A nanomaterial for imaging mitochondrial microRNA

Abstract

The application belongs to the technical field of biosensors, and provides a kind of nanometer material for mitochondrion microRNA imaging, which is ZIF-90 nanoparticle wrapped SMD probe;The SMD probe is obtained by hybridization of nucleotide sequences such as SD1 chain, SD2 chain and Substrate chain shown in SEQ ID NO:1-3.The nanometer material utilizes the mitochondrion targeting and ATP response characteristics of ZIF-90, realizes the precise positioning of live cell mitochondrion and endogenous stimulation response release, and can specifically recognize mitomiR-340.The sensor has the advantages of simple operation, fast reaction, high imaging contrast and strong specificity, etc., can make up for the defects and deficiencies of existing mitochondrion microRNA imaging method, and realize high spatiotemporal resolution precise imaging.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology and relates to the construction of a sensor for detecting mitochondrial miR-340. Background Technology

[0002] Mitochondria, as the core hub of cellular energy metabolism and programmed cell death regulation, are closely related to the occurrence, development, and metastasis of malignant tumors due to their functional abnormalities. Mito miRs play a key role in oncogenesis by regulating mitochondrial genome stability, OXPHOS, and apoptosis signaling pathways. Due to the mitochondrial membrane barrier effect and its low abundance, subcellular localization and in situ detection of mito miRs has always faced technical bottlenecks. Therefore, overcoming the technical barriers of subcellular organelle-level detection by designing mitochondrial-targeted nanocarriers can successfully achieve high spatiotemporal resolution and precise imaging of live-cell mito miRs. Current research focuses on subcellular precision diagnosis and treatment, aiming to overcome the bottlenecks in the diagnosis and treatment of TNBC heterogeneity by integrating dynamic subcellular imaging technology with multimodal mitochondrial targeted regulation. Summary of the Invention

[0003] To address the bottleneck in subcellular localization in situ detection technology for mitochondrial miR-340, this invention proposes a fluorescence-based sensor for detecting mitochondrial miR-340 that features high spatiotemporal resolution, high sensitivity, low cost, and fast detection speed.

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] A nanomaterial for mitochondrial microRNA imaging, abbreviated as ZM, is a ZIF-90 nanoparticle encapsulating an SMD probe; the SMD probe is obtained by hybridization of SD1 strand, SD2 strand and Substrate strand; The nucleotide sequences of the SD1 chain, SD2 chain and Substrate chain are shown in SEQ ID NO:1-3, and the 12th nucleotide of the SD1 chain is modified with the fluorescent group FAM, the 17th nucleotide of the SD2 chain is modified with the fluorescent group TAMRA, and the 14th nucleotide of the Substrate chain is riboadecanoside.

[0006] The SMD probe was obtained by thermally denaturing and annealing equimolar amounts of SD1, SD2, and Substrate chains in solution.

[0007] A method for preparing the above-mentioned nanomaterial includes the following steps: mixing zinc salt, 2-formaldehyde-imidazolium (2-ICA) and SMD probe in N,N-dimethylformamide (DMF), then adding trioctylamine (TOA) to pH 6.0-7.0, stirring the reaction and separating the solid, which is the nanomaterial ZM.

[0008] The molar ratio of zinc ions to 2-ICA in the zinc salt is 1:2-4.

[0009] The molar ratio of the SMD probe to zinc ions in the zinc salt is 1:100-200.

[0010] A reagent kit prepared based on the above-mentioned nanomaterial ZM.

[0011] The aforementioned ZM nanomaterial kit can be used to detect mitochondrial microRNA 340.

[0012] An imaging method for mitochondria includes the following steps: The live cells to be tested were co-incubated with the nanomaterials, and then fluorescence imaging was performed; The fluorescence imaging is achieved by detecting the FRET effect, and the FRET signal intensity is positively correlated with the content of mitochondrial microRNA.

[0013] A drug for treating tumors, wherein the active ingredient of the drug is the aforementioned nanomaterial. The tumor cells overexpress miR340.

[0014] The tumor is a triple-negative breast cancer cell that overexpresses mitomiR-340. This type of cell also has the characteristics of significantly higher mitochondrial ATP concentration than normal breast cells, high expression of glucose transporter GLUT1, and negative mitochondrial membrane potential that can target and enrich ZIF-90 nanomaterials.

[0015] The detection principle of this invention is as follows: Figure 1 As shown, the sequences of each nucleic acid element are as follows: Substrate: 5'-TGC ACC TCA T / rA / G GCC TCG CT-3'; SD 1: 5'-AAT CAG TCT CA / i6FAMdT / CGG TCG AAA ATG AGG TGC A -3'; SD 2: 5'- AGC GAG GCCT CC GAG C / iTAMRAdT / TT GCT TTA TAA-3'; In materials containing the aforementioned nucleic acid elements, the underlined portions of SD 1 and SD 2 are complementary to both ends of the Substrate, respectively, forming SMDs; and with Zn 2+ ZIF-90 nanoparticles (ZM) encapsulating SMD probes were synthesized in situ with 2-ICA. The above nanoparticles can undergo the following reaction: Without the target mio miR-340, the FAM group on the SD1 chain and the TAMRA group on the SD2 chain of the SMD probe encapsulated within ZIF-90 are too far apart to undergo FRET. When the sensor comes into contact with cells, the ZIF-90 shell carries a positive charge in the slightly acidic environment of the tumor (pH approximately 6.5-6.8). The positively charged ZM nanoparticles are efficiently endocytosed by the negatively charged cell membrane through electrostatic adsorption, forming endosomes. Subsequently, in the strongly acidic environment of lysosomes (pH 4.5-5.0), the ZIF-90 framework exerts a proton sponge effect, continuously absorbing protons, causing the lysosomal membrane to rupture and enabling lysosomal escape. The extremely high ATP levels near the mitochondrial surface react with the Zn in ZIF-90. 2+ Competitive binding causes escaped ZM to passively approach and contact mitochondria. Once ZM enters the cell and targets the mitochondria, the high concentration of ATP within the mitochondria acts as a "igniter" to bind with Zn in the ZIF-90 backbone. 2+ Competitive coordination leads to the disintegration of ZIF-90, resulting in the controlled release of the SMD probe. When the target mito miR-340 is present in the system, the SD1 and SD2 chains in the SMD probe bind to mito miR-340, causing the dividing DNAzymes to move closer to each other in space. At the same time, the distance between the FAM and TAMRA groups is shortened, resulting in a highly efficient fluorescence resonance energy transfer (FRET) effect. This leads to fluorescence quenching of the donor FAM and enhancement of the fluorescence signal of the acceptor TAMRA, achieving "on-off" regulation of the signal. Spatially close DNAzymes in Zn 2+ With the assistance of the SMD probe, the probe specifically identifies and cleaves the ribo-adenine site (rA) contained in the substrate chain. After the rA site is broken, the original structure of the SMD probe is destroyed, the spatial distance between the fluorescent group and the quenching group increases significantly, the intermolecular fluorescence quenching effect is greatly relieved, and the system recovers and releases a significant fluorescence signal. The qualitative and quantitative detection of miR-340 is achieved by measuring the changes in fluorescence signal intensity. After being cleaved, the Substrate releases ASO1 and ASO2, which target GLUT 1 mRNA, thereby interfering with its RNA and inducing apoptosis, thus completing the treatment process.

[0016] In normal cells, the extracellular pH of tissues is neutral at 7.3-7.4. ZM has a low degree of protonation and a weak surface positive charge, resulting in electrostatic adsorption and endocytosis efficiency with the cell membrane that is far lower than in the weakly acidic tumor microenvironment of pH 6.5-6.8. Secondly, the ATP concentration in the mitochondria of normal cells is significantly lower than that in triple-negative breast cancer cells, which is insufficient to competitively disrupt the ZIF-90 coordination scaffold, preventing the release of SMD probes. Furthermore, mitomiR-340 is underexpressed in the mitochondria of normal breast cells, so even a small amount of ZM entering the cells cannot initiate FRET signaling changes, DNAzyme cleavage, or subsequent therapeutic cascades. Therefore, ZM can achieve tumor cell-specific targeted imaging and intervention therapy.

[0017] The present invention has the following advantages: The biosensor provided by this invention utilizes the mitochondrial targeting and ATP-responsive properties of ZIF-90 to achieve precise localization of mitochondria in living cells and release in response to endogenous stimuli; it utilizes the target recognition and self-assembly properties of DNAzyme to achieve specific recognition of mitomiR-340; and it uses FRET technology to achieve high signal-to-noise ratio signal output. This sensor has advantages such as simple operation, fast response speed, high imaging contrast, and strong specificity, and can overcome the deficiencies and shortcomings of existing mitochondrial microRNA imaging methods, achieving precise imaging with high spatiotemporal resolution. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the experiment. Figure 2 The images are: ZIF-90 (A) TEM image, ZM (B) TEM image and SEM image (C); Figure 3 The fluorescence response spectrum is shown to demonstrate the feasibility of the sensor in vitro. Figure 4 The image shows the FRET fluorescence response of the sensor to different concentrations of mitomiR-340. Figure 5 This is a graph showing the sensor's specific detection results for the target miRNA; Figure 6 This is a confocal microscope image of mitochondria imaged by the sensor in different cell lines. Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0020] Example 1: Construction of ZM nanomaterials The SD1 chain (5' end modified with FAM), SD2 chain (3' end modified with TAMRA), and substrate chain were commercially synthesized according to SEQ ID NO:1-3.

[0021] (1) Preparation of SMD probes Add 10 μL of 100 μM SD 1, 10 μL of 100 μM SD 2 and 10 μL of 100 μM Substrate to HEPES buffer (pH=7.4) to a final volume of 100 μL. Incubate at 95°C for 5 min, then allow to cool naturally to room temperature to obtain the SMD probe. Store at 4°C for later use.

[0022] (2) Construction of ZM nanomaterials (i) Add 2-formaldehyde-imidazolium (2-ICA, 40 mg, 0.42 mmol) to N,N-dimethylformamide (DMF) (9 mL). Heat the solution to 50 °C with stirring until the 2-ICA is completely dissolved, then cool to room temperature; (ii) Mix 100 μL of SMD probe solution (10 μM) and 1 mL of DMF solution of Zn(COOH)2·6H2O (33 mg, 0.15 mmol); (iii) The mixed solution prepared in step (ii) was added dropwise to the 2-ICA solution prepared in step (i), and the mixture was stirred for 10 min. Then, trioctylamine (TOA, 500 μL) was added. After stirring at 25 °C for 12 h, the mixture was centrifuged at 17800 × g for 10 min to separate the solid particles. The solid particles were washed once with DMF and twice with ethanol, and the product was dried at 40 °C to obtain ZM nanomaterials.

[0023] The above steps do not involve adding SMD probes to synthesize ZIF-90 nanoparticles.

[0024] The TEM of ZIF-90 and ZM, such as Figure 2 As shown in Figures A and B, ZM exhibits a regular rhombic dodecahedral structure, similar to ZIF-90. The addition of contents did not affect its microstructure. This was confirmed by SEM images (…). Figure 2 The particle diameter in the middle (C) is relatively concentrated, and the size after encapsulation is about 125 nm.

[0025] (3) Feasibility verification of ZM nanomaterial detection The mimic of mio miR-340 was synthesized (5'-TTA TAAAGC AAT GAG ACT GAT T-3'); ATP and mio miR-340 mimic were considered as Input A and Input B, respectively.

[0026] ZM nanomaterials with a concentration of 200 μg / mL were used, with a single sample addition volume of 20 μL; the ATP concentration in the system was 5 mM, with an addition volume of 20 μL; the mito miR-340 mimic concentration was 100 nM, with an addition volume of 20 μL.

[0027] Add reagents according to the following groups: a) Without adding ATP and mito miR-340, denoted as (0, 0); b) Add only ATP (Input A exists, Input B does not exist), denoted as (1, 0); c) Add only mio miR-340 (Input B exists, Input A does not exist), denoted as (0, 1); d) Add ATP and mitomiR-340 simultaneously (both Input A and Input B are present), denoted as (1, 1); Fluorescence response spectra were collected using a fluorophotometer. The detection conditions were: excitation wavelength of 488 nm, scanning emission wavelength range of 500-700 nm, and fluorescence intensity at 520 nm (FAM) and 580 nm (TAMRA) was recorded respectively.

[0028] The results are as follows Figure 3 As shown, compared to other groups, the group with both ATP and mito miR-340 showed a significant fluorescence enhancement at 580 nm, while the fluorescence weakened at 520 nm, indicating a significant FRET effect. This demonstrates that the constructed ZM nanomaterial sensor can respond to ATP and mito miR-340, achieving detection and imaging functions.

[0029] Example 2 mito miR-340 detection limit The ZM nanomaterials prepared in Example 1 were used, and under the condition that other experimental conditions were kept consistent, the final concentrations of mito miR-340 were 0, 10 fM, 20 fM, 30 fM, 40 fM and 50 fM, respectively. The fluorescence intensity was measured according to the method in Example 1.

[0030] The results are as follows Figure 4 As shown: With the continuous increase of mito miR-340 concentration, the FRET signal (I 580 / I 520 The concentration gradually increases, and within the concentration range of 10-50 fM, I... 520 The logarithm (lgC) of the concentration of mito miR-340 showed a good linear negative correlation, and its linear regression equation was: I520 =-114080.985×lgC+1311160.142, where, I 520 The fluorescence intensity at 520 nm is denoted as α (au), C is the concentration of mito miR-340 (fM), and the correlation coefficient R is denoted as α. 2 =0.991, indicating that the sensor has excellent quantitative analysis capability within this concentration range. Calculated using the three-standard-deviation method, the limit of detection (LOD) for mitomiR-340 is as low as 3.3 fM, indicating that the prepared sensor is sensitive to changes in the concentration of mito miR-340 and has good sensitivity.

[0031] Example 3: Sensor response to different miRNA sequences Commercial synthesis was performed according to the following sequence: mito miR-340 mimic: 5'-TTA TAAAGC AAT GAG ACT GAT T-3'; Mismatch sequence Mut1: 5'-TTA TCAAGC AAT GAG ACT GAT T-3'; Mismatch sequence Mut2: 5'-TTA TCAAGC TAT GAG ACT GAT T-3'; Mismatch sequence Mut3: 5'-TTA TCAAGC TAT GAA ACT GAT T-3'; miR-195: 5'-UAGCAGCACAGAAAUAUUGGC-3'; miR-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3'; miR-155: 5'-UUAAUGCUAAUCGUGAUAGGGGU-3'; let-7a:5'-UGAGGUAGUAGGUUGUAUAGUU-3'.

[0032] Then, follow these steps to determine the specificity of the test: The ZM nanomaterials prepared in Example 1 were used, and under the condition that other experimental conditions were consistent, 100 nM non-target substances—mismatched sequences Mut1, Mut2, Mut3, miR-195, miR-21, miR155, let-7a, and a mixture of the above sequences—were used to replace 100 nM mio miR-340 mimic. Positive samples (mitomiR-340 mimic) and blank samples were set up, and fluorescence intensity was measured.

[0033] The results are as follows Figure 5 As shown, some designed mio miR-340 mismatch sequences (Mut 1, Mut 2, Mut 3) and common interfering RNA sequences miR-195, miR-21, miR-155, and let-7a cannot undergo SMD reconstruction to produce FRET fluorescence signals. Various RNA mixtures, including mio miR-340, can produce strong FRET fluorescence signals.

[0034] Example 4: Application of live-cell mitochondrial miR-340 imaging MDA-MB-231, MCF-7, and MCF-10A cells were co-incubated with ZM nanomaterials (200 μg / mL) for 8 h. Mitochondria were labeled with Mito Tracker Deep Red, and nuclei were labeled with Hoechst 33342. Imaging was then performed using confocal laser scanning microscopy. The detection parameters were: FAM channel excitation wavelength 488 nm, emission wavelength 500-550 nm; TAMRA channel excitation wavelength 543 nm, emission wavelength 560-650 nm.

[0035] The results are as follows Figure 6 As shown, in MDA-MB-231 cells with high mito miR-340 expression, the TAMRA (FRET) fluorescence signal and the fluorescence signal of the mitochondrial probe were clearly observed to perfectly overlap; while in MCF-7 cells with moderate mito miR-340 expression, the FRET signal was weak; and in MCF-10A cells with low mito miR-340 expression, the FRET signal was almost unobservable. This indicates that the constructed ZM nanomaterial sensor can precisely target mitochondria and achieve specific, high-resolution imaging of in situ mito miR-340 in mitochondria in different cells.

Claims

1. A nanomaterial for imaging mitochondrial microRNA, characterized in that, The nanomaterial is ZIF-90 nanoparticles encapsulating an SMD probe; the SMD probe is obtained by hybridizing SD1, SD2, and Substrate chains. The nucleotide sequences of the SD1 chain, SD2 chain and Substrate chain are shown in SEQ ID NO:1-3, and the 12th nucleotide of the SD1 chain is modified with the fluorescent group FAM, the 17th nucleotide of the SD2 chain is modified with the fluorescent group TAMRA, and the 14th nucleotide of the Substrate chain is riboadecanoside.

2. A method for preparing nanomaterials as described in claim 1, characterized in that, The process includes the following steps: Zinc salt, 2-formaldehyde-imidazole and SMD probe are mixed in N,N-dimethylformamide, then trioctylamine is added to bring the pH to 6.0-7.0, the reaction is stirred and the solid is separated, which is the nanomaterial.

3. The preparation method according to claim 2, characterized in that, The molar ratio of zinc ions to 2-formaldehyde-imidazole in the zinc salt is 1:2-4; the molar ratio of the SMD probe to zinc ions in the zinc salt is 1:100-200.

4. A kit comprising the nanomaterial as described in claim 1.

5. A method for imaging mitochondria, characterized in that, Includes the following steps: The live cells to be tested were co-incubated with the nanomaterials described in claim 1, and then fluorescence imaging was performed.

6. A drug for treating tumors, characterized in that, The active ingredient of the drug is the nanomaterial described in claim 1.

7. The medicament for treating tumors according to claim 6, characterized in that, The tumor cells overexpress miR340.

8. The medicament for treating tumors according to claim 7, characterized in that, The tumor was a triple-negative breast cancer cell.

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