Preparation method of DNA probe and application of DNA probe in super-resolution imaging of DNA of cells and tissue slices

Abstract

The invention belongs to the field of super-resolution imaging, and particularly discloses a preparation method of a DNA probe and application of the DNA probe in super-resolution imaging of cell and tissue slice DNA, rhodamine dye modified by different groups is connected with a Hoechst structure through a short connecting chain, and the self-flickering DNA probe is formed; the spiro opening and closing capability of the fluorescent dye in the probe is modified and adjusted through different groups, so that fine regulation and control on the performance of the DNA probe are realized, and different SMLM super-resolution imaging requirements are met; sMLM imaging of DNA in living cells, fixed cells, tissue frozen sections and clinical tissue paraffin sections is achieved by combining the SMLM technology with a self-scintillation probe with DNA specificity, and the resolution ratio is far higher than that of a traditional imaging technology.

Description

Technical Field

[0001] The present invention relates to the field of super-resolution imaging, in particular to a method for preparing a DNA probe and application of the same in super-resolution imaging of DNA in cells and tissue sections. Background Art

[0002] Single-molecule localization microscopy (SMLM) is a super-resolution microscopy technique widely used in biology to study cellular structures below the diffraction limit. While single-molecule localization microscopy (SMLM), including (d)STORM, PALM, and PAINT, can overcome the diffraction limit, it has several limitations when imaging DNA nanostructures in living cells and tissue sections. The main issues include:

[0003] 1. For cell samples:

[0004] 1) Poor cell permeability: Commonly used commercial organic dyes (such as Alexa 647 and Cy5) require highly reducing photoswitchable buffers that are incompatible with living cells.

[0005] 2) Requires complex photoswitchable buffers and high laser power for excitation imaging: This limits its application in living cells.

[0006] 3) Commonly used DNA-binding fluorescent dyes (such as Hoechst, DAPI, etc.) lack self-blinking ability, which limits their application in SMLM.

[0007] 4) 5-Ethynyl-2'-deoxyuridine (EdU), 5-ethynyl-2'-deoxycytosine (EdC), and their analogs can be combined with fluorescent dyes via click chemistry for super-resolution imaging after DNA labeling. However, their applicability is limited: EdU and EdC are primarily applicable to proliferating cells, limiting their application in non-proliferating cells. When EdU and EdC label proliferating cells, the efficiency of DNA labeling is affected by the cell cycle and proliferation rate, and the staining procedure is relatively complex.

[0008] 5) Limitations of 2D imaging: Biological structures are inherently three-dimensional, and 2D imaging may not provide comprehensive information. 3D biological structure resolution capabilities: The ability to resolve the native three-dimensional chromatin structure in living cells is required to provide richer and more comprehensive insights.

[0009] 2. For tissue samples:

[0010] 1) Limitations of HE staining: Although HE staining is widely used in pathological tissue sections, it often fails to provide sufficient cellular detail information and is sometimes insufficient to support accurate diagnosis or treatment planning.

[0011] 2) Fluorescence Imaging Challenges: Fluorescence imaging technology remains challenging in pathological tissues, particularly in the context of high-level chromatin structural changes during tumor development and progression. These changes are difficult to interpret using traditional widefield or confocal fluorescence microscopy and have therefore not been fully characterized in pathological tissues, primarily due to the lack of simple, rapid, and effective methods.

[0012] 3) Application limitations of super-resolution imaging: Existing fluorescent dyes and staining methods are limited in their application in super-resolution imaging (SMLM), especially in the imaging of chromatin structure in fixed paraffin-embedded (FFPE) and frozen tissue sections. Summary of the Invention

[0013] In order to solve the above technical problems, the present invention provides a method for preparing a DNA probe and its application in super-resolution imaging of DNA in cells and tissue sections.

[0014] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0015] One of the technical solutions of the present invention is a DNA probe having a structure as shown in Formula 1:

[0016]

[0017] Wherein: R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CF3)2.

[0018] A second technical solution of the present invention is a method for preparing a DNA probe, comprising the following steps:

[0019] S1. The compound represented by Formula IX (20.0 mg), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, IX-I: 31.3 mg; IX-II: 29.7 mg; IX-III: 26.3 mg; IX-IV: 28.3 mg) and N,N-diisopropylethylamine (DIEA, IX-I: 13.6 μL; IX-II: 12.9 μL; IX-III: 12.0 μL; IX-IV: 12.3 μL) were dissolved in 4 mL of DMF and stirred under a nitrogen atmosphere for 10 minutes; wherein:

[0020]

[0021] R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CH3)2;

[0022] S2. A solution of the compound represented by Formula VI (IX-I: VI = 28.9 mg; IX-II: VI = 27.4 mg; IX-III: VI = 24.2 mg; IX-IV: VI = 26.1 mg) in 0.5 mL of DMF is added dropwise to the reaction mixture at room temperature for 2 hours. After the reaction is completed, the solvent is removed by distillation under reduced pressure, and the residue is purified by silica gel column chromatography to obtain a DNA probe represented by Formula 1.

[0023] Furthermore, the preparation method of the compound represented by formula IX comprises:

[0024] S11. 6-Carboxytetramethylrhodamine (430.0 mg) was dissolved in anhydrous DMF (3 mL), followed by the addition of potassium carbonate (414.0 mg) and triethylamine (Et3N, 417.0 μL); the mixture was cooled in an ice bath, and allyl bromide (362.9 mg) was slowly added; the mixture was then warmed to room temperature and stirred for 24 hours. After the reaction was complete, the solvent was removed using a vacuum oil pump, and the residue was purified by silica gel column chromatography to obtain 450.0 mg of the compound represented by Formula VII:

[0025]

[0026] S12. Under a nitrogen atmosphere, 4-dimethylaminopyridine (DMAP, 5.2 mg) and H2N-R (CH2CH2CH2CH3NH2 = 31.0 mg; CF3CH2NH2 = 42.0 mg; (CF3)2CHNH2 = 70.9 mg; SO2N(CH3)2NH2 = 52.7 mg) were added to an ultra-dry dichloromethane solution containing the compound represented by Formula VII (100.0 mg). After stirring for 5 minutes, triethylamine (Et3N, 71.1 μL) was added to the mixture. Subsequently, an ultra-dry dichloromethane solution of phosphorus oxychloride (POCl3, 19.8 μL) was added to the reaction system, and the mixture was reacted at room temperature overnight. After completion of the reaction, the mixture was added dropwise to ice water, extracted with ethyl acetate (EA, 50 mL×3), washed with saturated sodium chloride solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 450.0 mg of the compound represented by Formula VIII:

[0027]

[0028] R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CH3)2;

[0029] S13. Dissolve the compound represented by formula VIII (40.0 mg) in 3 mL of anhydrous tetrahydrofuran, then add 1,3-dimethylbarbituric acid (VIII-I: 22.7 mg; VIII-II: 23.3 mg; VIII-III: 20.2 mg; VIII-IV: 22.8 mg) and tetrakistriphenylphosphine palladium (VIII-I: 16.8 mg; VIII-II: 17.2 mg; VIII-III: 14.9 mg; VIII-IV: 17.0 mg), and stir the reaction at room temperature for 1 hour; after the reaction is completed, remove the solvent by distillation under reduced pressure, and purify the residue by silica gel column chromatography to obtain the compound represented by formula IX.

[0030] Furthermore, the preparation method of the compound represented by formula VI comprises:

[0031] S21. Dissolve Hoechst dye hydrochloride (100 mg) in water, then add a solution of potassium carbonate (123 mg). Separate the precipitate by centrifugation, wash with water, and freeze-dry to obtain the free base of Hoechst dye.

[0032] S22. Potassium carbonate (98.4 mg) was dissolved in anhydrous DMF, and then the free base of Hoechst dye (80 mg) was added to a round-bottom flask and stirred. Subsequently, tert-butyl (2-bromoethyl) carbamate (84.7 mg) was added, and the mixture was reacted at 50° C. for 24 hours. After the reaction was completed, the solvent was removed by vacuum oil pump, and the residue was purified by silica gel column chromatography to obtain 45 mg of the compound represented by Formula V:

[0033]

[0034] S23. Dissolve the compound represented by formula V in 5 mL of an organic solvent, place the mixture in a round-bottom flask, and react for 1 hour. Then, remove the solvent by distillation under reduced pressure to obtain a compound represented by formula VI.

[0035] Preferably, the organic solvent consists of trifluoroacetic acid and dichloromethane in a volume ratio of 1:4.

[0036] The third technical solution of the present invention is the application of a DNA probe in super-resolution imaging of DNA in cells and tissue sections. The DNA probe is used to label DNA in living cells, fixed cells and tissue sections, and the self-scintillation of the DNA probe is used to achieve the positioning of DNA molecules and super-resolution imaging in super-resolution microscopy imaging.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1) Improved resolution: The present invention connects rhodamine dyes modified with different groups to the Hoechst structure via short linkers to form self-scintillation DNA probes. By adjusting the spirocyclization opening and closing ability of the fluorescent dye in the probe through different group modifications, the performance of the DNA probe can be finely controlled to meet different SMLM super-resolution imaging requirements. By combining SMLM technology with DNA-specific self-scintillation probes, SMLM imaging of DNA in living cells, fixed cells, frozen tissue sections, and clinical tissue paraffin sections can be achieved, with a resolution far higher than traditional imaging technologies.

[0039] 2) Enhanced imaging specificity: The specific binding of the Hoechst structure enhances the probe's labeling specificity for DNA.

[0040] 3) Reduced phototoxicity: Low-intensity illumination reduces phototoxicity to cells, making long-term imaging possible.

[0041] 4) Good cell permeability: No need to fix or perforate cells, and low toxicity to living cells.

[0042] 5) Real-time dynamic imaging of living cells: The probe allows real-time tracking and dynamic monitoring of DNA fiber movement in living cells.

[0043] 6) 3D imaging capability: Achieve three-dimensional imaging of chromatin structure, providing more comprehensive biological structural information.

[0044] 7) Wide range of applications: It is not only suitable for living cells and fixed cells, but also for tissue freezing and paraffin sections, and can be used in combination with other techniques.

[0045] 8) Combining artificial intelligence and live cell super-resolution imaging data offers new possibilities for identifying somatic and stem cells directly in living single cells. The combination of these technologies offers new insights into chromatin structure and function and has the potential to become a diagnostic method.

[0046] 9) Improve diagnostic accuracy: By revealing changes in chromatin structure, it helps to improve the diagnostic accuracy of clinical pathological tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Characteristics of the prepared DNA probe: A. Relationship between the normalized absorbance of 6-TAMRA and DNA probe (5 μM) at 565 nm and the dielectric constant in a mixture of water and hexadecane (volume ratio, 10 / 90-90 / 10); B. Fluorescence intensity of the DNA probe at 585 nm (5 μM) before and after incubation in PBS (pH = 7.4) containing 0.4% BSA or 0.4% SDS at room temperature for 4 hours; C. pH titration results of the DNA probe (5 μM) in BR buffer at room temperature for 4 hours; D. Transmission electron microscopy image of the DNA probe in water (2 μM); E. Fluorescence spectrum of the DNA probe (2.5 μM) before and after incubation in PBS (pH = 7.4) at room temperature for 4 hours; F. Confocal imaging of DNA treated with the probe (1 μM) in fixed cells for 1 hour; G. Representative single-frame images of the probe in live-cell SMLM imaging. HeLa cells were treated with probes (50 nM) for 1 hour before imaging; H, Representative single-molecule fluorescence time trajectories of probes 2 and 3 in live-cell SMLM imaging; I, Changes in the molecular survival fraction of probes 2 and 3 under 561 nm light irradiation over time; J, Distribution and single exponential fitting of the fluorescence on-time of probes 2 and 3; K, Distribution and single exponential fitting of the photons in each switching event of probes 2 and 3; L, SMLM images of cell nuclei stained by probes 2 and 3 reconstructed from different frames.

[0048] Figure 2 Absorption and fluorescence spectra of DNA probes (5 μM) before and after incubation with 0.4% BSA or 0.4% SDS in PBS (pH = 7.4) for 4 hours at room temperature.

[0049] Figure 3 This is the pH titration spectrum of the DNA probe (5 μM) after being exposed to BR buffer at room temperature for 4 h.

[0050] Figure 4 Dynamic light scattering (DLS) measurements of the size dispersion of DNA probes (2 μM) in water.

[0051] Figure 5Figure 3. Direct labeling of DNA with the scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst for STORM imaging of DNA ultrastructure in living and fixed hFib cells: A. Representative super-resolution (SR) images of nuclear DNA labeled with TMR-CF3-Hoechst (using SRVoronoi mosaic technique) and magnified DNA ultrastructure at the nuclear edge (enlarged area in red box), nucleoplasm (enlarged area in green box), and nucleolar periphery (enlarged area in blue box) in living hFib cells (top) and fixed hFib cells (bottom); B. Representative super-resolution (SR) images of nuclear DNA labeled with TMR-2CF3-Hoechst (using SR Voronoi mosaic technique) and the enlarged DNA ultrastructure of the nuclear edge (enlarged area in the red box), nucleoplasm (enlarged area in the green box) and nucleolar periphery (enlarged area in the blue box); C, Probability distribution of localization accuracy of living hFib cells (red histogram) and fixed hFib cells (black histogram) labeled with TMR-CF3-Hoechst; D, Probability distribution of super-resolution localization accuracy of living hFib cells (red histogram) and fixed hFib cells (black histogram) labeled with TMR-2CF3-Hoechst.

[0052] Figure 6 The self-scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst are used for direct labeling of DNA ultrastructure in living and fixed HeLa cells using STORM imaging.

[0053] Figure 7 Direct labeling with the self-scintillation dyes TMR-C4-Hoechst and TMR-SO2-Hoechst for STORM imaging of DNA ultrastructure.

[0054] Figure 8 To visualize 3D STORM super-resolution images of DNA in living hFib cells using TMR-CF3-Hoechst and ViSP software:

[0055] A, Representative three-dimensional STORM image of DNA labeled with TMR-CF3-Hoechst in living hFib cells, with the three-dimensional image color-coded by depth Z; B, Representative three-dimensional density map of DNA labeled with TMR-CF3-Hoechst in living hFib cells; C, Rotation sequence (0°, 45°, 90°, 135°, 180°) of the three-dimensional density map of DNA structures at the nuclear edge (enlarged area in the red box), nucleoplasm (enlarged area in the green box), and nucleolar periphery (enlarged area in the blue box); D, Rotation sequence (0°, 45°, 90°, 135°, 180°) of the three-dimensional STORM image of DNA structures cropped from the nuclear edge (enlarged area in the yellow box), with the three-dimensional image color-coded by depth Z (color scale bar).

[0056] Figure 9 Three-dimensional STORM super-resolution images of DNA visualized in live hyperacetylated hFib cells using TMR-CF3-Hoechst and ViSP software.

[0057] Figure 10 Time-lapse single-molecule localization microscopy (SMLM) imaging using TMR-CF3-Hoechst and TMR-2CF3-Hoechst in living HeLa cells: A. Sequential super-resolution (SR) images of DNA acquired using TMR-CF3-Hoechst at 0 minutes (white), 10 minutes (red), and 20 minutes (green); B. Sequential super-resolution (SR) images of DNA acquired using TMR-2CF3-Hoechst at 0 minutes (white), 10 minutes (red), and 20 minutes (green), with an excitation wavelength of 561 nm (power density ~0.33 kW / cm2). Each super-resolution image was reconstructed from 10,000 images (30 milliseconds per frame), corresponding to an acquisition time of 300 seconds; C. Results of DNA motion tracking analysis (under analysis).

[0058] Figure 11Figure 3 STORM imaging of DNA directly labeled with the scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst in live and fixed human fibroblasts (hFib) with and without hyperacetylation (TSA treatment): A, representative STORM density rendering images of DNA in live hFib cells labeled with TMR-CF3-Hoechst (left: intact nuclei; right: magnified area in red boxes); B, representative STORM density rendering images of DNA in fixed hFib cells labeled with TMR-CF3-Hoechst (left: intact nuclei; right: magnified area in red boxes) C, representative STORM density rendering images of DNA in live hFib cells labeled with TMR-2CF3-Hoechst (left: intact nuclei; right: magnified area in red boxes); D, representative STORM density rendering images of DNA in fixed hFib cells labeled with TMR-2CF3-Hoechst (left: intact nuclei; right: magnified area in red boxes); E, cumulative distribution plot of Voronoi polygon density in live hFib cells labeled with TMR-CF3-Hoechst (blue) (n=39) and TSA-treated (orange) (n=32). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ****P < 0.0001; F, Cumulative distribution of Voronoi polygon density in control (blue) (n = 32) and TSA-treated (orange) (n = 24) fixed hFib cells labeled with TMR-CF3-Hoechst. Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ****P < 0.0001; G, Cumulative distribution of Voronoi polygon density in control (blue) (n = 44) and TSA-treated (orange) (n = 39) living hFib cells labeled with TMR-2CF3-Hoechst. Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ***P = 0.0002; H, Cumulative distribution of Voronoi polygon density in fixed hFib cells labeled with TMR-2CF3-Hoechst in control (blue) (n = 31) and TSA-treated (orange) (n = 36). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ***P = 0.0001;

[0059] The interquartile range (25th–75th percentile) is shown in light shades, and the median is shown in thick, dark lines. Asterisks indicate statistically significant separation between medians based on an unpaired, two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

[0060] Figure 12Figure 3 STORM imaging of DNA direct labeling with the scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst in live and fixed HeLa cells with and without hyperacetylation (TSA treatment): A, representative STORM density rendering images of DNA distribution in live HeLa cells labeled with TMR-CF3-Hoechst (left: intact nuclei; right: magnified area in red boxes); B, representative STORM density rendering images of DNA distribution in live HeLa cells labeled with TMR-CF3-Hoechst (top) and TSA treatment (bottom). Representative STORM density rendering images of DNA distribution in HeLa cells (left: intact nuclei; right: magnified area in red boxes); C, Representative STORM density rendering images of DNA distribution in live HeLa cells labeled with TMR-2CF3-Hoechst (top) and TSA-treated (bottom) (left: intact nuclei; right: magnified area in red boxes); D, Representative STORM density rendering images of DNA distribution in fixed HeLa cells labeled with TMR-2CF3-Hoechst (top) and TSA-treated (bottom) (left: intact nuclei ; Right: red box magnified area); E, Cumulative distribution of Voronoi polygon density in live HeLa cells labeled with TMR-CF3-Hoechst (blue) (n=37) and TSA-treated (orange) (n=33), median and standard error mean (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; F, Cumulative distribution of Voronoi polygon density in fixed HeLa cells labeled with TMR-CF3-Hoechst (blue) (n=50) and TSA-treated (orange) (n=45) , median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, **P = 0.0033; G, Cumulative distribution of Voronoi polygon density in live HeLa cells labeled with TMR-2CF3-Hoechst (control group (blue)) (n = 62) and TSA-treated (orange) (n = 64), ****P < 0.0001; H, Cumulative distribution of Voronoi polygon density in fixed HeLa cells labeled with TMR-2CF3-Hoechst (control group (blue)) (n = 27) and TSA-treated (orange) (n = 40). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ***P = 0.0006;

[0061] The interquartile range (25th–75th percentile) is shown in light shades, and the median is shown in thick, dark lines. Asterisks indicate statistically significant separation between medians based on an unpaired, two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

[0062] Figure 13 The scintillation dye TMR-2CF3-Hoechst was used for direct DNA labeling STORM imaging of living human fibroblasts (hFib) and human fibroblast-induced pluripotent stem cells (hFib-iPSCs): A. Quantitative polymerase chain reaction (QPCR) was used to detect NANOG, OCT4, and SOX2 in hFib and hFib-iPSCs. mRNA expression; B. Expression of NANOG, OCT4, and SOX2 in hFib and hFib-iPSCs detected by immunofluorescence (IF); C. Representative STORM density-rendered images of DNA distribution in live hFib (top) and live hFib-iPSCs (bottom) labeled with TMR-2CF3-Hoechst (left: intact cell nuclei; right: magnified area in red boxes); D. Cumulative distribution of Voronoi polygon density in live hFib (blue) (n=24) and live hFib-iPSCs (orange) (n=58) labeled with TMR-2CF3-Hoechst, median, and standard error mean (SEM); unpaired two-tailed Student's t-test , ***P=0.0008; the light colors in the figure show the interquartile range (25th-75th percentile), and the thick dark line shows the median value; the asterisk indicates that the separation between the medians is statistically significant according to the unpaired two-tailed Student's t-test, and the super-resolution Voronoi mosaic image shows the difference in DNA density; E, the normalized confusion matrix (positive and negative class prediction images are marked in brackets) shows the performance of the model, and the accuracy of each class is shown on the main diagonal; F, the receiver operating characteristic (ROC) curve shows the performance of the model at all classification thresholds and the AUC value; G, the loss curve shows the change of the model loss value over time; H, the accuracy curve shows the ability of the model to correctly predict the training data during the training process.

[0063] Figure 14 The scintillation dye TMR-CF3-Hoechst is used to directly label DNA in living human fibroblasts (hFib) and human fibroblast-induced pluripotent stem cells (hFib-iPSCs): A, Representative STORM density-rendered images of DNA distribution in living hFib (top) and living hFib-iPSCs (bottom) using TMR-CF3-Hoechst labeling (left: intact cell nuclei; right: magnified area in red boxes);

[0064] B, Cumulative distribution of Voronoi polygon density in live hFib (blue) (n=30) and live hFib-iPSCs (orange) (n=98) labeled with TMR-CF3-Hoechst, median and standard error mean (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; light colors indicate the interquartile range (25th-75th percentile), and the thick dark line indicates the median; asterisks indicate statistically significant separation between medians according to an unpaired two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

[0065] Figure 15To perform whole-chromatin DNA imaging using TMR-CF3-Hoechst, OligoSTORM imaging was combined with the technique to observe the location of the OCT4 locus in chromatin structure: A. OligoSTORM images of the OCT4-TCF19 locus (20 kb) in hFib and hFib-iPSCs. Super-resolution images using conventional fluorescence imaging, Gaussian function rendering (red), and heat map density rendering; B. Cumulative distribution of Voronoi polygon density of the OligoSTORM OCT4-TCF19 locus (20 kb) in hFib (n = 45 loci) and hFib-iPSCs (n = 109 loci). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, *P=0.0257; C, Quantification of gyration radius length of OCT4-TCF19 OligoSTORM loci (20 kb) in hFib and hFib-iPSCs (n=45 and 109 loci, respectively). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; D, Conventional OligoSTORM image of the OCT4-TCF19 locus (20 kb), heat map density rendering of super-resolution imaging of DNA by TMR-CF3-Hoechst (color scale), and magnified images of the fine structure of DNA in the OCT4-TCF19 locus and its adjacent regions in hFib and hFib-iPSCs (red box); E, Cumulative distribution of Voronoi polygon density of DNA containing the OCT4-TCF19 locus and its adjacent regions in hFib cells (n=22 loci) and hFib-iPSCs (n=16 loci). Median and standard error of the mean (SEM); unpaired two-tailed Student's t-test, *P = 0.0482; Light shades indicate the interquartile range (25th-75th percentile), and the thick dark line indicates the median; asterisks indicate statistically significant separation between medians based on an unpaired two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

[0066] Figure 16DNA super-resolution imaging of GFAP-GFP transgenic zebrafish retinal slices using TMR-2CF3-Hoechst: A, conventional fluorescence image and super-resolution (SR) image of DNA in GFAP-GFP transgenic zebrafish retinal slices. From left to right, conventional fluorescence image of DNA, SR image, conventional fluorescence image of GFAP (labeling MGCs), SR image of DNA, and image of DNA and GFAPmerge (ONL-outer nuclear layer; OPL-outer plexiform layer; INL-inner nuclear layer; IPL-inner plexiform layer); B, Representative nuclear DNA of GFAP-labeled MGCs (yellow box) labeled by TMR-2CF3-Hoechst. SR images, other retinal neurons (red boxes) and super-resolution images magnified using super-resolution Voronoi mosaic images (green boxes), super-resolution Voronoi mosaic images show differences in DNA density; C, Cumulative distribution of Voronoi polygon density of MGCs (blue) and other retinal cells (orange) using TMR-2CF3-Hoechst (n = 6 for MGCs and n = 26 for other retinal cells), light colors show the interquartile range (25th-75th percentiles), and thick dark lines show the median values; asterisks indicate that the separation between medians is statistically significant according to the unpaired Student's t-test; D, Probability distribution of localization accuracy using TMR-2CF3-Hoechst in GFAP-GFP transgenic zebrafish retinal sections (n ​​= 22).

[0067] Figure 17 DNA super-resolution imaging of NMDA-damaged zebrafish retinal sections using TMR-CF3-Hoechst: A, from left to right, conventional fluorescence image of DNA, super-resolution (SR) image, conventional image of PCNA (labeling proliferating MGCs), and merged image of DNA SR image and PCNA (ONL - outer nuclear layer; OPL - outer plexiform layer; INL - inner nuclear layer; IPL - inner plexiform layer);

[0068] B, Representative nuclear DNA SR images of retinal photoreceptors (red boxes) and proliferating MGCs (yellow boxes) labeled with TMR-CF3-Hoechst, and magnified images using super-resolution Voronoi mosaic (green boxes); super-resolution Voronoi mosaic images show differences in DNA density); C, Probability distribution of localization accuracy using TMR-CF3-Hoechst in zebrafish retinal cryosections (n ​​= 19).

[0069] Figure 18Figure 3. Direct DNA labeling of human normal and colon cancer tissues and living normal colon cells and cancer cells using the scintillation dye TMR-CF3-Hoechst. A. Hematoxylin-eosin (H&E)-stained histological images of human normal and colon cancer tissues, STORM density-rendered images of DNA distribution imaged by TMR-CF3-Hoechst, representative STORM density-rendered images of DNA distribution in normal colon cells and colon cancer cells (green boxes), and magnified images using super-resolution Voronoi mosaic (red boxes). Super-resolution Voronoi mosaic images show differences in DNA density. B. Cumulative distribution of Voronoi polygon density by TMR-CF3-Hoechst in normal colon tissue (blue) (n>100) and colon cancer tissue (orange) (n>100). Light colors show the interquartile range (25th-75th percentiles), and thick dark lines show the median values. Asterisks indicate statistically significant separation between medians according to an unpaired Student's t-test. C. Probability distribution of localization accuracy using TMR-CF3-Hoechst labeling in human colon tissue (n=37); D, STORM density rendering image of DNA distribution in live human normal colon epithelial cells (HCoEpiC, top) and live human colon cancer cells (HCT116, bottom) labeled with TMR-CF3-Hoechst, showing differences in DNA density (left: intact cell nuclei; right: magnified area in red boxes); E, Cumulative distribution of Voronoi polygon density labeled with TMR-CF3-Hoechst in live human normal colon epithelial cells (HCoEpiC, blue) (n=48) and live human colon cancer cells (HCT116, orange) (n=46), P<0.0001, light colors show the interquartile range (25th-75th percentiles), and thick dark lines show the median values; asterisks indicate that the separation between the medians is statistically significant according to the unpaired Student's t-test, and the super-resolution Voronoi mosaic image shows differences in DNA density. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0071] Example 1

[0072] In this example, rhodamine fluorescent dyes modified with different groups are connected to the Hoechst structure through a short connecting chain to form a DNA probe. The reaction process is as follows:

[0073]

[0074] Referring to the above reaction process, the compound represented by formula V is first prepared. The specific preparation process is as follows:

[0075] 1) Preparation of Hoechst dye free base: Commercially available Hoechst dye hydrochloride (100 mg) was dissolved in water, and then a solution of potassium carbonate (123 mg) was added. The precipitate was separated by centrifugation, washed with water, and freeze-dried to obtain the Hoechst dye free base.

[0076] 2) Potassium carbonate (98.4 mg) was dissolved in anhydrous DMF, and the free base of the Hoechst dye (80 mg) was added to a round-bottom flask and stirred for several minutes. Tert-butyl (2-bromoethyl) carbamate (84.7 mg) was then added and the mixture was allowed to react at 50°C for 24 hours. After completion of the reaction, the solvent was removed using a vacuum oil pump, and the residue was purified by silica gel column chromatography to yield 45 mg of a yellow solid (compound represented by Formula V) in a 41.7% yield. The NMR characterization of the compound represented by Formula V was as follows: 1 HNMR (400 MHz, MeOD) δ8.23 (s, 1H), 8.03 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 8.6 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.16–7.02 (m, 4H), 4.07 (t, J = 5.4 Hz, 2H), 3.46 (t, J = 5.5 Hz, 2H), 3.27 (s, 4H), 2.85 (s, 4H), 2.51 (s, 3H), 1.46 (s, 9H); The mass spectrum of the compound represented by Formula V is characterized by: m / z [M+H] + calcd.for C 32 H 38 N7O3 + :568.3,found:568.4.

[0077] Next, the compound represented by formula VI was prepared. The specific preparation process is as follows:

[0078] Dissolve 45 mg of the compound of Formula V in 5 mL of a solvent (trifluoroacetic acid / dichloromethane = 1 / 4) in a round-bottom flask. After reacting for 1 hour, remove the solvent using a vacuum pump to obtain a colorless solid (the compound of Formula VI). This product was used directly in the subsequent DNA probe preparation reaction without further purification.

[0079] Then prepare the compound represented by formula VII, and the specific preparation process is as follows:

[0080] 6-Carboxytetramethylrhodamine (430.0 mg) was dissolved in anhydrous DMF (3 mL), followed by the addition of potassium carbonate (414.0 mg) and triethylamine (Et3N, 417.0 μL). The mixture was cooled in an ice bath, and allyl bromide (362.9 mg) was slowly added. The mixture was then warmed to room temperature and stirred for 24 hours. After the reaction, the solvent was removed using a vacuum oil pump, and the residue was purified by silica gel column chromatography to obtain 450.0 mg of a pink solid (compound represented by Formula VII) in a 95.5% yield. The compound represented by Formula VII was characterized by nuclear magnetic resonance (NMR) as follows: 1 H NMR (400 MHz, CDCl3) δ8.26 (dd, J = 8.0, 1.4 Hz, 1H), 8.08 (dd, J = 8.0, 0.6 Hz, 1H), 7.82 (s, 1H), 6.62 (d, J = 8.9 Hz, 2H), 6.50 (d, J = 2.5 Hz, 2H), 6.42 (dd, J = 8.9, 2.6 Hz, 2H), 6.02–5.90 (m, 1H), 5.34 (d, J = 17.2 Hz, 1H), 5.25 (d, J = 10.4 Hz, 1H), 4.76 (d, J = 5.9 Hz, 2H), 2.99 (s, 12H); The mass spectrum of the compound represented by Formula VII is characterized by: m / z calcd. for C 28 H 27 N2O5 + :471.1,found:471.3.

[0081] Then, the compound represented by formula VII is used to prepare the compound represented by formula VIII. The specific preparation process is as follows:

[0082] Under a nitrogen atmosphere, 4-dimethylaminopyridine (DMAP, 5.2 mg) and amines of various chemical structures (H2N-R) (CH2CH2CH2CH3NH2 = 31.0 mg; CF3CH2NH2 = 42.0 mg; (CF3)2CHNH2 = 70.9 mg; SO2N(CH3)2NH2 = 52.7 mg) were added to a solution of the compound represented by Formula VII (100.0 mg) in ultra-dry dichloromethane, where R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2, and SO2N(CH3)2. After stirring for 5 minutes, triethylamine (Et3N, 71.1 μL) was added to the mixture. Subsequently, a solution of phosphorus oxychloride (POCl3, 19.8 μL) in ultra-dry dichloromethane was added to the reaction system, and the reaction was allowed to proceed overnight at room temperature. After the reaction is completed, the mixture is added dropwise to ice water, extracted with ethyl acetate (EA, 50 mL×3), washed with saturated sodium chloride solution, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography to obtain the compound represented by Formula VIII. In this embodiment, the compound represented by Formula VIII includes the following four forms:

[0083] VIII-I: Allyl 2-butyl-3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylate. The compound represented by formula VIII-I was obtained as a pink solid in a yield of 40.4%. The NMR characterization of the compound represented by formula VIII-I was as follows: 1 HNMR(400MHz, CDCl3)δ8.15(d,J=7.8Hz,1H),7.96(d,J=7.9Hz,1H),7.69(s,1H),6.46–6.44(m,4H),6.33(d,J=8.9Hz,2H),6.00–5.90(m,1H),5.3 3(d,J=16.9Hz,1H),5.24(d,J=10.5Hz,1H),4.72(d,J=5.9Hz,2H),3.10( t,J=6.9Hz,2H),2.97(s,12H),1.16-1.03(m,4H),0.68(t,J=6.5Hz,3H). 13 C NMR (101MHz, CDCl3) δ166.9,165.6,153.5,152.9,151.4,135.4,133.7,131.9,129.6 ,128.6,125.1,122.8,118.8,108.7,106.0,98.8,66.0,65.0,40.3,30.1,20.3,13.5.

[0084] VIII-II: Allyl 3',6'-bis(dimethylamino)-3-oxo-2-(2,2,2-trifluoroethyl)spiro[isoindoline-1,9'-rhodamine]-6-carboxylate. The compound represented by formula VIII-II was obtained as a pink solid in a yield of 29.7%. The NMR characterization of the compound represented by formula VIII-II was as follows: 1 H NMR (400MHz, CDCl3) δ8.18(d,J=7.9Hz,1H),8.02(d,J=7.9Hz,1H),7.73(s,1H),6.48–6.40(m,4H),6.36(dd,J=8.8,1.8Hz,2H) ,6.01–5.89(m,1H),5.33(d,J=17.2Hz,1H),5.25(d,J=10.4Hz,1H),4.73(d,J=5.8Hz,2H),3.72(q,J=9.2Hz,2H),2.97(s,12H). 13CNMR (101 MHz, CDCl3) δ 168.3, 165.4, 154.0, 153.1, 151.6, 134.9, 133.3, 132.0, 130.0, 128.8, 125.7, 123.5, 123.3, 119.1, 109.7, 109.2, 100.3, 98.9, 66.3, 66.0, 40.4; The mass spectrum of the compound represented by formula VIII-II is characterized by: m / z [M+H] + calcd.for C 30 H 29 F3N3O4 + :552.2,found:552.3.

[0085] VIII-III: Allyl 3',6'-bis(dimethylamino)-2-(1,1,1,3,3,3-hexafluoropropyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylate (8-3). The compound represented by formula VIII-III was obtained as a pink solid in a yield of 35.0%. The NMR characterization of the compound represented by formula VIII-III was as follows: 1 H NMR (400MHz, CDCl3) δ8.23(d,J=7.9Hz,1H),8.03(d,J=8.0Hz,1H),7.78(s,1H),6.47–6.43(m,4H),6.36(d,J=11.3Hz,2H),6.02– 5.91(m,1H),5.34(d,J=17.2Hz,1H),5.26(d,J=10.4Hz,1H),4.75(d,J=5.9Hz,2H),3.94–3.86(quintJ=7.6Hz,1H),2.98(s,12H). 13 C NMR (101MHz, CDCl3) δ166.2,165.2,153.4,152.3,151.8,135.2,133.8,131. 8,130.2,129.7,125.9,123.7,119.1,109.1,103.9,98.3,68.2,66.3,40.2.

[0086] VIII-IV: Allyl 3',6'-bis(dimethylamino)-2-(N,N-dimethylsulfonyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylate. The compound represented by Formula VIII-IV was obtained as a pink solid in a yield of 53.2%. The NMR characterization of the compound represented by Formula VIII-IV was as follows: 1H NMR (400MHz, CDCl3) δ8.20(d,J=7.9Hz,1H),7.99(d,J=8.0Hz,1H),7.72(s,1H),6.59–6.46(m,4H),6.35(d,J=8.8Hz,2H ),6.03–5.89(m,1H),5.33(d,J=17.2Hz,1H),5.25(d,J=10.4Hz,1H),4.73(d,J=5.9Hz,2H),2.97(s,12H),2.72(s,6H). 13 CNMR (101MHz, CDCl3) δ166.3,165.0,153.2,135.9,131.7,130.0,129.0,128.5,126.2,123.7,119.2,108.2,98.9,66.3,40.3,37.9.

[0087] The compound represented by formula VIII is then used to prepare the compound represented by formula IX. The specific preparation process is as follows:

[0088] The compound represented by formula VIII (40.0 mg) was dissolved in 3 mL of anhydrous tetrahydrofuran, followed by the addition of 1,3-dimethylbarbituric acid (VIII-I: 22.7 mg; VIII-II: 23.3 mg; VIII-III: 20.2 mg; VIII-IV: 22.8 mg) and tetrakis(triphenylphosphine)palladium (VIII-I: 16.8 mg; VIII-II: 17.2 mg; VIII-III: 14.9 mg; VIII-IV: 17.0 mg). The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the compound represented by formula IX. In this embodiment, the compound represented by formula IX includes the following four forms:

[0089] IX-I: 2-Butyl-3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylic acid. The compound of Formula IX-I was obtained as a pink solid in a yield of 67.7%. The NMR characterization of the compound of Formula IX-I was as follows: 1 HNMR(400MHz, CDCl3)δ8.12(d,J=8.4Hz,1H),7.97(d,J=7.9Hz,1H),7.70(s,1H),6.47–6.41(m,4H) ,6.32(d,J=8.9,2H),3.09(t,J=6.9Hz,2H),2.96(s,12H),1.13–1.04(m,4H),0.67(t,J=6.9Hz,3H). 13C NMR (101 MHz, CDCl3) δ 166.9, 153.6, 152.9, 151.4, 130.1, 128.6, 125.6, 122.9, 112.8, 108.8, 105.9, 98.8, 65.2, 40.4, 40.3, 30.1, 20.3, 13.5; The mass spectrum of the compound represented by formula IX-I is characterized by: m / z [M+H] + calcd.for C 29 H 32 N3O4 + :486.2,found:486.3.

[0090] IX-II: 3',6'-bis(dimethylamino)-3-oxo-2-(2,2,2-trifluoroethyl)spiro[isoindoline-1,9'-rhodamine]-6-carboxylic acid. The compound represented by Formula IX-II was obtained as a pink solid with a yield of 78.2%. The NMR characterization of the compound represented by Formula IX-II was as follows: 1 HNMR(400MHz,DMSO)δ8.09(dd,J=7.9,1.2Hz,1H),8.00(d,J=7.9Hz,1H),7.43(s, 1H),6.44(d,J=6.9,4H),6.39–6.34(m,2H),3.75(q,J=10.0Hz,2H),2.92(s,12H). 13 C NMR (101 MHz, DMSO) δ 167.4, 166.3, 154.0, 152.4, 151.4, 135.6, 132.0, 129.8, 128.9, 128.3, 124.5, 123.5, 109.2, 104.1, 98.2, 65.0, 39.8; The mass spectrum of the compound represented by formula IX-II is characterized by: m / z [M+H] + calcd.for C 27 H 25 F3N3O4 + :512.2,found:512.2.

[0091] IX-III: 3',6'-bis(dimethylamino)-2-(1,1,1,3,3,3-hexafluoropropyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylic acid. The compound represented by Formula IX-III was obtained as a pink solid in a yield of 56.1%. The NMR characterization of the compound represented by Formula IX-III was as follows: 1H NMR (400MHz, DMSO) δ8.14(d,J=7.8Hz,1H),8.00(d,J=7.4Hz,1H),7.46(s,1 H),6.58–6.42(m,4H),6.42–6.31(m,2H),4.53–4.38(m,1H),2.92(s,12H). 13 C NMR (101 MHz, MeOD) δ 168.4, 159.0, 155.0, 153.7, 132.4, 130.5, 126.8, 124.0, 115.5, 110.4, 105.3, 99.5, 97.4, 70.0, 40.4; The mass spectrum of the compound represented by formula IX-III is characterized by: m / z [M+H] + calcd.for C 28 H 24 F6N3O4 + :580.2,found:580.3.

[0092] IX-IV: 3',6'-bis(dimethylamino)-2-(N,N-dimethylsulfonyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxylic acid. The compound of Formula IX-IV was obtained as a pink solid in a yield of 94.0%. The NMR characterization of the compound of Formula IX-IV was as follows: 1 H NMR (400 MHz, MeOD) δ8.19 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.60 (s, 1H), 6.56 (d, J = 8.8 Hz, 2H), 6.51–6.41 (m, 4H), 2.96 (s, 12H), 2.67 (s, 6H); The mass spectrum of the compound represented by Formula IX-IV is characterized by: m / z [M+H] + calcd.for C 27 H 29 N4O6S + :537.2,found:537.3.

[0093] Finally, a DNA probe was prepared using the compound represented by Formula IX. The specific preparation process is as follows:

[0094] The compound represented by formula IX (20.0 mg), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, IX-Ⅰ: 31.3 mg; IX-II: 29.7 mg; IX-III: 26.3 mg; IX-IV: 28.3 mg) and N,N-diisopropylethylamine (DIEA, IX-Ⅰ: 13.6 μL; IX-II: 12.9 μL; IX-III: 12.0 μL; IX-IV: 12.3 μL) were dissolved in 4 mL of DMF and stirred under a nitrogen atmosphere for 10 minutes; then, a solution of compound VI (IX-Ⅰ: VI = 28.9 mg; IX-II: VI = 27.4 mg; IX-III: VI = 24.2 mg; IX-IV: VI = 26.1 mg) in 0.5 mL of DMF was added dropwise to the reaction mixture. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a DNA probe. In this embodiment, the DNA probe includes the following four forms:

[0095] Probe I: 2-butyl-3',6'-bis(dimethylamino)-N-(2-(4-(5-(4-methylpiperazin-1-yl)-1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxamide. Probe I was obtained as a pink solid in a 10.4% yield; the NMR characterization of Probe I was as follows: 1 HNMR(400MHz,MeOD)δ8.32(s,1H),8.03–7.99(m,4H),7.94(d,J=7.9Hz,1H),7.85(d,J=8.0Hz,1H), 7.67(d,J=8.7Hz,1H),7.55(s,1H),7.34(d,J=8.6Hz,1H),7.28(s,1H),7.06(d,J=8.3Hz,2H),6.50 (s,2H),6.40(m,4H),4.15(t,J=5.3Hz,2H),4.02–3.89(m,2H),3.75–3.63(m,4H),3.28–3.10(m,4H ),3.07–3.03(t,J=6.4Hz,2H),3.01(s,3H),2.93(s,12H),1.10–1.05(m,4H),0.66(t,J=6.1Hz,3H). 13C NMR (101 MHz, MeOD) δ 169.1, 168.9, 162.1, 155.3, 154.5, 153.3, 148.5, 139.7, 135.0, 129.5, 129.4, 129.0, 125.3, 124.0, 123.7, 123.1, 116.1, 110.2, 106.7, 102.6, 99.9, 67.3, 67.1, 55.4, 50.4, 49.7, 49.5, 40.4, 31.3, 21.3, 13.8, 13.2; The mass spectrum of probe I is characterized by: m / z [M+H] + calcd.for C 56 H 59 N 10 O4 + :935.5, found:935.7; probe I was denoted as TMR-C4-Hoechst.

[0096] Probe II: 3',6'-bis(dimethylamino)-N-(2-(4-(5-(4-methylpiperazin-1-yl)-1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxo-2-(2,2,2-trifluoroethyl)spiro[isoindoline-1,9'-rhodamine]-6-carboxamide. Probe II was obtained as a pink solid with a yield of 26.6%. The nuclear magnetic resonance characterization of probe II was as follows: 1 HNMR (400MHz, DMSO) δ8.91(t,J=5.4Hz,1H),8.28(d,J=49.2Hz,1H),8.12–8.08(m,3H),8.05–7. 94(m,2H),7.65(dd,J=46.5,8.3Hz,1H),7.54(s,1H),7.45(s,1H),7.11(d,J=8.6Hz,2H),6.94( d,J=8.4Hz,1H),6.45–6.43(m,4H),6.36(d,J=9.5Hz,2H),4.15(t,J=5.7Hz,2H),3.73(q,J=9.6 Hz,2H),3.60(d,J=5.5Hz,2H),3.22–3.13(m,4H),2.92(s,12H),2.75–2.60(m,4H),2.36(s,3H). 13C NMR (101 MHz, DMSO) δ167.4, 165.2, 159.9, 153.8, 152.4, 151.3, 138.8, 130.9, 128.3, 128.2, 128.1, 123.1, 122.6, 122.5, 114.9, 109.2, 104.5, 98.2, 66.0, 65.0, 62.8, 54.5, 31.3, 29.0, 22.1, 14.0; The mass spectrum of probe II is characterized by: m / z [M+H] + calcd.for C 54 H 52 F3N 10 O4 + :961.4,found:961.4; probe II was denoted as TMR-CF3-Hoechst.

[0097] Probe III: 3',6'-bis(dimethylamino)-2-(1,1,1,3,3,3-hexafluoropropyl)-N-(2-(4-(5-(4-methylpiperazine-1-

[0098] (1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxamide. Probe III was obtained as a pink solid with a yield of 19.9%. The nuclear magnetic resonance characterization of probe II was as follows: 1 HNMR(400MHz,MeOD)δ8.18(s,1H),8.12(d,J=9.4Hz,1H),8.01(d,J=8.0Hz,1H),7.94–7.88(m,3H),7 .70(s,1H),7.64(d,J=8.5Hz,1H),7.49(d,J=8.8Hz,1H),7.13(d,J=2.0Hz,1H),7.03(dd,J=8.9,2.2H z,1H),6.96(d,J=8.9Hz,2H),6.45(d,J=1.0Hz,2H),6.43–6.36(m,4H),4.11(t,J=5.5Hz,2H),4.07– 3.99(m,1H),3.69(t,J=5.5Hz,2H),3.30–3.22(m,4H),2.92(s,12H),2.91–2.85(m,4H),2.54(s,3H). 13C NMR (101 MHz, MeOD) δ 168.7, 168.1, 162.1, 155.0, 153.6, 130.5, 129.5, 124.5, 123.1, 116.0, 112.5, 112.3, 110.7, 110.3, 105.0, 101.4, 99.4, 70.1, 55.9, 51.3, 40.3, 33.1, 30.8, 23.7, 14.4; The mass spectrum of probe III is characterized by: m / z [M+H] + calcd.for C 55 H 51 F6N 10 O4 + :1029.4,found:1029.4; probe III was denoted as TMR-2CF3-Hoechst.

[0099] Probe IV: 3',6'-bis(dimethylamino)-2-(N,N-dimethylsulfonyl)-N-(2-(4-(5-(4-methylpiperazine-1-

[0100] (1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxospiro[isoindoline-1,9'-rhodamine]-6-carboxamide. Probe IV was obtained as a pink solid with a yield of 40.8%. The nuclear magnetic resonance characterization of probe IV was as follows: 1 HNMR(400MHz,DMSO)δ8.95(s,1H),8.46(s,1H),8.16(d,J=8.6Hz,2H),8.10(dd,J=14.7,8.4Hz,2H),8 .01(d,J=8.0Hz,1H),7.90(d,J=8.5Hz,1H),7.72(d,J=9.0Hz,1H),7.49(s,1H),7.33(d,J=8.0Hz,1H) ,7.25(s,1H),7.16(d,J=8.7Hz,2H),6.53(d,J=8.3Hz,2H),6.47–6.34(m,4H),4.17(t,J=5.3Hz,2H), 3.92(d,J=11.0Hz,2H),3.71–3.42(m,4H),3.35–2.96(m,4H),2.92(s,12H),2.62(s,6H),2.50(s,3H). 13C NMR (101 MHz, DMSO) δ 165.7, 164.9, 160.7, 153.8, 153.6, 152.5, 151.1, 149.2, 148.4, 140.6, 139.8, 133.5, 129.6, 128.9, 128.6, 128.3, 123.7, 122.1, 120.3, 117.7, 116.8, 115.1, 114.4, 108.2, 106.3, 99.2, 98.1, 68.2, 66.0, 52.2, 46.4, 42.0, 37.4; The mass spectrum of probe IV is characterized by: m / z [M+H] + calcd.forC 54 H 56 N 11 O6S + :986.4,found:986.5; probe IV is denoted as TMR-SO2-Hoechst.

[0101] In order to characterize the prepared probes I to IV, the performance of the probes was evaluated by measuring the D50 value, ring opening and closing ability, pKa value, etc., as follows:

[0102] First, the D50 values ​​of probes I and IV were tested by plotting the relationship between the probe's absorption peak and the solvent's dielectric constant (see Figure 1 Compared with the parent 6-TAMRA (6-Carboxytetramethylrhodamine) dye (D50=12), all probes I to IV showed larger D50 values ​​(>40), indicating that they have a strong tendency to form ring-closed spirolactams.

[0103] Then, this example uses sodium dodecyl sulfate (SDS) as an inducing agent to examine the ring opening ability, as shown in FIG. Figure 1 B and Figure 2 As shown, all probes showed fluorescence enhancement in response to SDS, with the intensity difference being approximately three orders of magnitude. This indicates that the modification of rhodamine spirolactam dyes covers a wide range of ring opening capabilities, providing sufficient analogs to study structure-activity relationships. In addition, incubation of probes I-IV with BSA resulted in minimal fluorescence changes, indicating that the probes can effectively avoid interference from nonspecific interactions. This example then explored the pKa values ​​of the probe fluorescence (see Figure 1 C and Figure 3 All probes showed pKa values ​​below 4.5, further demonstrating their low fluorescence background under physiological conditions. Interestingly, probes I–III showed the ability to self-assemble into nanoaggregates, which provided higher fluorescence enhancement and better cell permeability (see Figure 1 D and Figure 4 ).

[0104] This example then studies the binding properties of probes I-IV to hairpin DNA (hpDNA) (see Figure 1 E in Figure 1). After incubation of hpDNA (30 μM) with the probes (2.5 μM), fluorescence enhancements ranging from 1.5 to 157.2 times were observed in this example. As expected, the fluorescence intensity of the probes after binding to hpDNA showed a three-order-of-magnitude difference, consistent with the results obtained with the addition of SDS. This example further utilized Probes I-IV to image DNA in fixed cells. All probes showed high specificity for DNA (see Figure 1 (F) The increase in fluorescence intensity follows the same trend as observed in hpDNA imaging. These results indicate that the loop-opening abilities of Probes I–IV are fine-tuned to label DNA over a wide range.

[0105] To gain a deeper understanding of the relationship between the ring-opening ability of the probes and their suitability for SMLM imaging, single-molecule photophysical studies were performed in this example. Only probes II and III exhibited good self-blinking properties in living cells at 50 nM without any additives (see Figure 1 G and H in Figure 3), indicating that spirolactam probes with moderate ring opening ability are most suitable for SMLM imaging. Both probes II and III exhibited extremely high photostability, as verified by molecular survival fraction analysis (see Figure 1 I, Probe II is 95% after 10,000 frames, and Probe III is 86%). The on-times of Probe II and Probe III are calculated to be 92 ms and 110 ms, respectively, which are suitable for SMLM (see Figure 1 More importantly, Probe II and Probe III showed high average photon numbers per switching event, 2200 and 2800, respectively (see Figure 1 It is worth noting that although the photostability of probe II is better than that of probe III, under the same imaging conditions, probe III has a higher temporal resolution (1.5 seconds) (see Figure 1 This is primarily due to the increased "ON" score (higher ring opening ability) of Probe III. These results confirm that Probes II and III are self-assembling, self-scintillation probes with high photostability and high temporal resolution, suitable for long-term and real-time chromatin SMLM applications. Therefore, Probes II and III will be used in subsequent studies.

[0106] Example 2: SMLM imaging of DNA probes

[0107] STORM super-resolution microscopy was used to image live and fixed cells (hFib and HeLa cells). The synthesized probes were able to effectively cross the living cell membrane and bind to DNA. Analysis of DNA density by Voronoi mosaic showed that the synthesized probes II (TMR-CF3-Hoechst) and III (TMR-2CF3-Hoechst) could specifically label compact heterochromatin regions (nuclear edge and nucleolar edge) and euchromatin regions with low DNA density (nucleoplasm) (see Figure 5 A, B and Figure 6 The other two synthetic probes I (TMR-C4-Hoechst) (see Figure 7 A, C) and probe IV (TMR-SO2-Hoechst) (see Figure 7 The super-resolution imaging effect of (B, D) in Figure 1 is poor. This example then evaluates the localization accuracy of TMR-CF3-Hoechst and TMR-2CF3-Hoechst probes. Localization accuracy directly affects the spatial resolution achievable in SMLM. The histogram shows that the probability distribution of the localization accuracy of the synthesized probe is about 20nm (see Figure 5 C, D and Figure 6 C, D in the above figure).

[0108] Furthermore, by using the synthetic DNA probe TMR-CF3-Hoechst, high-resolution 3D super-resolution imaging of DNA in living hFib cells and living hFib cells treated with histone hyperacetylation (Trichostatin A (TSA) histone deacetylase inhibitor) can be obtained. Since biological structures are inherently three-dimensional, SMLM imaging performed in two dimensions may be limited. Being able to resolve 3D biological structures will provide more informative and comprehensive insights. Therefore, in this example, after labeling with TMR-CF3-Hoechst, the native 3D chromatin structure of living hFib cells was visualized, including the hyperacetylation of histone tails induced by TSA treatment (see Figure 9 ) and untreated cases (see Figure 8 ). Traditional brightfield or confocal fluorescence microscopy often has difficulty resolving histone hyperacetylation, especially in three-dimensional imaging. Figure 9 A in (with histone hyperacetylation) and Figure 8 As shown in A (no histone hyperacetylation), this example clearly shows the image in different Z axes of living hFib cells, and this example also renders the DNA 3D density map of the STORM image of TMR-CF3-Hoechst labeled living hFib cells ( Figure 9 B and Figure 8B). The 3D density map of DNA structure in living hFib cells was further highlighted and visualized by rotationally cropping the nuclear rim (enlarged area in red box), the nucleoplasm (enlarged area in green box), and the nucleolar periphery (enlarged area in blue box). Figure 8 C) and hyperacetylated live hFib cells ( Figure 9 C) 3D structure of DNA. In addition, from the edge of the nucleus (the yellow box enlarges the area, Figure 8 The D in the equation is active hFib. Figure 9 A representative rotation sequence (0°, 45°, 90°, 135°, 180°) of a cropped 3D STORM image of a hyperacetylated living hFib cell (D in FIG) shows that this example captures the 3D ultrastructure of DNA with and without hyperacetylated histone tails in living cells at an unprecedented resolution.

[0109] At low laser power (power density ~0.33kW / cm2), TMR-CF3-Hoechst and TMR-2CF3-Hoechst can be used for time-lapse SMLM super-resolution imaging in living HeLa cells to track the movement of DNA fibers in the nucleus (see Figure 10 A, B, C in Figure 3). We also performed single particle tracking using the Trackpy Python package, which is a tool specifically designed for single particle tracking applications. Each frame of the video is analyzed to detect "blob-like" features, and the positions of these features are accurate to the sub-pixel level. The features identified in each frame are linked to construct the trajectory of the particle. Assuming that the particles perform Brownian motion, that is, the speed of the particles between adjacent frames is uncorrelated, the best prediction is that the position of the particle in the next frame is the same as its current position. The continuity of the particles is determined and the trajectory is established by searching for particles within a defined range (1.5 pixels in this study) in subsequent frames (see Figure 10 C, D in Figure 3). We calculated the mean square displacement (MSD) of the particles, which quantifies the average distance the particles move in a specific time. In two-dimensional diffusion, the MSD should theoretically increase linearly with time, but the experimentally observed MSD is lower than the theoretical line, indicating that the diffusion behavior of the particles is sub-diffusive. The MSD at the cell boundary is slightly higher than that inside the cell, indicating that the particles at the cell boundary have higher mobility (see Figure 10 E and F in the figure). Meanwhile, the violin plots of the DNA molecule velocity distribution analysis show the average velocity distribution of DNA molecules inside and at the cell boundary. It was found that the concentration of DNA molecules with higher average velocity at the cell boundary was higher than that inside the cell, further indicating that the mobility of the cell periphery is increased (see Figure 10 Single-particle tracking technology can provide a deeper understanding of the dynamic behavior of DNA molecules within cells, providing important data for studying biophysical processes within cells.

[0110] In addition, this example used the synthetic DNA probes TMR-CF3-Hoechst and TMR-2CF3-Hoechst to label the cells and observed a significant decrease in DNA compaction in living and fixed hFib and Hela cells after treatment with the histone deacetylase inhibitor Trichostatin A (TSA). Figure 11 as well as Figure 12 ).

[0111] In addition, this example uses the synthesized DNA probes TMR-CF3-Hoechst (see Figure S14) and TMR-2CF3-Hoechst ( Figure 13 ) markers, and the DNA compaction of living hFib-derived iPSCs was significantly reduced compared with hFib cells ( Figure 13 C, D and Figure 14 A, B in the figure), indicating an increase in open chromatin in iPSCs. This example previously developed a deep learning method, Artificial Intelligence for Nuclei (AINU), which can recognize specific nuclear markers, such as histones, RNA polymerase II, and DNA labeled by Edc click chemistry, to identify human cells and human induced pluripotent stem cells iPSCs at nanometer resolution in fixed cells. This example further explored whether AINU can accurately identify super-resolution images of living hFibs and induced pluripotent stem cells (iPSCs) after being trained with super-resolution images labeled with DNA probes (TMR-2CF3-Hoechst) synthesized by this example. This example found that AINU can identify DNA super-resolution images of all living human fibroblasts (hFib) and induced pluripotent stem cells (hiPSCs) with 100% accuracy ( Figure 13 E, F, G, H in the.

[0112] Application Example 1: DNA probe combined with OligoSTORM technology

[0113] To investigate whether DNA probes can be used in conjunction with OligoSTORM technology, this example first used OligoSTORM to image the OCT4-TCF19 gene pair in hFib-derived iPSCs and hFib cells at the nanoscale. These genes are active in iPSCs but repressed in hFib cells. As expected, this example found that OCT4-TCF19 is more densely packed in hFib cells and more loosely packed in hFib-iPSCs (see Figure 15 OligoSTORM data analysis showed that the localization density in iPSCs was reduced and the distribution was more dispersed (see Figure 15B, DNA density analysis), while the radius of the gyroids increased in iPSCs (see Figure 15 C in Figure 3), indicating that the chromatin is more loose. This example then combines DNA super-resolution images of the entire chromatin obtained using the DNA probe TMR-CF3-Hoechst with OligoSTORM imaging of OCT4-TCF19 to observe the chromatin structure of the OCT4-TCF19 gene locus and its adjacent regions in hFib and iPSCs (see Figure 15 D in this example). This example compares DNA compaction at the OCT4-TCF19 locus and its adjacent regions in hFib and hFib-iPSCs. Voronoi tessellation analysis of DNA super-resolution images showed increased DNA compaction in hFib, while DNA compaction was looser in iPSCs (see Figure 15 E, DNA density analysis).

[0114] Application Example 2: Application of TMR-CF3-Hoechst and TMR-2CF3-Hoechst to Zebrafish Retinal Cryosections

[0115] This example uses STORM technology to label frozen zebrafish retinal sections and uses the DNA probe hoechst-TMR-CF3 (see Figure 17 ) and hoechst-TMR-2CF3 (see Figure 16 ) for imaging. These probes exhibit excellent self-blinking properties, enabling this example to observe the fine structure of DNA at super-resolution, including photoreceptors in the outer nuclear layer, various neurons in the inner nuclear layer, and Müller glial cells. Compared to conventional imaging, this technique can reveal the nanostructure of DNA with a probability distribution of positioning accuracy of approximately 20 nanometers (see Figure 16 D in and Figure 17 In addition, this example also found that the DNA density of Müller glial cells is lower than that of other retinal neurons (see Figure 16 These findings provide a powerful tool for studying the regenerative capacity of the zebrafish retina, particularly the reprogramming and regeneration of Müller glia after injury.

[0116] Application Example 3: Application of TMR-CF3-Hoechst to Paraffin Sections of Human Clinical Colorectal Cancer Tissue

[0117] Direct observation of nanoscale nuclear structure and chromatin compaction in normal and colorectal cancer tissue sections. This example explores the possibility of using synthetic DNA probes for STORM imaging of chromatin structure in clinical samples. On commonly used formaldehyde-fixed paraffin-embedded (FFPE) tissue sections, this example found that the DNA probe hoechst-TMR-CF3 can maintain excellent self-scintillation ability. The use of this probe improves localization accuracy, allowing the spatial resolution in single-molecule localization microscopy (SMLM) to reach approximately 20 nanometers (see Figure 18 C in this example). This example directly visualizes the nanoscale nuclear structure and chromatin compaction of normal and cancerous colon tissue sections and finds that the density of genomic DNA is lower in colon cancer tissue, indicating that chromatin compaction has changed (see Figure 18 To further verify this result, this example compared the DNA density of normal colon epithelial cells HCoEpiC and colon cancer cell line HCT-116 after Hoechst-TMR-CF3 labeling and found that the DNA compaction degree of HCT-116 cells was lower than that of HCoEpiC (see Figure 18 D, E), which further confirms the findings of this example.

[0118] In summary, the SMLM technique combined with the DNA-specific self-scintillation probe of the present invention can achieve SMLM imaging of DNA in living cells, fixed cells, frozen tissue sections, and clinical tissue paraffin sections, with a resolution much higher than that of traditional imaging techniques.

[0119] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A DNA probe, characterized in that The DNA probe has a structure as shown in Formula 1: Wherein: R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CH3)2.

2. A method for preparing a DNA probe according to claim 1, characterized in that: The following steps are involved: S1. Dissolve 20.0 mg of the compound represented by Formula IX, 26.3 mg to 31.3 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 12.0 μL to 13.6 μL of N,N-diisopropylethylamine in 4 mL of DMF and stir under a nitrogen atmosphere for 10 minutes; the structural formula of the compound represented by Formula IX is: R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CH3)2; S2. A compound represented by Formula IX and a compound represented by Formula VI were prepared in a mass ratio of 24.2 mg to 28.9 mg:1, and a solution of the compound represented by Formula VI in 0.5 mL of DMF was added dropwise to the reaction mixture at room temperature for 2 hours. After the reaction was complete, the solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a DNA probe represented by Formula 1; wherein the structural formula of the compound represented by Formula VI is:

3. The method for preparing a DNA probe according to claim 2, wherein: The preparation method of the compound represented by formula IX comprises: S11. Dissolve 430.0 mg of 6-carboxytetramethylrhodamine in 3 mL of anhydrous DMF, then add 414.0 mg of potassium carbonate and 417.0 μL of triethylamine; cool the mixture in an ice bath, and slowly add 362.9 mg of allyl bromide; then warm the mixture to room temperature and stir for 24 hours. After the reaction, remove the solvent using a vacuum oil pump, and purify the residue by silica gel column chromatography to obtain 450.0 mg of the compound represented by Formula VII: S12. Under a nitrogen atmosphere, 5.2 mg of 4-dimethylaminopyridine and 31.0 mg-70.9 mg of H2N-R (CH2CH2CH2CH3NH2=31.0 mg; CF3CH2NH2=42.0 mg; (CF3)2CHNH2=70.9 mg; SO2N(CH3)2NH2=52.7 mg) were added to an ultra-dry dichloromethane solution containing 100.0 mg of the compound represented by Formula VII. After stirring for 5 minutes, 71.1 μL of triethylamine was added to the mixture. Subsequently, 19.8 μL of an ultra-dry dichloromethane solution of phosphorus oxychloride was added to the reaction system, and the mixture was reacted at room temperature overnight. After completion of the reaction, the mixture was added dropwise to ice water, extracted with 50 mL×3 of ethyl acetate, washed with saturated sodium chloride solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 450.0 mg of the compound represented by Formula VIII: R is one of CH2CH2CH2CH3, CH2CF3, CH(CF3)2 and SO2N(CH3)2; S13. Dissolve 40.0 mg of the compound represented by formula VIII in 3 mL of anhydrous tetrahydrofuran, then add 20.2 mg-23.3 mg of 1,3-dimethylbarbituric acid and 14.9 mg-17.2 mg of tetrakis(triphenylphosphine)palladium, and stir the reaction at room temperature for 1 hour; after the reaction is completed, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain the compound represented by formula IX.

4. The method for preparing a DNA probe according to claim 2, wherein: The preparation method of the compound represented by formula VI comprises: S21, dissolving 100 mg of Hoechst dye hydrochloride in water, then adding 123 mg of potassium carbonate solution, separating the precipitate by centrifugation, washing with water and freeze-drying to obtain the free base of Hoechst dye; S22. Dissolve 98.4 mg of potassium carbonate in anhydrous DMF, then add 80 mg of the free base of Hoechst dye to a round-bottom flask and stir, then add 84.7 mg of tert-butyl (2-bromoethyl) carbamate, and react at 50° C. for 24 hours. After the reaction is complete, remove the solvent using a vacuum oil pump, and purify the residue by silica gel column chromatography to obtain 45 mg of the compound represented by Formula V: S23. Dissolve the compound represented by formula V in 5 mL of an organic solvent, place the mixture in a round-bottom flask, and react for 1 hour. Then, remove the solvent by distillation under reduced pressure to obtain a compound represented by formula VI.

5. The method for preparing a DNA probe according to claim 4, wherein: The organic solvent consists of trifluoroacetic acid and dichloromethane in a volume ratio of 1:

4.

6. Use of the DNA probe according to claim 1 in super-resolution imaging of DNA in cells and tissue sections.

7. Use of the DNA probe according to claim 1 in super-resolution imaging of DNA in cells and tissue sections, characterized in that: The DNA probe is used for DNA labeling of living cells, fixed cells and tissue sections, and the self-scintillation of the DNA probe is utilized to realize the positioning of DNA molecules and super-resolution imaging in super-resolution microscope imaging.

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