A method for preparing a DNA probe and its application in super-resolution imaging of DNA from cells and tissue sections.

Abstract

This invention belongs to the field of super-resolution imaging, specifically disclosing a method for preparing a DNA probe and its application in super-resolution imaging of DNA in cells and tissue sections. The method involves linking rhodamine dyes modified with different functional groups to a Hoechst structure via short linker strands to form a self-scintillation DNA probe. By modifying the probe with different functional groups to adjust the spirocyclic opening and closing ability of the fluorescent dye, the performance of the DNA probe can be precisely controlled to meet different SMLM super-resolution imaging requirements. By combining SMLM technology with a DNA-specific self-scintillation probe, SMLM imaging of DNA in live cells, fixed cells, frozen tissue sections, and paraffin sections of clinical tissues can be achieved, with resolution far exceeding that of traditional imaging techniques.

Description

Technical Field

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

[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 SMLMs (including (d)STORM, PALM, PAINT, etc.) can overcome the diffraction limit, they have several limitations when imaging DNA nanostructures within living cells and tissue sections. The main problems 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 light-switching buffers, which are incompatible with living cells.

[0005] 2) It requires complex optical switching buffers and high laser power to excite imaging: this limits its application in living cells.

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

[0007] 4) 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytosine (EdC) and their analogues can be used for super-resolution imaging by binding to fluorescent dyes via click chemistry after labeling DNA, but their applicability is limited: EdU and EdC are mainly applicable to proliferating cells, which limits 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 steps are 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 capability: The ability to resolve the natural three-dimensional chromatin structure of living cells is needed 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 plans.

[0011] 2) Challenges of Fluorescence Imaging: Fluorescence imaging technology still faces challenges in pathological tissues, especially in detecting changes in high-level chromatin structure during tumor development. Changes in high-level chromatin structure are difficult to resolve under traditional wide-field or confocal fluorescence microscopy, and therefore have not been fully characterized in pathological tissues, mainly due to the lack of simple, rapid, and effective methods.

[0012] 3) Limitations of super-resolution imaging applications: The application of existing fluorescent dyes and staining methods in super-resolution imaging (SMLM) is limited, especially in imaging chromatin structure in fixed paraffin-embedded (FFPE) and frozen tissue sections. Summary of the Invention

[0013] To address the aforementioned technical problems, this invention provides a method for preparing a DNA probe and its application in super-resolution imaging of DNA from cells and tissue sections.

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

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

[0016]

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

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

[0019] S1. The compound shown in Formula IX (20.0 mg), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea 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 for 10 minutes under a nitrogen atmosphere; wherein:

[0020] ;

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

[0022] S2. A solution of the compound shown in 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 was added dropwise to the reaction mixture and carried out at room temperature for 2 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain the DNA probe of Formula 1.

[0023] Furthermore, the method for preparing the compound represented by formula IX includes:

[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 heated 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 give 450.0 mg of the compound shown in 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 shown in 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 reaction was carried out overnight at room temperature. After the reaction was completed, the mixture was added dropwise to ice water, extracted with ethyl acetate (EA, 50 mL × 3), washed with saturated sodium chloride solution, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 450.0 mg of the compound shown in Formula VIII.

[0027] ;

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

[0029] S13. The compound shown in 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 tetra(triphenylphosphine)palladium (VIII-I: 16.8 mg; VIII-II: 17.2 mg; VIII-III: 14.9 mg; VIII-IV: 17.0 mg). The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain the compound shown in Formula IX.

[0030] Furthermore, the method for preparing the compound represented by Formula VI includes:

[0031] S21. Dissolve Hearst 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 Hearst dye.

[0032] S22. Dissolve potassium carbonate (98.4 mg) in anhydrous DMF, then add the free base of Hoechst dye (80 mg) to a round-bottom flask and stir. Next, add tert-butyl (2-bromoethyl) carbamate (84.7 mg) and react at 50 °C for 24 hours. After the reaction is complete, remove the solvent using a vacuum oil pump. The residue is purified by silica gel column chromatography to obtain 45 mg of the compound shown in Formula V.

[0033] ;

[0034] S23. Dissolve the compound shown in Formula V in 5 mL of organic solvent and react it in a round-bottom flask for 1 hour. Remove the solvent by vacuum distillation to obtain the compound shown in Formula VI.

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

[0036] The third technical solution of the present invention is an application of a DNA probe in super-resolution imaging of DNA in cells and tissue sections. The DNA probe is used for DNA labeling of live cells, fixed cells and tissue sections. The self-scintillation of the DNA probe is used to realize the localization and super-resolution imaging of DNA molecules in super-resolution microscopy.

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

[0038] 1) Improved resolution: This invention links rhodamine dyes modified with different groups to the Hoechst structure through short linker chains to form a self-scintillation DNA probe; by modifying the fluorescent dyes in the probe with different groups, the spirocyclic opening and closing ability can be adjusted to achieve fine control of the DNA probe performance to meet different SMLM super-resolution imaging requirements; by combining SMLM technology with DNA-specific self-scintillation probes, SMLM imaging of DNA in live cells, fixed cells, frozen tissue sections, and paraffin sections of clinical tissues can be achieved, with a resolution far exceeding that of traditional imaging techniques.

[0039] 2) Enhanced imaging specificity: The specific binding of the Hoechst structure enhances the labeling specificity of the probe on 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 pore the cells, and it has low toxicity to live cells.

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

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

[0044] 7) Wide range of applications: It is not only suitable for live cell fixation, but also for tissue freezing and paraffin sectioning, 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 directly identifying somatic cells and stem cells in live single cells. This combination of technologies provides a new perspective for studying 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 clinicopathological tissues. Attached Figure Description

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

[0048] Figure 2 The absorption and fluorescence spectra of the DNA probe (5 μM) were obtained before and after incubation at room temperature in PBS (pH = 7.4) containing 0.4% BSA or 0.4% SDS for 4 hours.

[0049] Figure 3 The pH titration spectrum of the DNA probe (5 μM) after incubation in BR buffer at room temperature for 4 h is shown.

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

[0051] Figure 5Ultrastructure of DNA directly labeled with self-scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst for STORM imaging in live and fixed hFib cells: A. Super-resolution (SR) images of representative nuclear DNA labeled with TMR-CF3-Hoechst in live hFib cells (top) and fixed hFib cells (bottom) using SR Voronoi mosaic technique, and magnified DNA ultrastructure of the nuclear margin (red box magnified area), nucleoplasm (green box magnified area), and nucleolar periphery (blue box magnified area); B. Super-resolution (SR) images of representative nuclear DNA labeled with TMR-2CF3-Hoechst in live hFib cells (top) and fixed hFib cells (bottom) using SR Voronoi mosaic technique. Voronoi mosaic technique) and magnified DNA ultrastructure of the nuclear margin (red box magnified area), nucleoplasm (green box magnified area) and nucleolus periphery (blue box magnified area); C. Probability distribution of localization accuracy of live hFib cells (red histogram) and fixed hFib cells (black histogram) labeled with TMR-CF3-Hoechst; D. Probability distribution of super-resolution localization accuracy of live hFib cells (red histogram) and fixed hFib cells (black histogram) labeled with TMR-2CF3-Hoechst.

[0052] Figure 6 The ultrastructure of DNA was directly labeled with the self-scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst for STORM imaging in live and fixed HeLa cells.

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

[0054] Figure 8 Three-dimensional STORM super-resolution images of DNA visualized in live hFib cells using TMR-CF3-Hoechst and ViSP software:

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

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

[0057] Figure 10 To perform time-series single-molecule localization microscopy (SMLM) imaging in live HeLa cells using TMR-CF3-Hoechst and TMR-2CF3-Hoechst: A. DNA super-resolution (SR) images were acquired sequentially at 0 min (white), 10 min (red), and 20 min (green) using TMR-CF3-Hoechst; B. DNA super-resolution (SR) images were acquired sequentially at 0 min (white), 10 min (red), and 20 min (green) using TMR-2CF3-Hoechst, with an excitation wavelength of 561 nm (power density ~0.33 kW / cm2). Each super-resolution image was reconstructed from 10,000 images (30 ms per frame), corresponding to a 300-second acquisition time; C. DNA motion tracking analysis results (under analysis).

[0058] Figure 11STORM imaging of direct labeling DNA with and without hyperacetylation (TSA treatment) in live and fixed human fibroblasts (hFib) using the self-scintillation dyes TMR-CF3-Hoechst and TMR-2CF3-Hoechst: A. Representative STORM density rendering images of DNA in live hFib cells labeled with TMR-CF3-Hoechst (top) and TSA-treated (bottom) (left: intact cell nucleus; right: magnified area within red box); B. Representative STORM density of DNA in fixed hFib cells labeled with TMR-CF3-Hoechst (top) and TSA-treated (bottom). M. Density rendering image (left: intact cell nucleus; right: magnified area in red box); C. Representative STORM density rendering images of DNA in live hFib cells labeled with TMR-2CF3-Hoechst (top) and TSA-treated (bottom) (left: intact cell nucleus; right: magnified area in red box); D. Representative STORM density rendering images of DNA in fixed hFib cells labeled with TMR-2CF3-Hoechst (top) and TSA-treated (bottom) (left: intact cell nucleus; right: magnified area in red box); E. Cumulative distribution map of Voronoi polygon density in live hFib cells labeled with TMR-CF3-Hoechst (blue) (n = 39) and TSA-treated (orange) (n = 32). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; F, cumulative distribution of Voronoi polygon density in immobilized hFib cells using TMR-CF3-Hoechst labeled control group (blue) (n = 32) and TSA-treated (orange) (n = 24). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; G, cumulative distribution of Voronoi polygon density in live hFib cells using TMR-2CF3-Hoechst labeled control group (blue) (n = 44) and TSA-treated (orange) (n = 39). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, ***P=0.0002; H, cumulative distribution of Voronoi polygon density in fixed hFib cells using TMR-2CF3-Hoechst labeled control group (blue) (n = 31) and TSA-treated (orange) (n = 36). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, ***P=0.0001;

[0059] In the figure, light colors represent the interquartile range (25th-75th percentile), and thick dark lines represent the median value; asterisks indicate that the separation between medians is statistically significant according to an unpaired two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

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

[0061] In the figure, light colors represent the interquartile range (25th-75th percentile), and thick dark lines represent the median value; asterisks indicate that the separation between medians is statistically significant according to an unpaired two-tailed Student's t-test. Super-resolution Voronoi mosaic images show differences in DNA density.

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

[0063] Figure 14 Direct DNA labeling of live human fibroblasts (hFib) and human fibroblast-induced pluripotent stem cells (hFib-iPSCs) using the self-scintillation dye TMR-CF3-Hoechst: A. Representative STORM density rendering images of DNA distribution in live hFib (top) and live hFib-iPSCs (bottom) labeled with TMR-CF3-Hoechst (left: intact cell nucleus; right: magnified area within red box).

[0064] B. Cumulative distribution, median, and mean standard error (SEM) of Voronoi polygon density in live hFib (blue) (n = 30) and live hFib-iPSCs (orange) (n = 98) labeled with TMR-CF3-Hoechst; unpaired two-tailed Student's t-test, ****P < 0.0001; light colors in the figure show the quartile range (25th–75th percentile), and thick dark lines show the median value; asterisks indicate that the separation between medians is statistically significant according to the 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 used to observe the location of the OCT4 gene locus in the chromatin structure: A. OligoSTORM images of the OCT4-TCF19 gene locus (20 kb) in hFib and hFib-iPSCs. Super-resolution images of conventional fluorescence images, Gaussian function rendering (red), and hotspot density rendering; B. Cumulative distribution of Voronoi polygon density of the OCT4-TCF19 gene locus (20 kb) in hFib (n = 45 loci) and hFib-iPSCs (n = 109 loci) using OligoSTORM. Median and mean standard error (SEM); unpaired two-tailed Student's t-test, *P = 0.0257; C, quantitative statistics of gyration radius length of OCT4-TCF19 OligoSTORM loci (20 kb) in hFib and hFib-iPSCs (n = 45 and 109 loci respectively). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, ****P<0.0001; D. Conventional OligoSTORM image of the OCT4-TCF19 gene locus (20 kb), heatmap density rendering of DNA super-resolution imaging by TMR-CF3-Hoechst (color bar), and magnified images of the fine structure of the OCT4-TCF19 gene locus and its adjacent DNA region in hFib and hFib-iPSCs (red box); E. Cumulative distribution of Voronoi polygon density of DNA containing the OCT4-TCF19 gene locus and its adjacent region in hFib cells (n = 22 loci) and hFib-iPSCs (n = 16 loci). Median and mean standard error (SEM); unpaired two-tailed Student's t-test, *P = 0.0482; light colors in the figure show the interquartile range (25th–75th percentile), and thick dark lines show the median value; asterisks indicate that the separation between medians is statistically significant according to the 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 sections using TMR-2CF3-Hoechst: A. Conventional fluorescence and super-resolution (SR) images of DNA in GFAP-GFP transgenic zebrafish retinal sections, from left to right: conventional fluorescence image of DNA, SR image, conventional fluorescence image of GFAP (labeled MGCs), SR image of DNA, and image of DNA and GFAP merged (ONL - outer nuclear layer; OPL - outer plexiform layer; INL - inner nuclear layer; IPL - ...). A) Inner plexiform layer); B) Representative nuclear DNASR images of GFAP-labeled MGCs (yellow boxes) labeled with TMR-2CF3-Hoechst, other retinal neurons (red boxes), and super-resolution images magnified using super-resolution Voronoi mosaic images (green boxes), which 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, n=26 for other retinal cells), with light colors showing the quartile range (25-75 percentiles) and thick dark lines showing the median value; asterisks indicate statistically significant separation between medians according to an 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 (labeled proliferating MGCs), and a combined SR and PCNA image of DNA (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) labeled with TMR-CF3-Hoechst, proliferating MGCs (yellow boxes), and magnified images using super-resolution Voronoi mosaic (green boxes), showing differences in DNA density; C. Probability distribution of localization accuracy using TMR-CF3-Hoechst in zebrafish retinal frozen sections (n ​​= 19).

[0069] Figure 18For direct DNA labeling using the self-scintillation dye TMR-CF3-Hoechst in human normal and colon cancer tissues, as well as living normal colon cells and cancer cells: A. Hematoxylin-eosin (H&E) stained histological images of human normal and colon cancer tissues, STORM density rendering images of DNA distribution imaged by TMR-CF3-Hoechst, STORM density rendering images of DNA distribution in representative normal colon cells and colon cancer cells (green boxes), and magnified images using super-resolution Voronoi mosaic (red boxes), the super-resolution Voronoi mosaic images showing differences in DNA density; B. In normal colon tissue (blue) (n > 100) and colon cancer tissue (orange) (n > 100) In the image, the cumulative distribution of Voronoi polygon density by TMR-CF3-Hoechst is shown, with light colors indicating the quartile range (25th-75th percentile) and thick dark lines indicating the median values; an asterisk indicates that the separation between medians is statistically significant 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 images of DNA distribution in live human normal colonic epithelial cells (HCoEpiC, top) and live human colon cancer cells (HCT116, bottom) labeled by TMR-CF3-Hoechst, showing the difference in DNA density (left: intact cell nuclei; right: magnified area in red box); E. In live human normal colonic epithelial cells (HCoEpiC, blue) (n = 48) and live human colon cancer cells (HCT116, orange) (n = In 46), the cumulative distribution plot of Voronoi polygon density labeled by TMR-CF3-Hoechst, P<0.0001, light color shows the interquartile range (25-75 percentile), thick dark line shows the median value; asterisk indicates that the separation between the medians is statistically significant according to the unpaired Student's t test, and super-resolution Voronoi mosaic image shows the difference in DNA density. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0071] Example 1

[0072] In this embodiment, rhodamine fluorescent dyes modified with different groups are linked to the Hoechst structure via short linker strands to form a DNA probe. The reaction process is as follows:

[0073] .

[0074] Following the reaction process described above, the compound shown in formula V was first prepared. The specific preparation process is as follows:

[0075] 1) Preparation of free base of Hearst dye: Dissolve commercially available Hearst 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 free base of Hearst dye.

[0076] 2) 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 for several minutes. Tert-butyl (2-bromoethyl) carbamate (84.7 mg) was then added, and the mixture was reacted at 50 °C 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 give 45 mg of a yellow solid (the compound shown in Formula V), with a yield of 41.7%. The NMR characterization of the compound shown in Formula V was as follows: 1 ¹H NMR (400 MHz, MeOD) δ 8.23 ​​(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 spectrometry characterization of the compound shown in Formula V is: m / z [M+H] + calcd. for C 32 H 38 N7O3 + : 568.3, found: 568.4.

[0077] Secondly, the compound shown in formula VI was prepared, and the specific preparation process is as follows:

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

[0079] Then, the compound shown in formula VII was prepared, 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 heated to room temperature and stirred for 24 hours. After the reaction was complete, the solvent was removed by a vacuum oil pump, and the residue was purified by silica gel column chromatography to give 450.0 mg of a pink solid (compound shown in Formula VII), with a yield of 95.5%. The NMR characterization of compound shown in Formula VII was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.26 (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 spectrometric characterization of the compound shown in formula VII is: m / z calcd. forC 28 H 27 N2O5 + : 471.1, found: 471.3.

[0081] Subsequently, the compound shown in formula VII was used to prepare the compound shown in formula VIII. The specific preparation process is as follows:

[0082] Under a nitrogen atmosphere, 4-dimethylaminopyridine (DMAP, 5.2 mg) and amines with different 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 an ultra-dry dichloromethane solution containing the compound shown in Formula VII (100.0 mg), 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, an ultra-dry dichloromethane solution of phosphorus oxychloride (POCl3, 19.8 μL) was added to the reaction system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the mixture was added dropwise to ice water, extracted with ethyl acetate (EA, 50 mL × 3), washed with saturated sodium chloride solution, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the compound shown in formula VIII. In this embodiment, the compound shown in formula VIII includes the following four forms:

[0083] VIII-I: Allyl 2-butyl-3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-rhodane]-6-carboxylic acid ester. The compound shown in Formula VIII-I was obtained as a pink solid in 40.4% yield; the NMR characterization of the compound shown in Formula VIII-I was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 7.8 Hz, 1H), 7.96 (d, J =7.9 Hz, 1H), 7.69 (s, 1H), 6.46 – 6.44 (m, 4H), 6.33 (d, J = 8.9 Hz, 2H), 6.00 – 5.90 (m, 1H), 5.33 (d, J = 16.9 Hz, 1H), 5.24 (d, J = 10.5 Hz, 1H), 4.72 (d, J = 5.9 Hz, 2H), 3.10 (t, J = 6.9 Hz, 2H), 2.97 (s, 12H), 1.16-1.03(m, 4H), 0.68 (t, J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 166.9, 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'-rhodane]-6-carboxylic acid ester. The compound shown in Formula VIII-II was obtained as a pink solid in 29.7% yield; the NMR characterization of the compound shown in Formula VIII-II was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.73 (s, 1H), 6.48 – 6.40 (m, 4H), 6.36 (dd, J =8.8, 1.8 Hz, 2H), 6.01 – 5.89 (m, 1H), 5.33 (d, J = 17.2 Hz, 1H), 5.25 (d, J= 10.4 Hz, 1H), 4.73 (d, J = 5.8 Hz, 2H), 3.72 (q, J = 9.2 Hz, 2H), 2.97 (s,12H). 13 C NMR (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 spectrometry characterization of the compound shown in Formula VIII-II is as follows: m / z [M+H] + calcd. forC 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'-rhodane]-6-carboxylic acid ester (8-3). The compound shown in Formula VIII-III was obtained as a pink solid in 35.0% yield; the NMR characterization of the compound shown in Formula VIII-III was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​(d, J =7.9 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.78 (s, 1H), 6.47 – 6.43 (m, 4H), 6.36 (d, J = 11.3 Hz, 2H), 6.02 – 5.91 (m, 1H), 5.34 (d, J = 17.2 Hz, 1H), 5.26 (d, J = 10.4 Hz, 1H), 4.75 (d, J = 5.9 Hz, 2H), 3.94 – 3.86 (quint J=7.6Hz, 1H), 2.98 (s, 12H). 13 C NMR (101 MHz, CDCl3) δ 166.2, 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'-rhodane]-6-carboxylic acid ester. The compound shown in Formula VIII-IV was obtained as a pink solid in 53.2% yield; the NMR characterization of the compound shown in Formula VIII-IV was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 8.0 Hz, 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 = 17.2 Hz, 1H), 5.25 (d, J = 10.4Hz, 1H), 4.73 (d, J = 5.9 Hz, 2H), 2.97 (s, 12H), 2.72 (s, 6H).13 C NMR (101MHz, CDCl3) δ 166.3, 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 shown in formula VIII is reused to prepare the compound shown in formula IX. The specific preparation process is as follows:

[0088] The compound shown in 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 the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the compound shown in Formula IX. In this embodiment, the compound shown in Formula IX includes the following four forms:

[0089] IX-I: 2-Butyl-3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-rhodane]-6-carboxylic acid. The compound shown in Formula IX-I was obtained as a pink solid in 67.7% yield; the NMR characterization of the compound shown in Formula IX-I was as follows: 1 H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 7.9 Hz,1H), 7.70 (s, 1H), 6.47 – 6.41 (m, 4H), 6.32 (d, J = 8.9, 2H), 3.09 (t, J =6.9 Hz, 2H), 2.96 (s, 12H), 1.13 – 1.04 (m, 4H), 0.67 (t, J = 6.9 Hz, 3H). 13CNMR (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 spectrometric characterization of the compound shown in Formula IX-I is as follows: 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'-rhodane]-6-carboxylic acid. The compound of formula IX-II was obtained as a pink solid in 78.2% yield; the NMR characterization of the compound of formula IX-II was as follows: 1 H NMR (400 MHz, DMSO) δ 8.09 (dd, J = 7.9, 1.2 Hz, 1H), 8.00(d, J = 7.9 Hz, 1H), 7.43 (s, 1H), 6.44 (d, J = 6.9, 4H), 6.39 – 6.34 (m,2H), 3.75 (q, J = 10.0 Hz, 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 spectrometric characterization of the compound shown in Formula IX-II is as follows: 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'-rhodane]-6-carboxylic acid. The compound shown in Formula IX-III was obtained as a pink solid in 56.1% yield; the NMR characterization of the compound shown in Formula IX-III was as follows: 1H NMR (400 MHz, DMSO) δ 8.14 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 7.4 Hz, 1H), 7.46 (s, 1H), 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 spectrometric characterization of the compound shown in formula IX-III is as follows: 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'-rhodane]-6-carboxylic acid. The compound shown in Formula IX-IV was obtained as a pink solid in 94.0% yield; the NMR characterization of the compound shown in Formula IX-IV was as follows: 1 ¹H NMR (400 MHz, MeOD) δ 8.19 (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 spectrometry characterization of the compound shown in formula IX-IV is as follows: m / z [M+H] + calcd.for C 27 H 29 N4O6S + : 537.2, found: 537.3.

[0093] Finally, DNA probes were prepared using the compound shown in Formula IX. The specific preparation process is as follows:

[0094] Compound IX (20.0 mg), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea 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 for 10 minutes under a nitrogen atmosphere. Then, compound VI (IX-I: VI = 28.9 mg; IX-II: VI = 27.4 mg; IX-III: VI = 24.2 mg; IX-IV: VI = 28.3 mg) was dissolved in 4 mL of DMF and stirred for 10 minutes under a nitrogen atmosphere. A solution of 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 complete, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the DNA probe. In this example, the DNA probe included 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'-rhodane]-6-carboxamide. Probe I was obtained as a pink solid with a yield of 10.4%. The NMR characterization of Probe I was as follows: 1H NMR (400 MHz, MeOD) δ 8.32 (s, 1H), 8.03 – 7.99 (m, 4H), 7.94 (d, J = 7.9 Hz, 1H), 7.85 (d,J = 8.0 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.55 (s, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.28 (s, 1H), 7.06 (d, J = 8.3 Hz, 2H), 6.50 (s, 2H), 6.40 (m, 4H), 4.15(t, J = 5.3 Hz, 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 = 6.4 Hz, 2H), 3.01 (s, 3H), 2.93 (s, 12H), 1.10 –1.05 (m, 4H), 0.66 (t, J = 6.1 Hz, 3H). 13 C 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; Mass spectrometry characterization of probe I: m / z [M+H] + calcd. for C 56 H 59 N 10 O4 + : 935.5, found: 935.7; Probe I is 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'-rhodane]-6-carboxamide. Probe II was obtained as a pink solid with a yield of 26.6%. The NMR characterization of Probe II was as follows:1 HNMR (400 MHz, DMSO) δ 8.91 (t, J = 5.4 Hz, 1H), 8.28 (d, J = 49.2 Hz, 1H), 8.12 – 8.08 (m, 3H), 8.05 – 7.94 (m, 2H), 7.65 (dd, J = 46.5, 8.3 Hz, 1H),7.54 (s, 1H), 7.45 (s, 1H), 7.11 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.4 Hz,1H), 6.45 – 6.43 (m, 4H), 6.36 (d, J = 9.5 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 3.73 (q, J = 9.6 Hz, 2H), 3.60 (d, J = 5.5 Hz, 2H), 3.22 – 3.13 (m, 4H), 2.92 (s, 12H), 2.75 – 2.60 (m, 4H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 167.4, 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; Mass spectrometry characterization of probe II: m / z [M+H] + calcd. for C 54 H 52 F3N 10 O4 + : 961.4, found: 961.4; Probe II is designated as TMR-CF3-Hoechst.

[0097] Probe III: 3',6'-bis(dimethylamino)-2-(1,1,1,3,3,3-hexafluoropropyl)-N-(2-(4-(5-(4-methylpiperazin-1-yl)-1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxospiro[isoindoline-1,9'-rhodan]-6-carboxamide. Probe III was obtained as a pink solid with a yield of 19.9%; Probe II was characterized by NMR as follows: 1H NMR (400 MHz, MeOD) δ 8.18 (s, 1H), 8.12 (d, J = 9.4 Hz, 1H), 8.01 (d, J= 8.0 Hz, 1H), 7.94 – 7.88 (m, 3H), 7.70 (s, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.03 (dd, J = 8.9, 2.2Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.45 (d, J = 1.0 Hz, 2H), 6.43 – 6.36 (m,4H), 4.11 (t, J = 5.5 Hz, 2H), 4.07 – 3.99 (m, 1H), 3.69 (t, J = 5.5 Hz, 2H), 3.30 – 3.22 (m, 4H), 2.92 (s, 12H), 2.91 – 2.85 (m, 4H), 2.54 (s, 3H). 13 C 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; Mass spectrometry characterization of probe III: m / z [M+H] + calcd. forC 55 H 51 F6N 10 O4 + : 1029.4, found: 1029.4; Probe III is designated as TMR-2CF3-Hoechst.

[0098] Probe IV: 3',6'-bis(dimethylamino)-2-(N,N-dimethylsulfonyl)-N-(2-(4-(5-(4-methylpiperazin-1-yl)-1H,1'H-[2,5'-bibenzimidazole]-2'-yl)phenoxy)ethyl)-3-oxospiro[isoindoline-1,9'-rhodane]-6-carboxamide. Probe IV was obtained as a pink solid with a yield of 40.8%. The NMR characterization of probe IV was as follows: 1HNMR (400 MHz, DMSO) δ 8.95 (s, 1H), 8.46 (s, 1H), 8.16 (d, J = 8.6 Hz, 2H), 8.10 (dd, J = 14.7, 8.4 Hz, 2H), 8.01 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.5Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.49 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.25 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 6.53 (d, J = 8.3 Hz, 2H), 6.47 – 6.34(m, 4H), 4.17 (t, J = 5.3 Hz, 2H), 3.92 (d, J = 11.0 Hz, 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). 13 C 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 spectrometry characterization of probe IV is: m / z [M+H] + calcd. for C 54 H 56 N 11 O6S + : 986.4, found: 986.5; Probe IV is denoted as TMR-SO2-Hoechst.

[0099] To characterize the prepared probes I-IV, their performance was evaluated by measuring their D50 value, ring opening and closing ability, and pKa value, as detailed below:

[0100] First, the D50 values ​​of probes I-IV were tested by plotting the relationship between the absorbance peak of the probe and the dielectric constant of the solvent (see [reference]). Figure 1(A) Compared to the parent 6-TAMRA (6-Carboxytetramethylrhodamine) dye (D50 = 12), all probes I-IV showed larger D50 values ​​(> 40), indicating a strong tendency to form closed-ring spironolactams.

[0101] Then, in this embodiment, sodium dodecyl sulfate (SDS) is used as an inducing agent to check the ring-opening ability, such as... Figure 1 B and Figure 2 As shown, all probes exhibited fluorescence enhancement in response to SDS, with intensity differences of approximately three orders of magnitude. This indicates that the modification with rhodamine spironolactam dye covers a broad range of ring-opening capabilities, providing sufficient analogues to study structure-activity relationships. Furthermore, probes I-IV showed minimal fluorescence changes upon incubation with BSA, suggesting that the probes effectively avoid interference from nonspecific interactions. This example then explores 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 exhibited self-assembly capabilities to form nanoaggregates, which provided higher fluorescence enhancement and better cell permeability (see [link to relevant documentation]). Figure 1 D and Figure 4 ).

[0102] This embodiment then investigates the binding characteristics of probes I-IV to hairpin DNA (hpDNA) (see [link to documentation]). Figure 1 (E in the original text). After incubation of hpDNA (30 μM) with the probe (2.5 μM), fluorescence enhancement was observed in this example ranging from 1.5 to 157.2-fold. As expected, the fluorescence intensity of the probe after binding to hpDNA showed a three-order-of-magnitude difference consistent with the results obtained with SDS addition. This example further utilized probes I-IV to image DNA in fixed cells. All probes showed high specificity for DNA (see E in the original text). Figure 1 (F in the image). The increase in fluorescence intensity followed the same trend observed in hpDNA imaging. These results indicate that the loop-opening ability of probes I-IV can be fine-tuned over a wide range to label DNA.

[0103] To gain a deeper understanding of the relationship between the ring-opening ability of the probes and their suitability for SMLM imaging, single-molecule photophysics studies were conducted in this embodiment. Only probes II and III exhibited good self-scintillation properties in 50 nM live cells without any additives (see [link to study]). Figure 1The presence of G and H in the data indicates that spironolactam probes with moderate ring-opening ability are best suited for SMLM imaging. Both probes II and III exhibit extremely high photostability, as verified by molecular survival fraction analysis (see G and H in the data). Figure 1 In the model, probes I and II achieved 95% completion after 10,000 frames of data collection, while probe III achieved 86%. The activation times for probes II and III were calculated to be 92 milliseconds and 110 milliseconds, respectively, suitable for SMLM (see [link to SMLM documentation]). Figure 1 (J in the text). More importantly, probes II and III showed high average photon numbers per switching event, 2200 and 2800 respectively (see J in the text). Figure 1 The K in the model gives them good positioning accuracy. It is worth noting that although probe II has better photostability than probe III, probe III has a higher temporal resolution (1.5 seconds) under the same imaging conditions (see [reference needed]). Figure 1 The increase in the "L" value (in the original text) is primarily due to the increased "ON" fraction (higher ring-opening capability) of probe III. These results confirm that probes II and III are self-assembled self-scintillation probes with high photostability and high temporal resolution, capable of covering long-term / real-time chromatin SMLM applications. Therefore, probes II and III will be used in subsequent studies in the following examples.

[0104] Example 2: SMLM Imaging of DNA Probes

[0105] Imaging was performed using STORM super-resolution microscopy in live and fixed cells (hFib and HeLa cells). The synthesized probes effectively penetrated the cell membrane and bound to DNA. DNA density analysis using Voronoi mosaicism showed that the synthesized probes II (TMR-CF3-Hoechst) and III (TMR-2CF3-Hoechst) specifically labeled compact heterochromatin regions (nuclear margins and nucleolar margins) and low-DNA-density euchromatin regions (nucleoplasm) (see [link to documentation]). Figure 5 A, B and Figure 6 (A and B in the text). The other two synthesized probes, I (TMR-C4-Hoechst) (see...),... Figure 7 (A, C) and probe IV (TMR-SO2-Hoechst) (see A, C) and probe IV (TMR-SO2-Hoechst) (see Figure 7 The super-resolution imaging performance of B and D in the model is not good. This embodiment then evaluates the localization accuracy of the TMR-CF3-Hoechst and TMR-2CF3-Hoechst probes. Localization accuracy directly affects the achievable spatial resolution in SMLM. The histogram shows that the probability distribution of the synthesized probe localization accuracy is approximately 20 nm (see [reference]). Figure 5 C, D and Figure 6 (C and D in the text).

[0106] Furthermore, using the synthesized DNA probe TMR-CF3-Hoechst, high-resolution 3D super-resolution imaging of DNA in live hFib cells and in live hFib cells treated with histone hyperacetylation (Trichostatin A (TSA), a histone deacetylase inhibitor) can be obtained. Since biological structures are inherently three-dimensional, two-dimensional SMLM imaging can be limited. The ability to resolve 3D biological structures provides more informative and comprehensive insights. Therefore, this embodiment visualizes the native 3D chromatin structure in live hFib cells after labeling with TMR-CF3-Hoechst, including the case of TSA-induced histone tail hyperacetylation (see [link to documentation]). Figure 9 ) and unprocessed cases (see Figure 8 Traditional bright-field or confocal fluorescence microscopy often struggles to resolve histone hyperacetylation, especially in three-dimensional imaging. For example... Figure 9 A (with histone hyperacetylation) and Figure 8 As shown in A (no histone hyperacetylation), this embodiment clearly displays images along different Z axes of live hFib cells. Furthermore, this embodiment also renders DNA 3D density maps of TMR-CF3-Hoechst-labeled STORM images in live hFib cells. Figure 9 B and Figure 8 (B in the text). The 3D density map of the DNA structure, further highlighted by rotational cutting from the nuclear edge (red box magnification), nucleoplasm (green box magnification), and nucleolar periphery (blue box magnification), further enhances the visualization of live hFib cells (…). Figure 8 C in the middle) and highly acetylated live hFib cells ( Figure 9 The 3D structure of DNA (C) is shown. Furthermore, from the nuclear edge (enlarged area within the yellow box), Figure 8 In this context, D stands for active hFib. Figure 9 The representative rotation sequences (0°, 45°, 90°, 135°, 180°) of the cropped 3D STORM image of hyperacetylated hFib cells (D in the image represents hyperacetylated live hFib cells) show that this embodiment captures 3D ultrastructural images of hyperacetylated DNA with and without histone tails in live cells at an unprecedented resolution.

[0107] At low laser power (power density ~0.33 kW / cm²), TMR-CF3-Hoechst and TMR-2CF3-Hoechst can be used for time-series SMLM super-resolution imaging in live HeLa cells to track the movement of DNA fibers within the cell nucleus (see [link]). Figure 10(A, B, and C in the original text). We also used the Trackpy Python package for single-particle tracking, a tool specifically designed for single-particle tracking applications. We detected "blob-like" features by analyzing each frame of the video and pinpointed their locations to the sub-pixel level. The features identified in each frame were chained together to construct the particle's trajectory. Assuming the particles undergo Brownian motion, meaning their velocities are uncorrelated between adjacent frames, the best prediction is that the particle's position in the next frame is the same as its current position. Particle continuity was determined and trajectories were established by searching for particles within a defined range (1.5 pixels in this study) in subsequent frames (see [link to documentation]). Figure 10 (See C and D in the original text). We calculated the mean square displacement (MSD) of the particles to quantify the average distance the particles travel over a specific time interval. 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 [reference]). Figure 10 (E and F in the diagram). Simultaneously, a violin plot of DNA molecule velocity distribution analysis shows the average velocity distribution of DNA molecules inside and at the cell boundary. The concentration of DNA molecules with higher average velocities at the cell boundary was found to be higher than inside the cell, further indicating increased fluidity around the cell (see [reference]). Figure 10 (G, H, I). Single-particle tracking technology can provide insights into the dynamic behavior of DNA molecules within cells, offering crucial data for studying intracellular biophysical processes.

[0108] In addition, this embodiment, using the synthesized DNA probes TMR-CF3-Hoechst and TMR-2CF3-Hoechst, observed a significant reduction in DNA compaction in both live and fixed hFib and HeLa cells after treatment with the Trichostatin A (TSA) histone deacetylase inhibitor (see [link to documentation]). Figure 11 as well as Figure 12 ).

[0109] Additionally, this embodiment utilizes the synthesized DNA probe TMR-CF3-Hoechst (see...). Figure 14 ) and TMR-2CF3-Hoechst ( Figure 13 ) markers were used to observe a significant decrease in DNA compactness between live hFib-derived iPSCs and hFib cells. Figure 13 C, D and Figure 14The values ​​A and B in the diagram indicate an increase in open chromatin in iPSCs. This embodiment previously developed a deep learning method, Nuclear Artificial Intelligence (AINU), capable of recognizing specific nuclear markers, such as histones, RNA polymerase II, and DNA chemically labeled via Edc click, to identify human cells and human induced pluripotent stem cells (iPSCs) at nanometer-scale resolution in fixed cells. This embodiment further investigates whether AINU, after being trained on super-resolution images labeled with the DNA probe (TMR-2CF3-Hoechst) synthesized in this embodiment, can accurately recognize super-resolution images of live hFibs and induced pluripotent stem cells (iPSCs). This embodiment found that AINU can recognize DNA super-resolution images of all living human fibroblasts (hFibs) and induced pluripotent stem cells (hiPSCs) with 100% accuracy. Figure 13 (E, F, G, H in the text).

[0110] Application Example 1: Combining DNA probes with OligoSTORM technology

[0111] To investigate whether DNA probes could 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 was more densely packed in hFib cells and more loosely packed in hFib-iPSCs (see [link to original document]). Figure 15 (A in the text). OligoSTORM data analysis shows that the location density in iPSCs is reduced and the distribution is more dispersed (see A in the text). Figure 15 (B, DNA density analysis), while the gyroscope radius in iPSCs increased (see B). Figure 15 The presence of C indicates that the chromatin is more loosely distributed. Then, in this example, DNA super-resolution images of the entire chromatin obtained using the DNA probe TMR-CF3-Hoechst were combined with OCT4-TCF19 images obtained by OligoSTORM imaging to observe the chromatin structure of the OCT4-TCF19 gene locus and its adjacent regions in hFib and iPSCs (see [link to documentation]). Figure 15 (See D in the original text). This example compares DNA compression at the OCT4-TCF19 gene locus and its adjacent regions in hFib and hFib-iPSCs. Voronoi mosaic analysis of DNA super-resolution images showed increased DNA compression in hFib, while DNA compression in iPSCs was more loose (see D in the original text). Figure 15 E in DNA density analysis).

[0112] Application Example 2: TMR-CF3-Hoechst and TMR-2CF3-Hoechst applied to frozen sections of zebrafish retina

[0113] This embodiment uses STORM technology to label frozen zebrafish retinal slices and uses the DNA probe hoechst-TMR-CF3 (see [link to documentation]). Figure 17 ) and hoechst-TMR-2CF3 (see Figure 16 Imaging was performed using these probes. These probes exhibited excellent self-scintillation properties, enabling this embodiment 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 localization accuracy of approximately 20 nanometers (see [link to image]). Figure 16 D in and Figure 17 (C in the example). Furthermore, this embodiment also found that the DNA density of Müller glial cells is lower than that of other retinal neurons (see C in the example). Figure 16 (B and C in the original text). These findings provide a powerful tool for studying the regenerative capacity of zebrafish retinas, especially in the reprogramming and regeneration of Müller glial cells after injury.

[0114] Application Example 3: TMR-CF3-Hoechst applied to paraffin sections of human clinical colorectal cancer tissue

[0115] Direct observation of nanoscale nuclear structure and chromatin compression 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 maintained excellent self-scintillation ability. The use of this probe improved localization accuracy, enabling a spatial resolution of approximately 20 nanometers in single-molecule localization microscopy (SMLM) (see [link to documentation]). Figure 18 (See C in this example). This embodiment directly visualizes the nanoscale nuclear structure and chromatin compression of normal and cancerous colon tissue sections, revealing a lower density of genomic DNA in colon cancer tissue, indicating altered chromatin compression (see C in this example). Figure 18 (A and B in the text). To further verify this result, this example compared the DNA density of normal colonic epithelial cells HCoEpiC and colon cancer cell line HCT-116 after hoechst-TMR-CF3 labeling, and found that the DNA compression degree of HCT-116 cells was lower than that of HCoEpiC (see A and B in the text). Figure 18 The presence of D and E in the figures further confirms the findings of this embodiment.

[0116] In summary, by combining SMLM technology with the DNA-specific self-scintillation probe of this invention, SMLM imaging of DNA in live cells, fixed cells, frozen tissue sections, and paraffin sections of clinical tissues can be achieved with a resolution far exceeding that of traditional imaging techniques.

[0117] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions 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: Where R is one of CH2CF3 and CH(CF3)2.

2. A method for preparing a DNA probe as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve 20.0 mg of the compound shown in Formula IX, 26.3 mg-31.3 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 12.0 μL-13.6 μL of N,N-diisopropylethylamine in 4 mL of DMF and stir for 10 minutes under a nitrogen atmosphere; wherein the structural formula of the compound shown in Formula IX is: ； R is one of CH2CF3 and CH(CF3)2; S2. A solution of the compound shown in Formula VI in 0.5 mL of DMF was added dropwise to the reaction mixture and carried out at room temperature for 2 hours. The mass ratio of the compound shown in Formula VI to the compound shown in Formula IX was 1:(24.2-28.9). After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain the DNA probe of Formula 1. The structural formula of the compound shown in Formula VI is: 。 3. The method for preparing the DNA probe according to claim 2, characterized in that, The method for preparing the compound represented by Formula IX includes: S11. 430.0 mg of 6-carboxytetramethylrhodamine was dissolved in 3 mL of anhydrous DMF, followed by the addition of 414.0 mg of potassium carbonate and 417.0 μL of triethylamine. The mixture was cooled in an ice bath, and 362.9 mg of allyl bromide was slowly added. The mixture was then heated 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 shown in Formula VII. ； S12. Under a nitrogen atmosphere, 5.2 mg of 4-dimethylaminopyridine and 42.0 mg of CF3CH2NH2 or 70.9 mg of (CF3)2CHNH2 were added to an ultra-dry dichloromethane solution containing 100.0 mg of the compound shown in 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 reaction was carried out overnight at room temperature. After the reaction was completed, the mixture was added dropwise to ice water, extracted with 50 mL × 3 ethyl acetate, washed with saturated sodium chloride solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 450.0 mg of the compound shown in Formula VIII. ； R is one of CH2CF3 and CH(CF3)2; S13. Dissolve 40.0 mg of the compound shown in 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 tetra(triphenylphosphine)palladium, and stir the reaction at room temperature for 1 hour. After the reaction is complete, remove the solvent by vacuum, and purify the residue by silica gel column chromatography to obtain the compound shown in Formula IX.

4. The method for preparing the DNA probe according to claim 2, characterized in that, The preparation method of the compound represented by Formula VI includes: S21. Dissolve 100 mg of Hearst dye hydrochloride in water, then add 123 mg of potassium carbonate solution, separate the precipitate by centrifugation, wash with water and freeze dry to obtain the free base of Hearst 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. Next, 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. The residue is purified by silica gel column chromatography to obtain 45 mg of the compound shown in Formula V. ； S23. Dissolve the compound shown in Formula V in 5 mL of organic solvent and react it in a round-bottom flask for 1 hour. Remove the solvent by vacuum distillation to obtain the compound shown in Formula VI.

5. The method for preparing the DNA probe according to claim 4, characterized in that, The organic solvent is composed of trifluoroacetic acid and dichloromethane in a volume ratio of 1:

4.

6. The use of the DNA probe as described in claim 1 in the preparation of probes for the localization and super-resolution imaging of DNA molecules.

7. The application of the DNA probe according to claim 6 in the preparation of probes for the localization and super-resolution imaging of DNA molecules, characterized in that, The DNA probe is used for DNA labeling of live cells, fixed cells, and tissue sections. The self-scintillation of the DNA probe is used to achieve the localization and super-resolution imaging of DNA molecules in super-resolution microscopy.

Images