A mitochondrial DNA fluorescent probe, preparation method and biological imaging application thereof

Abstract

The present invention relates to the field of biomaterial technology, and in particular to a mitochondrial DNA fluorescent probe, a preparation method and a biological imaging application thereof. The chemical structure of the mitochondrial DNA fluorescent probe is: It can be well used for STED imaging, providing a new way for multifunctional photostable materials to achieve target-targeted STED super-resolution imaging at low power, and solving the problem of the lack of mitochondrial DNA-targeted fluorescent probes for super-resolution imaging in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a mitochondrial DNA fluorescent probe, a preparation method and biological imaging application thereof. Background Art

[0002] Since the discovery of DNA as genetic material, the study of the structural diagram of cellular DNA has become very important. Changes in the copy number and function of mitochondrial DNA (mtDNA) directly affect the energy conversion, brain function, metabolic regulation, cell carcinogenesis, immune activation, aging rate and life span of the body's internal systems. Therefore, changes in mtDNA content and function can be used as biomarkers for many diseases. In order to better observe the structure of DNA in cells, fluorescence microscopy is a key tool for imaging. However, due to the diffraction limit of light, the spatial resolution of optical microscopes is less than 250nm, which cannot meet the needs of subcellular scale observation. In recent years, the progress of super-resolution fluorescence microscopy (SRM) has broken this technical bottleneck. Among them, stimulated emission depletion microscopy (STED) and structured illumination microscopy (SIM) are two widely used methods for SRM. SIM is based on the illumination of samples with structured light, and SIM combines imaging speed with improved resolution, which means that this technology is the best choice for live cell imaging, 3D imaging and thick tissue sections. By selectively quenching the stray light of fluorescent probes with depletion lasers, STED obtains a higher resolution of about 30nm, but requires fluorescent probes with strict photostability or specific photoexcitation characteristics.

[0003] Most commercial nuclear and mtDNA dyes are organic dyes that cannot meet the requirements of SRM imaging due to their small Stokes shift, photobleaching, and short emission lifetime. In particular, probes for mtDNA in living cells are extremely rare. Commercially available mitochondrial dyes, such as MitoTracker probes and JC-1, are not specifically targeted to mtDNA. Therefore, there is an urgent need to develop new fluorescent probes for mtDNA staining for SRM. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes a mitochondrial DNA fluorescent probe, a preparation method and its biological imaging application, aiming to solve the problem that the prior art lacks a mitochondrial DNA targeted fluorescent probe suitable for super-resolution imaging.

[0005] In order to achieve the above-mentioned object of the invention, one of the technical solutions adopted by the present invention is:

[0006] Provided is a mitochondrial DNA fluorescent probe, wherein the mitochondrial DNA fluorescent probe has mitochondrial-related targeting ability by introducing a triphenylamine luminescent group and optimizing two pyridinium cation side arms of the skeleton; by introducing hydroxybenzofuranone to expand its function, mitochondrial DNA-specific targeting can be achieved, and it is applied to super-resolution imaging; the chemical structure of the mitochondrial DNA fluorescent probe is shown in formula (I): Named: MitoNA,

[0007] Where X is selected from F - , Cl - Br - ,I - PO4 3- 、NO3 - R1, R2, R3, R4 are selected from one of OH, NH4, COOH; Y is independently selected from one of a positively charged alkyl chain and triphenylphosphine, and n is the number of alkyl chains.

[0008] Furthermore, n is any one of the number of alkyl chains: 0, 1, 2, 3, ...

[0009] The second technical solution adopted by the present invention is:

[0010] A method for preparing the above-mentioned mitochondrial DNA fluorescent probe is provided, comprising the following steps:

[0011] S1, dissolving compound 1 and compound 2 in a mixed solution of toluene and water, adding tetrakis(triphenylphosphine)palladium catalyst, heating for reaction, cooling to room temperature, separating the liquids, removing the organic solvent by vacuum distillation, and separating and purifying by silica gel column to obtain compound 3;

[0012] S2, compound 3 and compound 4 were dissolved in anhydrous ethanol, and potassium hydroxide was added to heat the reaction. After cooling to room temperature, the mixture was separated and purified by a silica gel column to obtain an orange-yellow powder compound 5.

[0013] S3 and compound 5 were subjected to alkylation reaction and recrystallization to obtain reddish brown powder MitoNA. The reaction formula is as follows:

[0014]

[0015] Furthermore, in step S1, the volume ratio of toluene to water is 3-10:1; the molar ratio of compound 1 to compound 2 is 1.2-1.8:1; and the molar ratio of compound 1 to tetrakis(triphenylphosphine)palladium is 1:0.05-0.12.

[0016] Furthermore, the feed ratio of compound 5 in step S3 to the alkylation reaction is 1:16-64 equivalents.

[0017] Furthermore, the heating reaction in steps S1 and S2 and the alkylation reaction in step S3 are all carried out at 70-120° C. for 6-8 hours.

[0018] The third technical solution adopted by the present invention is:

[0019] Provided is the application of mitochondrial DNA fluorescent probe as a super-resolution dynamic imaging target marker.

[0020] Furthermore, the mitochondrial DNA fluorescent probe exhibits the ability to specifically recognize mitochondrial DNA in spectral titration and circular dichroism titration.

[0021] Furthermore, the mitochondrial DNA fluorescent probe is used in super-resolution dynamic imaging of living cells for non-diagnostic purposes.

[0022] Furthermore, the mitochondrial DNA fluorescent probe can target cell mitochondrial DNA at low power to achieve STED super-resolution imaging.

[0023] Furthermore, the mitochondrial DNA labeled with the mitochondrial DNA fluorescent probe is wrapped by the mitochondrial inner membrane and distributed in the mitochondrial inner membrane, achieving a resolution of 117nm at a power loss of 0.25mW and having the ability to track the dynamic changes of mitochondrial DNA and mitochondria over a long distance.

[0024] Furthermore, the mitochondrial DNA fluorescent probe described above has the ability to specifically identify mitochondrial DNA in cells; the cells do not repel common ions in the biological environment; the excitation wavelength of the fluorescent probe is 488nm, and the emission wavelength is 505-550nm.

[0025] Furthermore, the living cells are incubated with the mitochondrial DNA fluorescent probe for 2-5 hours, and the probe can stain the mitochondrial DNA in the cells.

[0026] The above solution of the present invention can bring the following beneficial effects:

[0027] 1. The mitochondrial DNA fluorescent probe of the present invention is obtained through a simple coupling reaction and an addition reaction, has a long emission spectrum in a PBS buffer solution, and the tail of the emission peak is located near 775 nm, which can be well used for STED imaging.

[0028] 2. In in vitro experiments, the mitochondrial DNA fluorescent probe MitoNA of the present invention not only exhibits good binding ability with different double-stranded DNAs and mitochondria in the fluorescence spectrum, but also has the ability to induce changes in the chirality of mitochondrial DNA.

[0029] MitoNA has good co-localization ability with commercial probes Picogreen and POLG2-mCherry in cell imaging. During STED super-resolution imaging, it can be well observed that MitoNA-labeled mitochondrial DNA is wrapped by and distributed in the mitochondrial inner membrane. While achieving a resolution of 117nm at a low power loss of 0.25mW, it can also track the dynamic changes of mitochondrial DNA and mitochondria over a long distance.

[0030] 3. The mitochondrial DNA fluorescent probe of the present invention provides a new way for multifunctional photostable materials to achieve target-targeted STED super-resolution imaging at low power, solving the problem of the lack of mitochondrial DNA-targeted fluorescent probes for super-resolution imaging in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1 This is the nuclear magnetic hydrogen spectrum of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 1 of the present invention.

[0033] Figure 2 This is the nuclear magnetic carbon spectrum of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 1 of the present invention.

[0034] Figure 3 This is a high-resolution mass spectrum of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 1 of the present invention.

[0035] Figure 4 This is the absorption spectrum of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 2 of the present invention in different solvents.

[0036] Figure 5 This is the emission spectra of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 2 of the present invention in different solvents.

[0037] Figure 6 This is a fluorescence intensity diagram of the mitochondrial DNA fluorescent probe MitoNA-1's ability to bind to DNA in Example 3 of the present invention.

[0038] Figure 7 This is a diagram of the chirality change of mtDNA15653 induced by MitoNA-1 in Example 3 of the present invention.

[0039] Figure 8This is an ion interference diagram of the main ions in the cell microenvironment on the mitochondrial DNA fluorescent probe MitoNA-1 in Example 4 of the present invention.

[0040] Fig. 9 This is the confocal localization image of the mitochondrial DNA fluorescent probe MitoNA-1 in Example 5 of the present invention and the commercial probe Mitochondria Deep Red (MTDR) in COS-7 cells, the confocal localization image of MitoNA-1 in COS-7 cells and the commercial mitochondrial inner membrane probe (PKMO) used for STED imaging, and the co-localization image with POLG2pcDNA3.1-mCherry-C2 protein.

[0041] Fig.10 The image comparison diagram of the mitochondrial DNA fluorescent probe MitoNA-1 and PKMO in stimulated depletion microscopy (STED) and common confocal microscopy (CLSM) and their corresponding resolutions in Example 6 of the present invention; wherein, Fig.10 In the figure, a and b are the contrast images of MitoNA-1 and PKMO under stimulated depletion microscopy (STED) and the corresponding resolutions, respectively; in the figure, c and d are the contrast images of MitoNA-1 and PKMO under conventional confocal microscopy (CLSM) and the corresponding resolutions, respectively. DETAILED DESCRIPTION

[0042] In order to more clearly explain the overall concept of the present invention, the following is a detailed description in the form of examples in conjunction with the accompanying drawings of the specification. The embodiments described below are only preferred implementations of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the implementation methods based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0043] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0044] Embodiment 1:

[0045] Preparation of mitochondrial DNA fluorescent probe MitoNA-1, the compound structure is as follows:

[0046]

[0047] The specific synthetic route is as follows:

[0048]

[0049] The specific synthesis steps are as follows:

[0050] S1. Dissolve compound 1 and compound 2 in a mixed solution of toluene and water (volume ratio: 10:1), add tetrakis(triphenylphosphine)palladium catalyst and heat to react at 90°C for 8 hours, cool to room temperature, separate the liquids, remove the organic solvent by vacuum distillation, and then separate and purify on a silica gel column to obtain compound 3; wherein the molar ratio of compound 1 to compound 2 is 1.5:1; the molar ratio of compound 1 to tetrakis(triphenylphosphine)palladium is 1:0.8.

[0051] S1. Compound 3 (429 mg, 0.1 mmol), compound 4 (200 mg, 0.8 mmol), and saturated potassium hydroxide aqueous solution were mixed in 10 mL of anhydrous ethanol and reacted at 80°C for 4 hours under nitrogen protection. After the reaction was completed, the mixture was filtered while hot, the filter cake was dissolved with dichloromethane, and the compound 5 was separated by column chromatography (dichloromethane / methanol = 10 / 1, v / v) with a yield of 90%.

[0052] S3. Compound 5 (280 mg, 0.05 mmol) was dissolved in 3 mL of anhydrous acetonitrile, and 16 eq of iodomethane was added to react at 90°C for 5 h. After slowly cooling to room temperature, a large amount of anhydrous ether was added overnight to obtain a large amount of precipitate. After suction filtration, 300 mg of reddish-brown powder MITONA-1 was obtained with a yield of 85%. 1 H NMR (400MHz, DMSO-d6) δ11.21 (s, 1H), 8.97 (d, J = 6.6Hz, 4H), 8.50–

[0053] 8.45(m,4H),8.16–8.11(m,4H),8.01(d,J=8.5Hz,2H),7.65(d,J=8.4Hz,1H) ,7.31(dd,J=15.4,8.6Hz,6H),6.83(s,1H),6.78–6.72(m,2H),4.32(s,6H). 13 CNMR(101MHz,Methanol-d4)δ145.01,129.59,124.61,123.87,77.82,77.50,77.18,48.07,47.85,47.64,34.58.HRMS:calculated for C 39 H 31 N3O3 2+ ,[MI] + :294.6177(m / z), found 294.6204.

[0054] The results of the H-NMR spectrum, C-NMR spectrum and high-resolution mass spectrum of the mitochondrial DNA fluorescent probe MitoNA-1 are shown in Figures 1 to 3 .

[0055] Embodiment 2:

[0056] Taking the mitochondrial DNA fluorescent probe MitoNA-1 prepared in Example 1 as an example, the photophysical properties of the mitochondrial DNA fluorescent probe were characterized. The specific steps are as follows:

[0057] The compound MitoNA-1 was prepared into a mother solution with a concentration of 2 mmol, and it was diluted into a 2 μM solution with different solvents, such as toluene, ethyl acetate, tetrahydrofuran, 1,4-dioxane, dichloromethane, acetone, N,N-dimethylformamide, anhydrous acetonitrile, and dimethyl sulfoxide, and the absorption and fluorescence spectra were characterized. Figure 4 , Figure 5 The mitochondrial DNA fluorescent probe MitoNA-1 has different absorption peaks in different solvents, fluctuating around 420nm to 470nm. At the same time, the strongest emission peak is at 570nm, and it shows a strong fluorescence intensity in tetrahydrofuran solution, which is about 50 times higher than in other solvents. In addition, the peak position of the emission peak does not change with the change of the excitation wavelength, which also proves that the mitochondrial DNA fluorescent probe has excitation independence.

[0058] Example 3: Testing of the binding ability of mitochondrial DNA fluorescent probe MitoNA-1 to double-stranded DNA.

[0059] Using Tris at pH 7.8 as solvent, MitoNA-1 was prepared into a solution with a concentration of 2 μmol / mL, and titrated with mtDNA and double-stranded DNA of different base sequences according to 0.2 equivalent value, and the DNA spectrum titration was characterized by fluorescence spectrophotometer. Figure 6-7 , indicating that it has the ability to specifically recognize mitochondrial DNA in spectral titration and circular dichroism titration.

[0060] Embodiment 4:

[0061] Using Tris with a pH of 7.8 as a solvent, MitoNA-1 was prepared into a solution with a concentration of 2 μmol / mL as a control group. Subsequently, 20 times the equivalent of common ions in the body were added to perform fluorescence spectrum tests. The fluorescence intensity values ​​were then statistically analyzed in columns, and each group of data was tested in parallel three times. The in vitro simulation of the coexistence of ions in the body and mitochondrial DNA fluorescent probes showed that MitoNA-1 did not react with common ions in the body and had good coexistence. Figure 8 shown.

[0062] Embodiment 5:

[0063] Inoculate 1×10 4 COS-7 cells of 1.5 density were placed in two confocal dishes and cultured in a cell culture incubator with 5% CO2 at 37°C for 48 hours. Then, 2 μM MitoNA-1 was added and incubated with the COS-7 cells for 4 hours. 200 nM mitochondrial deep red probe (MTDR) was added and incubated for 10 min. Then, 250 nM mitochondrial inner membrane probe PKMO (PK Mito Orange) for STED imaging was added and incubated for 40 min. Then, the culture medium was discarded and replaced with PBS buffer solution for cell imaging under a STEDYCON super-resolution microscope.

[0064] Proof of co-localization of mitochondrial DNA fluorescent probe with mitochondria. In COS-7 cells, compound MitoNA-1 was co-localized with the commercial mitochondrial deep red probe (MTDR) and the mitochondrial inner membrane probe PKMO (PK MitoOrange) for STED imaging. Fig. 9 As shown, mitochondrial DNA is tightly distributed inside the mitochondrial filamentous structure, confirming that the compound MitoNA-1 has the targeting ability to stain the genetic material inside mitochondria.

[0065] Using COS-7 cells, POLG2 pcDNA3.1-mCherry-C2 and Lipo 2000 liposomes were modified evenly at a volume ratio of 1:500 for cell transfection experiments. MitoNA-1 probe was added 48 hours after transfection for cell imaging. The excitation wavelength of MitoNA-1 is 488nm, and the emission wavelength is 500-560nm. The excitation wavelength of POLG2 pcDNA3.1-mCherry-C2 protein is 561nm, and the emission wavelength is 580-640nm. Fig. 9 As shown, the green channel and the red channel have a good co-localization coefficient, thereby confirming that the mitochondrial DNA compounds provided by the present invention specifically stain mitochondrial DNA.

[0066] Embodiment 6:

[0067] The mitochondrial DNA fluorescent probe was used for super-resolution imaging of mitochondrial DNA in cells. Taking COS-7 cells as an example, COS-7 cells were measured at 1×10 4Density seeded into confocal dish, and incubated at 37 degrees, 5% CO2 for 12 hours, after adding culture medium containing 1μM MitoNA-1 for 2 hours, add commercial fluorescent probe PKMO for 45 minutes, wash 3 times with PBS, wash away the residual PKMO small molecules for STED imaging. Confocal (CLSM) and STED imaging were performed under the laser with excitation wavelength of 488nm for MitoNA-1, 561nm for PKMO, emission wavelength of 575-625nm, and loss wavelength of 775nm. Fig.10 As shown in the figure, under CLSM conditions, not only the morphology of mitochondria is not clearly observed, but also the DNA has agglomeration phenomenon and still cannot be observed clearly in detail. Under STED, not only can the mitochondrial inner ridges be clearly seen, but also the mitochondrial DNA can be seen to be irregularly distributed and embedded in the mitochondrial inner ridges.

[0068] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A mitochondrial DNA fluorescent probe, characterized in that: Its chemical structure is shown in formula (I): Wherein, X is I; R1, R2, R4 are H, R3 is OH; and Y is methyl.

2. The method for preparing a mitochondrial DNA fluorescent probe according to claim 1, characterized in that: The reactions include:

3. The method for preparing a mitochondrial DNA fluorescent probe according to claim 2, characterized in that: The steps are as follows: S1, dissolving compound 1 and compound 2 in a mixed solution of toluene and water, adding tetrakis(triphenylphosphine)palladium catalyst and heating for reaction, cooling to room temperature, separating the liquids, removing the organic solvent by vacuum distillation, and separating and purifying by silica gel column to obtain compound 3; S2, compound 3 and compound 4 were dissolved in anhydrous ethanol, and potassium hydroxide was added to heat the reaction. After cooling to room temperature, the mixture was separated and purified by a silica gel column to obtain an orange-yellow powder compound 5; After alkylation reaction between S3, compound 5 and YX, reddish brown powder MitoNA was obtained by recrystallization.

4. The method for preparing a mitochondrial DNA fluorescent probe according to claim 3, characterized in that: In step S1, the volume ratio of toluene to water is 3-10:1; the molar ratio of compound 1 to compound 2 is 1.2-1.8:1; and the molar ratio of compound 1 to tetrakis(triphenylphosphine)palladium is 1:0.05-0.

12.

5. The method for preparing a mitochondrial DNA fluorescent probe according to claim 3, characterized in that: In step S3, the feed ratio of compound 5 to YX is 1:16-64 equivalents.

6. The method for preparing a mitochondrial DNA fluorescent probe according to claim 3, characterized in that: The heating reaction in steps S1 and S2 and the alkylation reaction in step S3 are all carried out at 70-120° C. for 6-8 hours.

7. Use of the mitochondrial DNA fluorescent probe as claimed in claim 1 in preparing a super-resolution dynamic imaging targeting marker.

8. The use according to claim 7, characterized in that: The mitochondrial DNA fluorescent probe can target cell mitochondrial DNA at low power to achieve STED super-resolution imaging.

9. The use according to claim 7, characterized in that: The mitochondrial DNA labeled by the mitochondrial DNA fluorescent probe is wrapped by the mitochondrial inner membrane and distributed in the mitochondrial inner membrane, achieving a resolution of 117nm at a power loss of 0.25mW, and having the performance of long-range tracking of the dynamic changes of mitochondrial DNA and mitochondria.

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