DNA sensor for monitoring lysosomal zinc ion-induced ATP depletion

Abstract

This invention discloses a method for studying the mechanism by which the mucoprotein channel 1 (TRPML1) regulates the outflow of zinc ions from lysosomes and induces changes in mitochondrial ATP. This method uses 13 nm AuNPs as loading materials to construct a logic-gated sensor for pH and zinc ion regulation within lysosomes and for TRPML1 mRNA in the cytoplasm. It also uses 5 nm AuNPs as loading materials to construct a sensor targeting mitochondrial ATP for cell imaging. This method enables the detection of mRNA and zinc ions in the cytoplasm, lysosomes, and mitochondria. 2+ ATP-associated ATP sensors, Zn 2+ Three sensors—a sensor, an mRNA sensor, and an X-ray sensor—are used simultaneously to image cells, aiming to answer questions about the mechanisms of ATP changes in mitochondria under the regulation of zinc ion efflux from lysosomes by TRPML1.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry detection technology, specifically relating to an adenosine triphosphate (ATP) sensor that efficiently targets mitochondria and responds to ATP within mitochondria; and to lysosome pH and Zn. 2+ Logic-gated sensors responding to zinc ions within lysosomes; and intracytoplasmic TRPML1 mRNA sensors responding to specific intracytoplasmic TRPML1 mRNA; ATP sensors, Zn 2+ The three sensors—a sensor, an mRNA sensor, and another—simultaneously enter the cell for imaging, aiming to answer questions about the mechanisms of ATP changes in mitochondria under the regulation of zinc ion efflux from lysosomes by TRPML1. Background Technology

[0002] Currently, adenosine triphosphate (ATP) is the primary energy source for organisms. ATP is not only an energy molecule but also plays a crucial role in cell signaling. In cancer, ATP acts as a cell signaling molecule, participating in the regulation of cell proliferation, survival, migration, and invasion. In cancer and neurodegenerative diseases, abnormal cell survival and death signals often involve ATP regulation. However, ATP is regulated by many factors, such as ion concentration, metabolic activity, and cellular physiological functions like differentiation, proliferation, and death, all of which affect the production and demand of ATP within cells. Intracellular ion homeostasis is closely related to mitochondrial function; imbalances in intracellular ion homeostasis can lead to mitochondrial damage, thereby affecting ATP production.

[0003] Zinc exists in organisms as ions (Zn). 2+ Zn exists in the form of a trace element essential for living organisms. 2+ Zn plays a crucial role in physiological and cellular processes such as development, metabolism, DNA synthesis, and transcriptional regulation. 2+ Zinc is a cofactor for many mitochondrial enzymes, including some involved in ATP synthesis. For example, certain enzymes and protein complexes in mitochondria require zinc ions to maintain normal catalytic activity, thereby promoting ATP production. The vast majority of free zinc ions in the cytoplasm are in the nM range; therefore, changes in cytoplasmic ion homeostasis would theoretically affect changes in mitochondrial ATP levels. Studies in neurodegenerative diseases and cancers such as breast cancer have shown that zinc... 2+ Zn accumulates in lysosomes, reaching concentrations in the micromolar to millimole range. 2+ The release of Zn is closely related to the lipoprotein channel 1 (TRPML1), a popular target for cancer therapy. This non-cationic channel is mostly located on the late lysosomal membrane and acts as a Zn... 2+Plasma exchange channels. TRPML1 is upregulated to varying degrees in cancer and can control Zn in lysosomes. 2+ Inflow and outflow. To investigate the Zn in lysosomes-mitochondria 2+ To understand the relationship between ATP and Zn, we designed three sensors: an ATP sensor, a Zn sensor, and a Zn sensor. 2+ The simultaneous entry of three sensors—a sensor, an mRNA sensor, and another—into cells for imaging aims to answer questions about the mechanisms underlying changes in mitochondrial ATP levels under TRPML1-regulated lysosomal zinc ion efflux. All three sensors exhibited excellent enzyme and time stability, which is of significant importance and practical value for studying signaling pathways within tumors and for precision cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a three-sensor simultaneous imaging method for cells, utilizing analytical chemical and biological sensors to address biological signaling pathway mechanisms. The biosensors include an adenosine triphosphate (ATP) sensor for efficient targeting of mitochondria, responding to ATP within the mitochondria; and lysosomes for pH and Zn. 2+ Logic-gated sensors respond to zinc ions within lysosomes; and intracytoplasmic TRPML1 mRNA sensors respond to specific TRPML1 mRNA within the cytoplasm. ATP sensors, Zn 2+ The sensor, mRNA sensor, and three other sensors simultaneously enter the cell for imaging, in order to answer questions about the mechanism by which TRPML1 regulates the outflow of zinc ions from lysosomes and induces changes in ATP in mitochondria.

[0005] The objective of this invention is achieved through the following technical solution.

[0006] ATP, Zn 2+ The construction and application of mRNA biosensors are characterized by the following synthesis steps:

[0007] (1) Preparation of SH-DNA strand modified 13 nm AuNPs: 9 μL 1 mM L1 was added to 1 μL 500 mM acetate buffer (pH 5.2) and 1.5 μL 100 mM TCEP for 1 h activation, 3 mL AuNPs (freshly prepared) were added and shaken at room temperature for 16 h, 30 μL 500 mM Tris acetate (pH 8.2) was added, and the mixture was shaken slowly. 300 μL 1 M NaCl was added dropwise to each vial and the mixture was aged in salt for at least one day. Finally, AuNPs@SH-DNA were obtained and stored at room temperature for later use.

[0008] (2) Preparation of SH-AP chain modified 5 nm AuNPs: 9 μL 1 mM SH-AP was added to 1 μL 500 mM acetate buffer (pH 5.2) and 1.5 μL 100 mM TCEP for 1 h activation, 3 mL AuNPs (freshly prepared) were added and shaken at room temperature for 16 h, 30 μL 500 mM Tris-acetate (pH 8.2) was added and shaken slowly, 300 μL 1 M NaCl was added dropwise to each vial, and the salt was aged for at least one day. Finally, AuNPs@SH-L1 were obtained and stored at room temperature for later use.

[0009] (3) Zn 2+ Sensor preparation: Centrifuge 3 mL of 13 nm AuNPs@SH-DNA, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to a final volume of 1.5 mL, add 10 μL of 100 μM I3 and incubate at 37 ℃ for 2 h, add 3 μL of 100 μM Cy3 strand and incubate at 37 ℃ for 2 h, centrifuge and bring the final volume to 150 μL, Zn... 2+ The final concentration of the sensor was 200 nM.

[0010] (4) Preparation of ATP sensor: 3 mL of 5 nm AuNPs@SH-AP, add 2 mg SH-PEG-TPP (MW:2438), incubate at room temperature for 2 h, centrifuge at 18000 rpm for 30 min, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to adjust the volume to 1 mL, add 2 μL of 100 μM FA, incubate at 37 ℃ for 2 h, centrifuge to adjust the volume to 300 μL, the final concentration of ATP sensor is 700 nM.

[0011] (5) Preparation of mRNA sensor: Centrifuge 3 mL of 13 nm AuNPs@SH-L1, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to a final volume of 1.5 mL, add 3 μL of 100 μM L2, incubate at 37 ℃ for 2 h, centrifuge to a final volume of 150 μL, the final concentration of TRPML1 mRNA sensor is 200 nM.

[0012] The working principle of this invention is as follows:

[0013] (1) Zn 2+The sensor's triplet consists of three functional DNA single strands: SH-DNA, a 5'-modified Cy3 fluorophore on the Q-strand, and an i-motif (I3), along with 13 nm AuNPs. In neutral conditions and without zinc ions, the Cy3 fluorophore on the 5' of the Q-strand is close to the AuNPs, quenching the Cy3 fluorescence and resulting in a low background fluorescence signal. Under acidic conditions, the i-motif folds to form a quadruplex, but without the target compound, the Cy3 fluorescence is also quenched. Under neutral conditions, the i-motif does not fold, and in the presence of zinc ions, there is no fluorescence recovery because the target compound does not cleave the DNA-zyme structure. Only under acidic conditions, with the presence of target compound zinc ions, does the i-motif fold to form a G-quadruplex, creating the DNA-zyme structure, and the active site is cleaved by zinc ions, generating abundant fluorescence.

[0014] (2) The ATP sensor consists of two functional DNAs: SH-AP, a 5' modified FAM fluorophore of the ATP aptamer, a mitochondrial-targeting small molecule SH-PEG-TPP, and 5 nm AuNPs. Since the FAM fluorophore modified on the 5' of the ATP aptamer chain is complementary to the SH-AP chain and is quenched by AuNPs, it results in an extremely low fluorescence background signal. The sensor specifically targets mitochondria, and in the presence of the target ATP, the ATP aptamer will specifically recognize it and there will be a fluorescence rebound.

[0015] (3) The mRNA sensor consists of two functional DNAs, namely the 5' modified Cy5 fluorophores of SH-L1 and L2 and 13 nm AuNPs. Since the Cy5 fluorophore modified on the 5' of the L2 strand is complementary to the SH-L1 strand and is quenched by the AuNPs, it results in a very low fluorescence background signal. The sensor specifically recognizes TRPML1 mRNA. In the presence of the target TRPML1 mRNA, the L2 strand will specifically recognize it and there will be a fluorescence rebound.

[0016] (4) ATP sensor, Zn 2+ The sensor enters the cell and simultaneously images, using the SA1 activator to activate the TRPML1 channel, releasing zinc ions from the lysosome, thereby monitoring the Zn content in the lysosome. 2+ and ATP in mitochondria;

[0017] (5) Zn 2+ Both the sensor and the mRNA sensor enter the cell for imaging.

[0018] The ATP sensor and Zn designed in this invention 2+Three sensors—a sensor, an mRNA sensor, and an X-ray sensor—are used simultaneously to image cells, aiming to answer questions about the mechanisms of ATP changes in mitochondria under the regulation of zinc ion efflux from lysosomes by TRPML1.

[0019] The fluorescence detection conditions were set as follows: excitation wavelength: 540 nm, excitation slit: 5 nm, emission slit: 5 nm, voltage: 1000 V; excitation wavelength: 635 nm, excitation slit: 5 nm, emission slit: 10 nm, voltage: 750 V; excitation wavelength: 488 nm, excitation slit: 5 nm, emission slit: 5 nm, voltage: 800 V; the cell imaging settings were: excitation wavelengths of 488 nm, 514 nm, and 633 nm. Attached Figure Description

[0020] [ Figure 1 ATP sensor, Zn 2+ The sensor and mRNA sensor respond to ATP and Zn, respectively. 2+ A schematic diagram of the principle of mRNA and a schematic diagram of the three-sensor entry into the cell in response to the target substance.

[0021] [ Figure 2 Gel electrophoresis validates the construction of the ATP sensor (A), Zn 2+ Construction of sensors (B) and construction of mRNA sensors (C), ATP sensors, Zn 2+ Fluorescence feasibility analysis of the sensor and mRNA sensor (D, E, F).

[0022] [ Figure 3 ATP sensor, Zn 2+ Transmission electron microscopy images (A1, B1, C1), UV-Vis absorption spectra (A2, B2, C2), particle size (A3, B3, C3), and potential (A4, B4, C4) of the sensor and mRNA sensor.

[0023] [ Figure 4 ATP sensor, Zn 2+ Fluorescence spectra (A1, B1, C1) and fluorescence linearity (F / F0, F) spectra (A2, B2, C2) of the sensor and mRNA sensor in response to different concentrations of target analytes.

[0024] [ Figure 5 ATP sensor (A1), Zn 2+ Reproducibility of the sensor (B1) and mRNA (C1) within the linear range was investigated. ATP sensor, Zn... 2+ Selectivity of the sensor and mRNA sensor for different ATP analogs, ions, and mRNA (A2, B2, C2).

[0025] [ Figure 6 ATP sensor (A), Zn 2+ Sensor (B) and mRNA sensor (C) respond to ATP and Zn in serum (1%) and Tris-NaCl buffer. 2+ An examination of the performance of TRPML1 mRNA.

[0026] [ Figure 7 ATP sensor, Zn 2+ Stability study of deoxyribonuclease I (DNase I) in sensors and mRNA sensors (A1, B1, C1); ATP sensor; Zn 2+ Temporal stability studies of the sensor and mRNA sensor (A2, B2, C2).

[0027] [ Figure 8 ATP sensor (A), Zn 2+ Cytotoxicity analysis of MCF-7 cells by sensor (B) and mRNA (C) sensors.

[0028] [ Figure 9 ] ATP sensor targeting mitochondrial colocalization analysis diagram (A), Zn 2+ Sensor logic-gated cell imaging (B), mRNA (C), sensor mimic and inhibitor cell imaging (C), Zn 2+ Cell imaging map (D) showing the regulation of ATP sensor by sensor, and the regulation of Zn by mRNA sensor. 2+ Imaging image (E) of sensor modulation and the effect of mRNA sensor on Zn 2+ Cellular imaging of the sensor and ATP sensor (F).

[0029] [ Figure 10 The DNA sequence required for this experiment. Detailed Implementation Plan

[0030] Here, specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but do not limit the scope of application or extension of the present invention.

[0031] Example 1: ATP sensor, Zn 2+ Design principles of sensors and mRNA sensors.

[0032] (1) such as Figure 1 As shown in A, Zn 2+ The sensor consists of a functional DNA strand and AuNPs. Zn 2+The sensor's triplet consists of three functional DNA strands: SH-DNA, a 5'-modified Cy3 fluorophore on the Q-strand, and an i-motif, along with 13nm AuNPs. Under neutral conditions and in the absence of zinc ions, the Cy3 fluorophore on the 5' of the Q-strand is close to the AuNPs, quenching the Cy3 fluorescence and resulting in a low background fluorescence signal. Under acidic conditions, the i-motif folds to form a quadruplex, but without the target compound, the Cy3 fluorescence is also quenched. Under neutral conditions, the i-motif does not fold, and in the presence of zinc ions, there is no fluorescence recovery because no DNA-zyme structure bound to the target compound is formed. Only under acidic conditions, with the presence of target compound zinc ions, does the i-motif fold to form a G-quadruplex, creating a DNA-zyme structure with cleavage activity, resulting in a large amount of fluorescence at the cleavage site.

[0033] (2) such as Figure 1 As shown in B, the ATP sensor consists of a DNA strand SH-AP, a 5' modified FAM fluorophore of the ATP aptamer, a mitochondrial-targeting small molecule SH-PEG-TPP, and 5 nm AuNPs. Because the FAM fluorophore modified on the 5' of the ATP aptamer strand is complementary to the SH-AP strand and is quenched by the AuNPs, it results in an extremely low fluorescence background signal. This sensor specifically targets mitochondria, and in the presence of the target ATP, the ATP aptamer will specifically recognize it, resulting in a fluorescence rebound.

[0034] (3) such as Figure 1 As shown in C, the mRNA sensor consists of 5' modified Cy5 fluorophores on the DNA strands SH-L1 and L2, and 13nm AuNPs. Since the Cy5 fluorophore modified on the 5' of the L2 strand is complementary to the SH-L1 strand and is quenched by the AuNPs, it results in an extremely low fluorescence background signal. This sensor specifically recognizes TRPML1 mRNA. In the presence of the target TRPML1 mRNA, the L2 strand will specifically recognize it, and fluorescence will rebound.

[0035] (4) such as Figure 1 As shown in D, the three sensors enter the cell simultaneously: generating light signals in the cytoplasm, lysosomes, and mitochondria, respectively.

[0036] Example 2: ATP sensor, Zn 2+ Feasibility analysis of the construction of sensors and mRNA sensors and the response targets.

[0037] The ATP sensor and Zn were verified by polyacrylamide gel electrophoresis. 2+ Construction of sensors, including mRNA sensors. Figure 2Lanes 1-5 in A represent ATP aptamer, SH-AP chain, ATP aptamer + SH-AP chain, ATP aptamer + SH-AP chain + 5 mM ATP, and ATP aptamer + SH-AP chain + 20 mM ATP, respectively. The new bands generated in lanes 4 and 5 represent ATP aptamer / ATP binding bands, respectively. Figure 2 Lanes 1-9 in section B are: SH-DNA, I, Q-strand, S+I (SI), S+Q, S+I+Q, S+I+Q (pH=5), S+I+Q (Zn) 2+ S+I+Q(Zn) 2+ Lane 4 produces a new band representing the hybridization of S and I double strands; lane 5 produces a band resulting from the hybridization of I and Q strands; lane 6 produces a triplet resulting from the hybridization of S, I, and Q. Under acidic conditions, lane 7 forms two bands: one representing the i-motif and the other representing the DNAzyme structure formed by S and Q, demonstrating that the i-motif can detach under acidic conditions. Under neutral conditions and in the presence of zinc ions, as shown in lane 8, no new bands are formed, demonstrating that no false positives occur under non-acidic conditions and in the presence of zinc ions. In contrast, lane 9, under acidic conditions and in the presence of zinc ions, produces a band with a DNAzyme structure smaller than that in lane 7, and also produces a band identical to that in lane 2. Therefore, it successfully demonstrates that the i-motif in the triplet can be replaced under acidic conditions, and that zinc ions can cleave the DNAzyme. Thus, the gel electrophoresis results prove that strand substitution reactions only occur under acidic conditions and in the presence of zinc ions. Figure 2 In cell C, lanes 1-5 represent L2, SH-L1, L2+SH-L1, L2+SH-L1+1μM TRPML1 mRNA, and L2+SH-L1+2μM TRPML1 mRNA, respectively. Lanes 4 and 5 can displace L2 in the presence of the target compound. Therefore, the gel electrophoresis results can indicate the presence of the ATP sensor and Zn... 2+ The successful development of sensors and mRNA sensors.

[0038] We also used fluorescence to detect ATP sensors and Zn 2+ Feasibility analysis of fluorescence quenching for sensors and mRNA sensors. For example... Figure 2 As shown in D, E, and F, the fluorescence was quenched when modified FAM, Cy3, and Cy5 were added, respectively, indicating that quenching of AuNPs is feasible.

[0039] Example 3: ATP sensor, Zn 2+Transmission electron microscopy, ultraviolet spectroscopy, particle size, and potential characterization of the sensor and mRNA sensor construction.

[0040] By controlling the number of DNA links on the surface of AuNPs, the size of DNA sensors can be tuned to assemble size-controllable DNA sensors, meeting the application requirements of DNA sensors in different systems and environments. The interaction between AuNPs and DNA is influenced by various intermolecular forces, such as electrostatic interactions, hydrophobic forces, DNA base stacking, and chemical bonds. The Au-S covalent bonding in chemical bonds is a stable and irreversible connection between AuNPs and DNA. UV-vis spectroscopy and zeta potential were used to investigate the connection between AuNPs and thiol-containing DNA. As shown in Figures A1, A2, A3, and A4, the individual TEM images of the ATP sensor show that the spherical AuNPs have a uniform size distribution concentrated at 5 nm. The maximum absorption of the 5 nm AuNPs is 515 nm, which redshifts to 520 nm after SH-AP functionalization, and to 525 nm after SH-PEG-TPP functionalization. After SH-AP functionalization, due to the influence of the negative charge of the oligonucleotide phosphate backbone, the zeta potential of AuNPs gradually increases from -27.90 mV to -38.76 mV and then to -2.93 mV after SH-AP and SH-PEG-TPP are gradually connected; the hydrated particle size also increases from 11.7 nm to 21 nm. As shown in Figures B1, B2, B3, and B4, Zn 2+ TEM images of the sensor alone show that the spherical AuNPs are uniformly distributed and concentrated at 13 nm. The maximum absorption of the 13 nm AuNPs is 520 nm, which red-shifts to 525 nm after SH-DNA functionalization. Due to the negative charge of the oligonucleotide phosphate backbone, the zeta potential of the AuNPs after SH-DNA linkage increases from -16.23 mV to -25.36 mV, and the hydrated particle size also increases from 15.7 nm to 24.4 nm. As shown in Figures C1, C2, C3, and C4, TEM images of the mRNA sensor alone show that the spherical AuNPs are uniformly distributed and concentrated at 13 nm. The maximum absorption of the 13 nm AuNPs is 520 nm, which red-shifts to 525 nm after SH-L1 functionalization. Due to the negative charge of the oligonucleotide phosphate backbone, the zeta potential of the AuNPs after SH-L1A linkage increases from -16.23 mV to -23.18 mV, and the hydrated particle size also increases from 13.5 nm to 21.0 nm. Therefore, the results combining UV-vis spectroscopy and zeta potential indicate that SH-DNA was successfully attached to the surface of AuNPs.

[0041] Example 4: ATP sensor, Zn 2+ Sensors and mRNA sensors detect different concentrations of ATP and Zn.2+ Sensitivity analysis of TRPML1 mRNA.

[0042] At a 200 nM ATP sensor, 20 nM Zn 2+ Sensors, 20 nM mRNA sensors with different concentrations of ATP and Zn 2+ The sensitivity of the sensor was determined by evaluating TRPML1 mRNA. Figure 4 As shown in A1, the measured fluorescence signal gradually increased with increasing ATP concentration. Within the range of 10-200 μM, the fluorescence intensity showed a good linear relationship with the target concentration. Figure 4 A2), its linear equation is F / F0 = 0.0074C + 1.11, R 2 =0.9949. (For example...) Figure 4 As shown in B1, with Zn 2+ As the concentration of the target analyte increases, the measured fluorescence signal also gradually increases. Within the range of 0-200 μM, the fluorescence intensity shows a good linear relationship with the target analyte concentration. Figure 4 B2), its linear equation is F / F0 = 0.0081C + 1.16, R 2 =0.9956. For example... Figure 4 As shown in C1, the measured fluorescence signal gradually increased with increasing TRPML1 mRNA concentration. Within the range of 1-200 nM, the fluorescence intensity showed a good linear relationship with the target concentration. Figure 4 C2), its linear equation is F = 12.27C + 782.23, R 2 =0.9984, where F0 represents the fluorescence intensity of the sensor alone, F represents the fluorescence intensity after the addition of the target analyte, and C represents the fluorescence intensity of the target analyte ATP and Zn. 2+ The detection limits for TRPML1 mRNA concentrations, calculated based on the 3σ / k theory, were 1.1 μM, 0.2 μM, and 8.2 pM, respectively. These results indicate that the ATP sensor and Zn... 2+ The sensor, specifically the mRNA sensor, exhibits excellent detection performance, with high detection sensitivity and low detection limit.

[0043] Example 5: ATP sensor, Zn 2+ Reproducibility and selectivity analysis of sensors and mRNA sensors.

[0044] To investigate ATP sensors, Zn 2+ The sensor and mRNA sensor are respectively detecting ATP and Zn within the linear range. 2+To assess the reproducibility of TRPML1 mRNA, we used ATP at concentrations of 0, 1, 50, and 100 μM, and Zn at concentrations of 0, 50, 100, and 200 μM. 2+ Reproducibility analysis was performed in six parallel experiments with ML1 mRNA at concentrations of 0, 10, 50, 100, and 200 nM. The results are as follows: Figure 5 As shown in A1, B1, and C1, the ATP sensor and Zn... 2+ The fluorescence ratios (F / F0) of the sensor and mRNA sensor remained relatively consistent despite varying concentrations of the target analyte, indicating good reproducibility of this strategy for the target (probe concentrations were 100 nM). Further research is needed on the ATP sensor and Zn... 2+ Sensors, mRNA sensors for ATP, Zn 2+ Regarding the selectivity of ML1 mRNA, we selected 200 μM ATP and five other analogues (ADP, GTP, CTP, UTP, AMP), and 100 μM Zn. 2+ And eight other ions (Ca 2+ K + Fe 2+ Ni 2+ Ba 2+ Cu 2+ Cd 2+ Mg 2+ Selective analysis was performed using ML1 mRNA at a concentration of 200 nM and four other mRNAs (CADM2, miR-10b, miR-125b, and vimentin mRNA). The results are as follows: Figure 5 As shown in A2, B2, and C2, the fluorescence ratio F / F0 of this sensor is maximized only in the presence of the target, indicating that the strategy has high selectivity for the target.

[0045] Example 6: ATP sensor, Zn 2+ Comparative analysis of the sensor and mRNA sensor in Tris-NaCl buffer and serum (1%).

[0046] Comparative experiments using the biosensor in Tris-NaCl buffer and serum (1%) can determine whether the sensor can be used for detection in real samples. Figure 6 As shown in A, B, and C, different concentrations of ATP and Zn... 2+ TRPML1 mRNA, ATP sensor, Zn 2+The detection performance of both the sensor and the mRNA sensor in serum was not significantly different from that in Tris-NaCl buffer, indicating that the sensor can effectively detect the target analytes in actual samples.

[0047] Example 7: ATP sensor, Zn 2+ Deoxyribonuclease I (DNase I) and time stability analysis of the sensor and mRNA sensor.

[0048] Enzyme stability in biosensors is crucial for accurate target detection. Therefore, we investigated the deoxyribonuclease I of this sensor. Figure 7 As shown in A1, B1, and C1, the probe ATP sensor and Zn 2+ The sensor and mRNA sensor remained stable in DNase I (2 U / mL) for 3 h, 3 h, and 4 h, respectively. We also investigated the temporal stability of the probe, which maintained a very stable structure for 7 days. Figure 7 A2, B2, C2). The above results indicate that the ATP sensor and Zn described in this invention... 2+ The DNase I and time stability of the sensor and mRNA sensor are very satisfactory.

[0049] Example 8: ATP sensor (A), Zn 2+ Cytotoxicity analysis of MCF-7 cells by sensor (B) and mRNA (C) sensors.

[0050] Biocompatibility of DNA sensors is a crucial factor in determining whether in vitro constructed sensors can be used for in vivo applications. MTT assays were used to reduce the effective components in living cells and quantify cell viability to assess the biocompatibility of DNA sensors. Different concentrations of ATP and Zn were investigated. 2+ Cell viability of MCF-7 cells 24 hours after sensor and mRNA sensor administration. The effect of the sensor on MCF-7 cell viability is as follows: Figure 8 As shown in A, B, and C, when the concentration of the ATP sensor is less than 15 nM, the survival rate of MCF-7 cells remains above 90%; Zn 2+ After incubating MCF-7 cells with both the sensor and mRNA sensor for 24 hours, the survival rate of MCF-7 cells remained above 90% even when the sensor concentration was less than 5 nM. Figure 8 (A, B, C). Therefore, the DNA sensor has a relatively small impact on MCF-7 cell viability. Furthermore, the ATP sensor and Zn sensor in the cell imaging experiment... 2+ The incubation time of the sensor in MCF-7 cells was much shorter than 24 hours. Based on the cell viability corresponding to both concentration and time, the effects of the ATP sensor and Zn were inferred. 2+The sensors, including the mRNA sensor, are not toxic to MCF-7 cells. Therefore, the ATP sensor and Zn... 2+ Sensors, especially mRNA sensors, have good biocompatibility, and their impact on cell behavior can be ignored.

[0051] Example 9: Analysis of ATP sensor targeting mitochondria for colocalization (A), Zn 2+ Sensor logic-gated cell imaging (B), mRNA (C), sensor mimic and inhibitor cell imaging (C), Zn 2+ Cell imaging map (D) showing the regulation of ATP sensor by sensor, and the regulation of Zn by mRNA sensor. 2+ Imaging image (E) of sensor modulation and the effect of mRNA sensor on Zn 2+ Cellular imaging of the sensor and ATP sensor (F).

[0052] The ATP sensor's ability to target cellular mitochondria was validated using the commercial dye Mito-tracker for mitochondrial co-localization. Imaging was performed after co-incubating MCF-7 cells with the ATP sensor and Mito-tracker. Figure 9 As shown in A, left Figure 1 The green glow pattern represents the green light emitted by the FAM in the ATP sensor; left. Figure 2 The red glow pattern is the red fluorescence of the Mito-tracker; left. Figure 3 The image shows an overlap of the green fluorescence of the FAM and the red fluorescence of the Mito-tracker in the ATP sensor; the intensity correlation diagram of the green and red channels in the right image also shows a high correlation, with an overlap rate of 87%. Therefore, the ATP sensor enters the cell via endocytosis and specifically targets mitochondria. To investigate Zn... 2+ The sensor measures the pH and Zn content within the lysosomal cavity. 2+ To assess the specificity of the imaging, chloroquine, which regulates intracellular pH, was used to increase the acidity to neutrality in lysosomes. TPEN chelated zinc ions in the cell and even in lysosomes, setting up four control groups ((1,1), (1,0), (0,1), (0,0)) to examine Zn. 2+ Sensors monitor pH and Zn within lysosomes 2+ Response to changes. Chloroquine (200 μM) and TPEN (50 μM) were added to the cell culture medium at their respective concentrations beforehand and incubated with MCF-7 cells. For example... Figure 9 As shown in B, only in the absence of chloroquine and TPEN, cells are generated by Zn. 2+ The sensor emitted the strongest yellow light, while no yellow light signal was detected in the other three cases. This indicates that Zn... 2+The sensor's luminescence intensity effectively imaged zinc ions within lysosomes and responded to pH changes. To investigate the imaging effect of the mRNA sensor in response to TRPML1 mRNA within cells, MCF-7 cells were co-incubated with a mimic and inhibitor for 12 h, followed by further incubation with the mRNA sensor for 4 h. Results are as follows: Figure 9 As shown in Figure C, the intracellular light intensity gradually increased after the addition of the mimic, while the fluorescence of the corresponding inhibitor control group was weaker. Therefore, the mRNA sensor can image the changes in intracellular TRPML1 mRNA concentration. To further investigate the Zn in lysosomes... 2+ The link between this signaling pathway that induces changes in mitochondrial ATP and Zn further expands the understanding of ATP sensors and Zn. 2+ Simultaneous sensor-cell imaging experiment. First, SA1 was used to pretreat MCF-7 cells for 2 hours, and then SA1 and TPEN were used to pretreat MCF-7 cells simultaneously for 2 hours. Then, Zn... 2+ The sensor and ATP sensor were co-incubated with treated MCF-7 cells for 6 h to simultaneously observe Zn. 2+ Imaging changes in ATP concentration within cells. From Figure 9 As can be observed, the yellow fluorescence of Cy3 gradually weakens with increasing SA1 concentration, due to the influence of Zn. 2+ Released into the cytoplasm, causing intracytoplasmic Zn 2+ The concentration of Zn changes, which in turn weakens the green fluorescence signal of ATP; when TPEN chelating agent is added, Zn 2+ The yellow fluorescence signal almost disappeared, and at low SA1 concentrations, the light signal of ATP was slightly weaker compared to the control group. The results indicate that the change in ATP is caused by Zn within the lysosomes. 2+ Caused by the outflow of Zn in lysosomes 2+ A decrease in mitochondrial ATP leads to a drop in mitochondrial ATP. Given the direct correlation between TRPML1 mRNA and TRPML1 channel function, this experiment further investigated the regulatory role of mRNA in the working mechanism of the TRPML1 lysosomal membrane ion protein channel. When the mRNA sensor enters the cell via endocytosis, it specifically captures TRPML1 mRNA. This process inhibits the ion flow function of TRPML1, thereby leading to a decrease in lysosomal Zn. 2+ The concentration gradually increased.

[0053] Since mRNA plays a crucial role in regulating protein transcription and translation, combining mRNA sensors with Zn... 2+The sensor was simultaneously delivered into the cell to investigate intracellular physiological processes and further validate the results experimentally. Two concentrations of the mRNA sensor were co-incubated with MCF-7 cells at 37 °C for different durations (24, 18, and 12 h), followed by the addition of Zn. 2+ The sensor was incubated for another 6 hours, and the results were as follows: Figure 9 As shown in E, the red fluorescence signal of Cy5 gradually increased with the prolonged time the mRNA sensor remained in MCF-7 cells. Zn was also observed... 2+ The yellow fluorescence signal of Cy3 corresponding to the sensor also gradually increased, indicating that Zn in lysosomes... 2+ The concentration changed. When the mRNA sensor concentration was 0.5 nM and incubated with MCF-7 cells, the yellow fluorescence signal of Cy3 was also observed to gradually increase over time. These experimental results indicate that the TRPML1 channel can indeed affect Zn in lysosomes. 2+ Inflow and outflow. To further investigate TRPML1 mRNA, Zn 2+ ATP, Zn, and Zn in lysosomes are regulated by TRPML1. 2+ The association with ATP in mitochondria. TRPML1 mRNA was regulated at different time points using an mRNA sensor, followed by the addition of Zn. 2+ The three sensors, including the ATP sensor, were co-incubated with MCF-7 cells to investigate their effects on TRPML1 mRNA and Zn2 in cells. 2+ Simultaneous imaging of ATP, ATP, and other components. For example... Figure 9 As shown in Figure F, three sensors simultaneously entered the cells as a blank control, and three strong light signals were observed. After the mRNA sensors were pre-incubated for 12 and 24 hours, Zn was added. 2+ Sensors and ATP sensors (such as) Figure 9 F(a), (b)), the imaging results clearly show that Zn 2+ The Cy3 fluorescence signal gradually increased, while the green fluorescence signal of ATP gradually decreased; after 24 h of regulation using mRNA sensors at different concentrations (0.25, 0.5 nM), as shown... Figure 9 As shown in F(c) and (d), a similar phenomenon was observed, namely Zn 2+ The Cy3 fluorescence signal was enhanced compared to the blank control group, while the ATP fluorescence signal was weakened. The experimental results indicate that regulation of the TRPML1 channel induces the release of Zn in lysosomes. 2+ The accumulation of ATP in mitochondria leads to a decrease in ATP levels in downstream mitochondria. In summary, mitochondrial ATP can be reduced via the upstream signaling molecule Zn. 2+ The regulation of Zn has changed, and Zn 2+Furthermore, they are regulated by specific TRPML1 mRNAs, which can not only provide the distribution of dynamic levels of different target signals in the signaling pathway network, but also provide new perspectives and clues for further revealing the biomolecular homeostasis mechanisms in cell signaling pathways.

[0054] Example 10: The DNA sequence required for this experiment.

Claims

1. A DNA sensor for monitoring lysosomal zinc ion-induced ATP depletion, characterized in that... A method for studying the mechanism by which lipoprotein channel 1 (TRPML1) regulates the outflow of zinc ions from lysosomes and induces changes in mitochondrial ATP is proposed. The sensor is constructed using: a lysosome pH and zinc ion logic-gated cell imaging method, a mitochondrial ATP-targeted cell imaging method, and an intracellular TRPML1 mRNA fluorescence sensor. The sensor is prepared using 13 nm and 5 nm AuNPs as loading carriers, and DNA strands are immobilized on the cell surface via Au-S. The specific steps include: (1) Preparation of 5 nm gold nanoparticles: Before the reaction, all instruments were soaked in aqua regia (HCl:HNO3 volume ratio of 3:1) for more than 12 h, and then washed with a large amount of ultrapure water. 0.5 mL of HAuCl4 (25 mM) was added to 22.5 mL of ultrapure water and stirred for 1 min (600 rpm). Then, 0.5 mL of trisodium citrate solution (38.8 mM) was added and stirred for another 2 min. Then, 0.25 mL of 0.075 wt% sodium borohydride (NaBH4) was added and stirred for another 5 min. The mixture was then placed in a 4 ℃ refrigerator for 2 h to fully decompose the sodium borohydride. (2) Preparation of 13 nm gold nanoparticles: Before the reaction, all instruments were soaked in aqua regia (HCl:HNO3 volume ratio of 3:1) for more than 12 h, and then washed with a large amount of ultrapure water. 25 mL of HAuCl4 (1 mM) was added to a single-necked round-bottom flask and refluxed at 140 °C. When the solution boiled, 2.5 mL of trisodium citrate solution (38.8 mM) was quickly added and refluxed for 15 min to stop the reaction. (3) Preparation of SH-DNA strand modified 13nm AuNPs: Add 1 μL of 500 mM acetate buffer (pH 5.2) and 1.5 μL of 100 mM TCEP to 9 μL of 1 mM L1 and activate for 1 h. Add 3 mL of AuNPs (prepared fresh) and shake at room temperature for 16 h. Add 30 μL of 500 mM Tris-acetate (pH 8.2), shake slowly, and add 300 μL of 1 M NaCl dropwise to each vial. Salt age for at least one day to obtain AuNPs@SH-DNA. Store at room temperature for later use. (4) Preparation of SH-DNA strand modified 5nm AuNPs: 9 μL 1 mM SH-DNA was added to 1 μL 500 mM acetate buffer (pH 5.2) and 1.5 μL 100 mM TCEP for 1 h activation, 3 mL AuNPs (freshly prepared) were added and shaken at room temperature for 16 h, 30 μL 500 mM Tris-acetate (pH 8.2) was added and shaken slowly, 300 μL 1 M NaCl was added dropwise to each vial, and the salt was aged for at least one day. Finally, AuNPs@SH-L1 were obtained and stored at room temperature for later use. (5) Zn 2+ Sensor preparation: Centrifuge 3 mL of 13 nm AuNPs@SH-DNA, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to a final volume of 1.5 mL, add 10 μL of 100 μM I3 and incubate at 37 ℃ for 2 h, add 3 μL of 100 μM Cy3 strand and incubate at 37 ℃ for 2 h, centrifuge and bring the final volume to 150 μL, Zn... 2+ The final concentration obtained by the sensor was 200 nM; (6) Preparation of ATP sensor: 3 mL of 5 nm AuNPs@SH-AP, add 2 mg SH-PEG-TPP (MW:2438), incubate at room temperature for 2 h, centrifuge at 18000 rpm for 30 min, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to adjust the volume to 1 mL, add 2 μL of 100 μM FA, incubate at 37 ℃ for 2 h, centrifuge to adjust the volume to 300 μL, the final concentration of ATP sensor is 700 nM; (7) Preparation of mRNA sensor: Centrifuge 3 mL of 13 nm AuNPs@SH-L1, add Tris-NaCl (50 mM Tris, 150 mM NaCl) buffer to a final volume of 1.5 mL, add 3 μL of 100 μM L2, incubate at 37 ℃ for 2 h, centrifuge to a final volume of 150 μL, the final concentration of TRPML1 mRNA sensor is 200 nM; (8) Fluorescence measurement experiment: Take 50 μL Zn 2+ Sensors and target Zn at different concentrations 2+ The mixture was prepared to a final volume of 200 μL. The system was incubated at 37 °C for 90 min in a buffer solution of 50 mM Tris and 150 mM NaCl. After the reaction was complete, the fluorescence signal of the mixture was detected by a fluorescence spectrometer. The measurement conditions were as follows: excitation wavelength: 540 nm, fluorescence emission acquisition range: 560 nm-650 nm, excitation slit: 5 nm, emission slit: 5 nm, voltage: 1000 V. Take 80 μL of mRNA sensor and buffer system containing different concentrations of target TRPML1 mRNA, with a final volume of 200 μL; incubate the system at 37 ℃ for 60 min, the buffer is 50 mM Tris, 150 mM NaCl. After the reaction, the fluorescence signal of the mixture is detected by fluorescence spectrometry. The measurement conditions are: excitation wavelength: 635 nm, fluorescence emission acquisition range: 655 nm-760 nm, excitation slit: 5 nm, emission slit: 10 nm, voltage: 750 V. 40 μL of the ATP sensor and different concentrations of the target ATP were mixed in a buffer system, with a final volume of 200 μL. The system was incubated at 37 °C for 90 min. The buffer solution was 50 mM Tris and 150 mM NaCl. After the reaction was completed, the fluorescence signal of the mixture was detected by a fluorescence spectrometer. The measurement conditions were: excitation wavelength: 480 nm, fluorescence emission acquisition range: 500-650 nm, excitation slit: 5 nm, emission slit: 5 nm, voltage: 800 V.

2. The method for studying the mechanism by which lipoprotein channel 1 (TRPML1) regulates the outflow of zinc ions from lysosomes and induces changes in mitochondrial ATP, as described in claim 1, is characterized in that... DNA sequence: SH-DNA: 5'-CGTCCATCTACTCCGAGCCGGTCGAAATAGTGAGTTTTTTT-SH-3'; i-motif (I3): 5'-CCCCAACCCCAACGACCGGCTCGAACCCCAACCC-3'; Q-Strand: 5'-Cy3-ACTCACTATrAGGTAGAGATGGACG-3'; SH-L1: 5'-ATCCAGGAGTGTAATTTTTTTT-SH-3'; L2: 5'-Cy5-TTACACTCCTGGATGTGGG-3'; ATP aptamer: 5'-FAM-ACCTGGGGAGTATTGCGGAGGAAGGT-3'; SH-AP: 5'-ACCTTCCTCCGCAATACTCCCCCAGGTTTTTTTTTT-SH-3'.

3. A method for studying the mechanism by which lipoprotein channel 1 (TRPML1) regulates the outflow of zinc ions from lysosomes and induces changes in mitochondrial ATP, as described in claim 1, characterized in that: Steps 4, 5, and 6 (1) Zinc ion sensors enter the subcellular organelles of the cell lysosome and only produce fluorescence under acidic conditions and in the presence of zinc ions; (2) The ATP sensor specifically targets mitochondria to image ATP cells in mitochondria; (3) The mRNA sensor specifically recognizes intracellular TRPML1 mRNA; (4) Regulation of zinc ion sensor by mRNA sensor at 24, 18 and 12 h; (5) Regulation of the ATP sensor by the zinc ion sensor under the action of the activator; (6) The mRNA sensor simultaneously regulates the zinc ion sensor and ATP sensor at 24, 18 and 12 h.

4. The application of the method for preparing the DNA fluorescence sensor according to claim 1 to study the intracellular zinc ion and ATP signaling mechanisms mediated by the intracellular TRPML1 mucolipin channel in the cytoplasm, lysosomes, and mitochondria, characterized in that: mRNA sensors can inhibit the translation of TRPML1 mucoprotein channel protein. TRPML1 mucoprotein channel, as a non-cation-selective channel on the lysosome, can control Zn 2+ The efflux of plasma induces the accumulation of zinc ions in lysosomes over time; the efflux of zinc ions from lysosomes disrupts the intracellular Zn... 2+ Homeostasis can induce a decrease in mitochondrial ATP; mRNA sensors inhibit the translation of TRPML1 mucoprotein channel protein, which can lead to the accumulation of zinc ions and a decrease in ATP.

5. The method for studying the mechanism by which lipoprotein channel 1 (TRPML1) regulates the outflow of zinc ions from lysosomes and induces changes in mitochondrial ATP, as described in claim 1, is characterized in that... This biosensor uses pH response to image Zn in cells. 2+ Highly efficient targeted mitochondrial ATP cell imaging; sensitive imaging of low abundance TRPML1 mRNA in the cytoplasm; the three sensors exhibit high sensitivity, detection speed, and excellent temporal stability in in vitro fluorescence; sensitive imaging within cells and simultaneous imaging with all three sensors; constituting a biological signaling pathway monitoring system encompassing the cytoplasm, lysosomes, and mitochondria, including ATP sensors and Zn... 2+ The three sensors—a sensor, an mRNA sensor, and another—simultaneously enter the cell for imaging, aiming to answer questions about the mechanisms of ATP changes in mitochondria under the regulation of zinc ion efflux from lysosomes by TRPML1.

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