved by APE1. The remaining components were identical to those of R-CHA. Specific sequences are shown in Table 2. The preparation conditions were exactly the same as in Example 1.

[0042] The components and reaction conditions tested in both control systems were the same as in Example 1.

[0043] The test was performed using a fluorescence spectrophotometer, and the results are as follows: Figure 6 As shown.

[0044] Depend on Figure 6 It was observed that only the R-CHA system exhibited significant fluorescence signal enhancement in the simultaneous presence of 12S rRNA and APE1. The mR-CHA and R-nCHA systems, however, did not produce significant signal responses under any conditions. This experiment confirms that the signal output of the system of this invention is strictly dependent on the specific activation of the R-H1 module by 12S rRNA and the specific cleavage of the AP-I module by APE1; both are indispensable, thus mechanistically demonstrating the effectiveness and high specificity of the described trigger-type signal amplification design.

[0045] Table 2 Example 7 This embodiment provides a method for constructing a mitochondrial-targeted signal amplification nanosystem (R-CHA / mNP), and the specific operation steps are as follows: (1) Preparation of nanocarriers Lanthanide nanoparticles (NaGdF4:Yb / Tm) were synthesized via a high-temperature thermal decomposition method. The specific procedure was as follows: a metal trifluoroacetate precursor (1 mmol CF3COONa, 0.70 mmol Yb(CF3COO)3, 0.29 mmol Gd(CF3COO)3, and 0.01 mmol Tm(CF3COO)3) was mixed with a solvent (40 mmol oleic acid, oleylamine, and 1-octadecene, molar ratio 1:1:2). After dehydration and deoxygenation under vacuum at 120 °C, the mixture was reacted at 310 °C under a nitrogen atmosphere for 30 min to synthesize α-core nanoparticles. Using the core particles as seeds, a β-shell was epitaxially grown under the same conditions (precursors: 0.145 mmol Gd(CF3COO)3, 0.35 mmol Yb(CF3COO)3, 0.005 mmol Tm(CF3COO)3, 0.5 mmol CF3COONa) for 50 min. The resulting product was washed with ethanol by centrifugation, dispersed in cyclohexane, and stored at 4°C for later use.

[0046] (2) Preparation of mitochondrial targeting ligand (PLL-TPP) 0.2 mmol of triphenylphosphine (TPP) was dissolved in phosphate-buffered saline (PBS, pH 7.4), and 0.2 mmol of EDC and 0.5 mmol of NHS were added. The mixture was stirred at 25 °C for 15 min to activate the solution. Subsequently, the activated solution was mixed with 10 mL of L-polylysine solution (1 mg / mL, PBS) and reacted at 25 °C for 24 h. The resulting reaction solution was purified using an ultrafiltration centrifuge tube with a 3 kDa molecular weight cutoff to remove free TPP, yielding the PLL-TPP complex, which was dispersed in sterile water and stored at 4 °C.

[0047] (3) Assembly of signal amplification nanosystems Take 25 mg of the above OA-terminated UCNPs and treat them with acidic ethanol / water solution (containing 0.05 M HCl). After sonication for 10 min, stir at room temperature for 2 h to completely remove the surface oleic acid ligands, obtaining hydrophilic ligand-free UCNPs. Mix them with 20 mg of polylysine in deionized water and stir at 25 °C for 24 h to form NPs-PLL complexes through electrostatic interaction. Remove excess PLL by centrifugation and washing.

[0048] The obtained NPs-PLL complex was incubated with the R-CHA probe system prepared in Example 1 at a molar ratio of 1:50 in buffer. The DNA probe was loaded by electrostatic adsorption to obtain the R-CHA / NP intermediate. The unloaded probe was removed by centrifugation.

[0049] Finally, R-CHA / NP was co-incubated with PLL-TPP solution for 2 h, and then electrostatically self-assembled into the mitochondrial-targeted modification layer. The final product, R-CHA / mNP, was obtained by centrifugation and redispersed in buffer or sterile water, and stored at 4°C in the dark for later use. Before cell experiments, R-CHA / mNP was added to an appropriate amount of DMEM medium without fetal bovine serum and diluted to a concentration of 100 nM for R-H1 and H2.

[0050] The mutant systems mR-CHA / mNP and R-nCHA / mNP were prepared using the method described above.

[0051] Example 8 This embodiment tests the intracellular sensing performance of the signal amplification system R-CHA prepared in Example 1. The specific operating steps are as follows: MCF-7 breast cancer cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a rate of 1×10⁻⁶. 5 Cells were seeded at a culture density of 35 mm confocal culture dishes (for flow cytometry analysis) and cultured in a 5% CO2 incubator at 37°C for 24 h. After the culture medium was removed, R-CHA / mNP, mR-CHA / mNP, and R-nCHA / mNP solutions prepared in Example 7 were added, and the cells were cultured for another 4 h. After the culture medium was removed, the cells were washed with PBS solution and the nuclei were stained with Mito-Tracker Green and Hoechst. The cells were then examined using a laser confocal scanning microscope.

[0052] In addition, to accurately quantify intracellular fluorescence signals, cell samples from parallel experiments were washed with PBS, digested with trypsin, and completely collected. Cells were resuspended in PBS buffer and filtered through a 300-mesh sieve to prepare a single-cell suspension. Immediate analysis was performed using flow cytometry. Cy5 fluorescence signals were collected using 488 nm laser excitation and a 670 / 30 nm filter channel. 10,000 cellular events were collected from each sample.

[0053] Depend on Figure 7 As shown in Figure a, cells treated with R-CHA / mNP exhibited numerous bright Cy5 fluorescent spots in their cytoplasm. The distribution of these spots highly overlapped with the mitochondrial network visualized by MitoTracker Green, indicating that the nanodevice successfully targeted and accumulated in the mitochondria, where it was activated to generate a fluorescent signal. In contrast, cells treated with mR-CHA / mNP or R-nCHA / mNP showed only extremely weak Cy5 fluorescence. Figure 7As shown in Figures b and c, the observed strong fluorescence signal is strictly dependent on the correct recognition and activation of the R-H1 module by 12S rRNA and the specific cleavage of the AP-I module by APE1. Furthermore, flow cytometry data showed that the mean fluorescence intensity of cells treated with R-CHA / mNP was 2.7 times and 2.6 times that of the mR-CHA / mNP group and the R-nCHA / mNP group, respectively, further quantitatively confirming the ability of the nanodevice to achieve APE1-specific imaging and signal amplification within the mitochondria of living cells.

[0054] In summary, this invention provides a signal amplification system for apopurinylpyrimidine endonuclease 1 activated by mitochondrial 12S rRNA and its mitochondrial-targeted nanocarrier. The system uses endogenous 12S rRNA from organelles as a specific activation switch, combined with a catalytic hairpin assembly signal amplification strategy, to achieve highly specific and sensitive detection of the low-abundance APE1 enzyme activity in mitochondria. After delivery via the mitochondrial-targeted nanocarrier, this system enables in-situ, real-time imaging of APE1 in mitochondrial compartments within living cells and has the potential to monitor dynamic changes in its activity, providing a powerful tool for studying mitochondrial-related physiological and pathological processes and drug mechanisms of action. The system has a clear design concept, high modularity, and reliable preparation and assembly methods, and has broad application prospects.

[0055] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A signal amplification system for detecting the activity of depurine-depyrimidine endonuclease 1 in mitochondria, characterized in that, The signal amplification system includes: a depurinylpyrimidine endonuclease 1 response module, an rRNA activation module, and a signal reporting and amplification module; The depurine-depyrimidine endonuclease 1 response module contains a DNA duplex AP-I with an AP site that can be specifically recognized and cleaved by depurine-depyrimidine endonuclease 1, and the duplex releases initiation chain I after enzymatic cleavage. The rRNA activation module contains a hairpin probe R-H1 with a mitochondrial rRNA recognition sequence. After the hairpin probe R-H1 binds to the mitochondrial rRNA, it releases the hairpin H1 through a strand displacement reaction. The signal reporting and amplification module contains a fuel hairpin H2 labeled with fluorescent reporter groups and quencher groups; The initiation chain I and the hairpin H1 together trigger the catalytic hairpin assembly reaction between the fuel hairpin H2, thereby achieving cascaded amplification and output of the signal.

2. The signal amplification system according to claim 1, characterized in that, The mitochondrial rRNA includes mitochondrial 12S rRNA; Preferably, the nucleic acid sequence of the mitochondrial 12S rRNA includes the sequence shown in SEQ ID NO.

6.

3. The signal amplification system according to claim 1 or 2, characterized in that, The DNA duplex AP-I is formed by hybridization of the initiating strand I with a complementary strand carrying at least two AP sites.

4. The signal amplification system according to any one of claims 1-3, characterized in that, The hairpin probe R-H1 is formed by partial hybridization of hairpin H1 with a closed strand containing a toehold region that specifically binds to mitochondrial rRNA.

5. The signal amplification system according to claim 4, characterized in that, The toehold region is 8-15 nucleotides in length.

6. The signal amplification system according to any one of claims 1-5, characterized in that, The fluorescent reporter group includes any one or a combination of at least two of Cy5, FAM, HEX, VIC or ROX, and the quencher group includes any one or a combination of at least two of BHQ1, BHQ2 or MGB.

7. A mitochondrial-targeted nanocarrier for delivering the signal amplification system according to any one of claims 1-6, characterized in that, The mitochondrial-targeting nanocarrier has a layered structure; Preferably, the mitochondrial-targeting nanocarrier uses lanthanide upconversion nanoparticles coated with poly-L-lysine as the core; the signal amplification system of any one of claims 1-6 is loaded onto the surface of the core by electrostatic adsorption to form the intermediate layer; and the outermost layer is a poly-L-lysine coating layer covalently linked with triphenylphosphine targeting molecules.

8. The mitochondrial-targeting nanocarrier according to claim 7, characterized in that, The lanthanide upconversion nanoparticles include NaGdF4:Yb / Tm nanoparticles.

9. The mitochondrial-targeting nanocarrier according to claim 7 or 8, characterized in that, The poly-L-lysine coating layer is covalently linked to triphenylphosphine via an amide reaction to form a poly-L-lysine-triphenylphosphine complex.

10. The use of the signal amplification system according to any one of claims 1-6 or the mitochondrial-targeting nanocarrier according to any one of claims 7-9 in the preparation of products for the detection or imaging of depurinylpyrimidine endonuclease 1 activity in mitochondria of living cells.

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