Spatial single-cell transcriptome sequencing method based on light-controlled cell labeling

Abstract

This invention discloses a spatial single-cell transcriptome sequencing method based on light-controlled cell labeling. The method uses ONPF-biotin as a probe and includes labeling the cells to be sequenced with ONPF-biotin; sorting the ONPF-biotin-labeled cells; and performing single-cell sequencing. This invention achieves efficient light-controlled cell labeling and spatial single-cell transcriptome sequencing, solving the problems of low labeling efficiency, limited number of labeling sites, high background signal, and dependence on genetic manipulation in existing methods. It provides a new generation solution for studying the spatial distribution and regulation of cells in different biological systems.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to a spatial single-cell transcriptome sequencing technology based on light-controlled cell labeling. Background Technology

[0002] Single-cell transcriptome sequencing is one of the core technologies in modern biology (scRNA-seq). Using single-cell sequencing, researchers can detect gene expression in each cell, thereby obtaining information such as cell type, quantity, and state. In multicellular organisms, the function and gene expression of each cell are regulated by its spatial location, but this spatial information is lost during the single-cell sequencing process. To address this challenge, researchers have developed various methods to achieve spatially resolved single-cell sequencing. The core of these methods is to fluorescently label cells at specific spatial locations, then isolate and sequence these labeled cells, thus adding spatial location information to the single-cell sequencing data. The Ido group used genetically encoded photoactivated fluorescent proteins to achieve spatial labeling and single-cell sequencing of cells in mouse tissues. This method relies on genetic manipulation and is difficult to apply. The Costantino group used the photobleaching principle of fluorescein to achieve spatial labeling of cells, but this method is inefficient and has high background signal. The ZipSeq technology developed by the Krummel group uses light to control DNA hybridization on the cell surface, achieving spatially specific DNA barcoding and single-cell sequencing. This method suffers from high background adsorption because it requires labeling single-stranded DNA in cells. The SCARI technology developed by Schumacher's group avoids single-stranded DNA labeling by using photoactivated genetic tags, but it cannot label multiple regions simultaneously, limiting its application.

[0003] Existing methods suffer from problems such as low labeling efficiency, limited number of labeling sites, high background signal, and dependence on genetic manipulation, which restricts their further application in fields such as biology and medicine. Summary of the Invention

[0004] Space-based single-cell transcriptome sequencing requires labeling cells at specific locations in space. However, existing methods suffer from low labeling efficiency, limited number of labeling sites, high background signal, and reliance on genetic manipulation. The aim of this method is to overcome these shortcomings using novel chemical approaches, achieving more efficient and practical space-based single-cell transcriptome sequencing.

[0005] To address the aforementioned problems with existing methods, this invention claims the application of ONPF-biotin in space-based single-cell transcriptome sequencing technology, wherein the structural formula of ONPF-biotin is as follows:

[0006]

[0007] This invention provides a probe for space-based single-cell transcriptome sequencing, the probe being ONPF-biotin, the structural formula of which is:

[0008] The present invention provides a method for preparing the probe of claim 2, comprising the step of adding AzONPF and DBCO-biotin to a mixed solution of dimethyl sulfoxide and water, reacting to obtain ONPF-biotin.

[0009] The molar ratio of AzONPF to DBCO-biotin is 1:1; the reaction conditions are room temperature and 2 hours.

[0010] The preparation method of AzONPF is as follows:

[0011] 1) Dissolve 2-azidoethanol in pyridine, add chloroformyl-(4-nitro)-phenol ester and stir until homogeneous. After reaction and purification, compound 1 is obtained. The structural formula of compound 1 is as follows:

[0012] 2) Compound 1 was dissolved in dichloromethane, and N-tertiary oxycarbonyl-1,2-ethylenediamine and triethylamine were added to the system. The reaction was carried out overnight at room temperature. After the reaction was completed, the product was further extracted and purified to obtain compound 2.

[0013] 3) Compound 2 was dissolved in dichloromethane, trifluoroacetic acid was added, and the mixture was reacted at room temperature to obtain compound 3;

[0014] 4) Methyl 2-hydroxy-5-formylbenzoate was dissolved in dimethylformamide, cesium carbonate was added, and a dimethylformamide solution of o-nitrobenzyl bromide was added under ice bath conditions. The system was then brought to room temperature and reacted overnight. After the reaction was completed, the product was further extracted and purified to obtain compound 4.

[0015] 5) Compound 4 was dissolved in methanol / tetrahydrofuran, sodium borohydride was slowly added under ice bath, and the reaction was continued with stirring. After the reaction was completed, the product was further extracted and purified to obtain compound 5.

[0016] 6) Compound 5 was dissolved in tetrahydrofuran, and an aqueous solution of lithium hydroxide was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the product was further extracted and purified. The resulting white crude product was redissolved in dimethylformamide, and compound 3, LN,N-diisopropylethylamine, hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The mixture was reacted overnight at room temperature. After the reaction was completed, the product was further extracted and purified to obtain a white solid, AzONPOH.

[0017] 7) Dissolve AzONPOH in dichloromethane, slowly add a dichloromethane solution of bis(2-methoxyethyl)aminotrifluoride under ice bath conditions, raise the system to room temperature and react overnight; after the reaction is complete, further extract and purify the product to obtain a white solid AzONPF.

[0018] The present invention also provides a spatial single-cell transcriptome sequencing method based on light-controlled cell labeling, wherein the probe used in the method is ONPF-biotin.

[0019] The method includes the following steps:

[0020] 1) Use ONPF-biotin to label the cells to be sequenced;

[0021] 2) Sorting and single-cell sequencing of ONPF-biotin-labeled cells.

[0022] The marking method in step 1) includes:

[0023] 11) Incubate the cells to be tested with HBSS solution containing ONPF-biotin;

[0024] 12) Remove the solution from the cells incubated in step 11) and irradiate them with a laser; the laser irradiation can be achieved by a laser scanning co-concentration microscope or by other devices containing a 405nm laser, such as a fluorescence microscope;

[0025] 13) After washing the irradiated cells twice, add culture medium containing streptavidin-fluorescent molecule conjugate and incubate at room temperature; after incubation, wash the cells twice more with HBSS solution.

[0026] 9. The method according to claim 8, characterized in that the labeling in step 1) is repeated multiple times, and steps 11)-13) are repeated after the last HBSS solution washing in step 13.

[0027] 10. The method according to claim 8, wherein step 2) specifically includes the following steps:

[0028] 21) Add trypsin to the labeled cells, incubate, remove the trypsin, blow the cells with PBS containing 5% fetal bovine serum and then perform flow cytometry sorting. Clear cell clustering can be observed in the flow cytometry signal. Select and separate the positive colonies.

[0029] 22) The positive cells were sorted into 96-well plates pre-filled with lysis buffer, one cell per well, and the cells were lysed.

[0030] 23) Subsequently, the RNA obtained from each well was reverse transcribed to obtain cDNA;

[0031] 24) Perform PCR amplification on the cDNA products obtained in step 3;

[0032] 25) The PCR products from each well were purified using magnetic beads and then used to construct next-generation sequencing libraries.

[0033] 26) The completed library was quality controlled using 5200Fragment Analyzer;

[0034] 27) After merging the quality-controlled libraries, next-generation sequencing is performed. Approximately 1.4 million reads are generated per sample for subsequent analysis to obtain the spatial single-cell transcriptome sequencing results.

[0035] This invention proposes using a photoactivated methylenebenzoquinone probe (ONPF-biotin) to label cells in specific regions, followed by single-cell sequencing after cell isolation. This method is named OpTAG-seq. Methylenebenzoquinone is a short-lived chemical intermediate with extremely high reactivity. After in situ generation, methylenebenzoquinone can efficiently covalently link to nucleophilic residues of cell surface proteins, achieving in situ cell labeling. This labeling method can be repeated, enabling labeling at multiple spatial locations within the same sample. Using flow cytometry, labeled cells are isolated into microplates for single-cell library construction and sequencing, thus achieving spatial single-cell transcriptome sequencing. In the embodiments of this invention, experiments demonstrate that ONPF-biotin can achieve efficient spatial cell labeling, and combined with flow cytometry sorting, enables spatial single-cell transcriptome sequencing.

[0036] This invention utilizes a novel chemical method to achieve highly efficient light-controlled cell labeling and spatial single-cell transcriptome sequencing, solving problems such as low labeling efficiency, limited number of labeling sites, high background signal, and dependence on genetic manipulation in existing methods. It provides a new generation solution for studying the spatial distribution and regulation of cells in different biological systems. Attached Figure Description

[0037] Figure 1 The synthetic route for ONPF-biotin;

[0038] Figure 2 To utilize OpTAG for multi-round light-controlled labeling of live cells;

[0039] Figure 3 The results of fluorescence imaging using OpTAG for multi-round optically controlled labeling of live cells;

[0040] Figure 4Flowchart for spatial single-cell transcriptome sequencing using OpTAG-seq;

[0041] Figure 5 The image shows the results of detecting cell labeling signals in labeled cells using flow cytometry.

[0042] Figure 6 To analyze gene expression regulation during cancer cell migration using OpTAG-seq.

[0043] Figure 7 To perform immunoblotting analysis on proteins in cell lysates after labeling live cells with ONPF-biotin.

[0044] Figure 8 The fluorescent signals on the cell surface after cell labeling using ONPF-Cy5 (Fig. a) and ONPF-biotin (Fig. b) as probe molecules are shown. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Example 1: Preparation of ONPF-biotin

[0048] ONPF-biotin is prepared according to the following steps, the preparation process of which is as follows: Figure 1 As shown, the structural formula of the ONPF-biotin is... The specific steps are as follows:

[0049] 1) Dissolve 500 mg of 2-azidoethanol in 10 mL of pyridine, add 1.27 mg of chloroformyl-(4-nitro)-phenol ester and stir until homogeneous. React overnight at room temperature. After the reaction is complete, remove the pyridine from the system by rotary evaporation, redissolve in 20 mL of ethyl acetate, wash successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dry the resulting organic phase with anhydrous sodium sulfate and remove the solvent again by rotary evaporation. The crude product is purified by high performance liquid chromatography (water / acetonitrile) to give a white solid compound 1 (563 mg, 39%), the structural formula of which is [insert structural formula here].

[0050] 2) 520 mg of compound 1 was dissolved in 15 mL of dichloromethane. 364 mg of N-tertiary oxycarbonyl-1,2-ethylenediamine and 865 μL of triethylamine were added to the system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the dichloromethane was removed by rotary evaporation, and the mixture was redissolved in 20 mL of water. The mixture was then extracted three times with 60 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and the solvent was removed again by rotary evaporation. The crude product was purified by high-performance liquid chromatography (water / acetonitrile) to give a white solid compound 2 (370 mg, 66%). The structural formula of compound 2 is [insert structural formula here].

[0051] 3) Dissolve 308 mg of compound 2 in 15 mL of dichloromethane, add 4 mL of trifluoroacetic acid to the system, and react at room temperature for 1 hour to obtain compound 3 (162 mg). Compound 3 is a light brown oily substance with the following structural formula: It can be used directly in subsequent steps.

[0052] 4) Dissolve 500 mg of methyl 2-hydroxy-5-formylbenzoate in 20 mL of dimethylformamide. Add 950 mg of cesium carbonate to the system, and add 5 mL of a dimethylformamide solution of o-nitrobenzyl bromide (580 mg) under ice bath conditions. The system is then brought to room temperature and reacted overnight. After the reaction is complete, 50 mL of water is added to quench the reaction, and the mixture is extracted with 3 × 40 mL of ethyl acetate. The organic phases are combined and washed with 2 × 25 mL of water and 2 × 30 mL of saturated sodium chloride solution. The mixture is dried over anhydrous sodium sulfate and the solvent is removed by rotary evaporation. The crude product is purified by column chromatography (petroleum ether / ethyl acetate) to give a white solid compound 4 (700 mg, 82%). The structural formula of compound 4 is [insert structural formula here].

[0053] 5) Dissolve 500 mg of compound 4 in 20 mL of methanol / tetrahydrofuran (1:9), slowly add 34 mg of sodium borohydride under ice bath conditions, and continue the reaction for 30 minutes with stirring. After the reaction is complete, quench the reaction by adding 10 mL of saturated ammonium chloride solution, and extract with 3 × 40 mL of ethyl acetate. Combine the organic phases and wash with 2 × 30 mL of saturated sodium chloride solution, dry with anhydrous sodium sulfate, and remove the solvent by rotary evaporation. The crude product is purified by column chromatography (petroleum ether / ethyl acetate) to give a white solid compound 5 (480 mg, 95%), the structural formula of which is [insert structural formula here].

[0054] 6) Dissolve 170 mg of compound 5 in 10 mL of tetrahydrofuran, add 5 mL of an aqueous solution of 257 mg lithium hydroxide, and react overnight at room temperature. After the reaction is complete, add 20 mL of 1 M hydrochloric acid solution to neutralize the reaction, and extract with 3 × 15 mL of ethyl acetate. Combine the organic phases and wash with 2 × 20 mL of saturated sodium chloride solution. Dry with anhydrous sodium sulfate and remove the solvent by rotary evaporation. The resulting white crude product is redissolved in 10 mL of dimethylformamide, and 100 mg of compound 3, 140 μL of N,N-diisopropylethylamine, 8.0 mg of hydroxybenzotriazole, and 153 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added. React overnight at room temperature. After the reaction was complete, 20 mL of 1 M hydrochloric acid solution was added to neutralize the reaction, and the mixture was extracted with 3 × 20 mL of ethyl acetate. The organic phases were combined and washed with 3 × 20 mL of saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (petroleum ether / ethyl acetate) to give a white solid AzONPOH (128 mg, 53%). The structural formula of AzONPOH is [insert structural formula here].

[0055] 7) Dissolve 110 mg AzONPOH in 8 mL of dichloromethane. Slowly add 2 mL of a dichloromethane solution of bis(2-methoxyethyl)aminotrifluoride (530 mg) under ice bath conditions. Bring the system to room temperature and react overnight. After the reaction is complete, quench the reaction with 20 mL of ice water and extract with 2 × 20 mL of dichloromethane. Combine the organic phases and wash with 3 × 15 mL of saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. Dry with anhydrous sodium sulfate and remove the solvent by rotary evaporation. Purify the crude product by column chromatography (dichloromethane / methanol) to obtain a white solid AzONPF (53 mg, 48%). The structural formula of AzONPF is [insert structural formula here].

[0056] 8) Add 10 mM AzONPF and 10 mM DBCO-biotin to a mixed solution of dimethyl sulfoxide and water, and react at room temperature for 2 hours to obtain a final concentration of 10 mM ONPF-biotin solution. The structural formula of the DBCO-biotin is as follows: The structural formula of ONPF-biotin is:

[0057]

[0058] Example 2: Labeling live cells using ONPF-biotin

[0059] 1. Dilute ONPF-biotin with HBSS solution (Beyotime, CO219) to a working solution concentration of 100 μM. After aspirating the culture medium from HeLa cells (ATCC, CCL-2) cultured in glass-bottom containers, wash twice with HBSS solution, add the working solution, and incubate at room temperature for 5 minutes.

[0060] 2. After incubation, aspirate the solution and place the cells on a Zeiss LSM710 laser scanning confocal microscope. With the aid of software, select the marked area and irradiate with laser using the "Bleach" mode (iterations = 250, laser intensity = 95%, other parameters are default).

[0061] 3. After irradiation, the cells were washed twice with PBS solution containing 5% fetal bovine serum (FBS) (FBS was purchased from Gibco, catalog number 10099; PBS solution was purchased from Solarbio, catalog number P1020. 95 mL of PBS solution was mixed with 5 mL of FBS to obtain a 5% FBS PBS solution). Then, the cells were incubated at room temperature for 5 minutes with a final concentration of 5 μg / mL of streptavidin-fluorescent molecular conjugate (Bioss, bs-0437P) in the PBS solution (i.e., each mL of 5% FBS PBS solution contained 5 μg of streptavidin-fluorescent molecular conjugate).

[0062] 4. After incubation, the cells will be washed twice more with HBSS solution.

[0063] 5. If multiple rounds of labeling are required, after the final HBSS wash, add the working solution to the cells and repeat steps 1-4. The cell labeling procedure and specific results are attached. Figure 2 and 3 As shown.

[0064] Example 3: Sorting and single-cell sequencing of ONPF-biotin-labeled cells

[0065] 1. After labeling, trypsin (Thermo, 25200056) was added to the cells at a final concentration of 0.25%, and the cells were incubated at 37°C for 5 min. The trypsin was removed, and the cells were dispersed with PBS containing 5% fetal bovine serum and then sorted by flow cytometry. Clear cell clustering was observed in the flow cytometry signal; positive colonies were selected and separated. Results are as follows: Figure 5 As shown, Figure 5 The image shows the results of flow cytometry detection of cell labeling signals after ONPF-biotin was used to label live cells. As can be seen from the image, positive colonies were those in which obvious labeling signals were detected by flow cytometry.

[0066] 2. The positive cells from step 1 were sorted into 96-well plates pre-filled with lysis buffer, one cell per well. Each well contained 4 μL of lysis buffer, which included 4 units of Ambion RNase inhibitor (Invitrogen, AM2684), 0.5% Triton X-100 (Sigma, T9284), 2.5 μM reverse transcription primers, and 2.5 mM dNTPs (NEB, N0447). The reverse transcription primer sequence was: 5'-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (V represents A / C / G, N represents A / C / G / T). After cell separation into the wells, the plates were heated to 72°C and held for 3 minutes to lyse the cells.

[0067] 3. Subsequently, the RNA obtained from each well was reverse transcribed. The reverse transcription system consisted of 6 μL RT Mix (containing 10 units of SuperScript II reverse transcriptase (Invitrogen, 18064014), 1 unit of Ambion RNase inhibitor, Superscript II first-chain buffer (Invitrogen, 18064014), 5 mM dithiothreitol, 1 M betaine, 6 mM magnesium chloride, and 1 M TSO primer) and 4 μL of lysis product. The TSO primer sequence was 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrGG-3'. The reverse transcription program was set to 42℃ for 90 minutes, followed by ten cycles of 50℃-42℃, and then 70℃ for 15 minutes.

[0068] 4. Perform PCR amplification on the cDNA product obtained in step 3. The primer sequence is: 5'-AAGCAGTGGTATCAACGCAGAGT-3'.

[0069] 5. PCR products were purified using AMPure XP SPRI magnetic beads (Beckman, A63882). For each cell, 5 ng of cDNA product was used for next-generation sequencing library construction. Library construction was performed using the Vazyme TruePrep DNA LibraryPrep Kit V2 (Vazyme, TD502).

[0070] 6. The constructed libraries were quality controlled using a 5200 Fragment Analyzer. After quality control, the merged libraries underwent next-generation sequencing, with approximately 1.4 million reads per sample for subsequent analysis. The spatial single-cell transcriptome sequencing analysis workflow for cancer cell migration is as follows: Figure 4 As shown, the results are as follows Figure 6 As shown. Figure 6'a' represents the spatial extent of the labeled cancer cells. Highly mobile cancer cells and less mobile cancer cells located further inward were labeled and sorted using the ONPF-biotin probe. Single-cell sequencing of these cells and dimensionality reduction analysis revealed two cell populations, such as... Figure 6 As shown in b. Through differential gene analysis, we identified many genes that were upregulated or downregulated in highly migratory cells, such as Figure 6 As shown in c. Overlaying these genes onto the UMAP map reveals that the genes upregulated in outer cells are indeed more numerous, but unevenly distributed, such as... Figure 6 As shown in d. Functional cluster analysis of genes significantly upregulated in highly migratory cells revealed a significant enrichment of pathways related to cell migration, wound response, and cell motility. Figure 6 e). Two genes were selected from the identified geogene list for immunofluorescence assays, and the results showed a high degree of consistency with the sequencing results. This demonstrates that the method of this invention can achieve sensitive and efficient single-cell transcriptome sequencing in space.

[0071] Example 4: Immunoblot analysis of ONPF-biotin-labeled cells

[0072] Immunoblot analysis of proteins in the cell lysates of the ONPF-biotin-labeled cells prepared in Example 2 showed that a large number of proteins carried biotin signals, such as... Figure 7 As shown.

[0073] Example 5: A comparative example using ONPF-Cy5 as the probe molecule.

[0074] 1. ONPF-Cy5 was prepared in the laboratory by chemical synthesis for later use. The ONPF-Cy5 has the following structural formula: Compounds.

[0075] 2. Following the steps in Example 2, replace ONPF-biotin with ONPF-Cy5 for cell labeling.

[0076] 3. The ONPF-Cy5-labeled cells prepared in step 2 were subjected to flow cytometry analysis to determine the fluorescence signal on the cell surface after labeling, following steps 1 and 2 in Example 3. The results showed that although the Cy5 signal was significantly enhanced after illumination, a high signal was also observed in the unilluminated negative control group. Figure 8a) This is likely due to the adsorption of dye molecules by living cells. In contrast, the ONPF-biotin obtained through probe structure optimization design in Example 3 can effectively label living cells after light exposure, and exhibits almost no background signal in the negative control without light exposure, successfully achieving light-controlled labeling of the surface of living cells. Figure 8 b)

[0077] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of ONPF-biotin as a probe for fluorescent labeling of cells at specific spatial locations in space-based single-cell transcriptome sequencing technology, wherein the structural formula of ONPF-biotin is: .

2. A probe for space-based single-cell transcriptome sequencing, characterized in that, The probe is an ONPF-biotin, and the structural formula of the ONPF-biotin is as follows: .

3. A method for preparing the probe according to claim 2, characterized in that, The process includes adding AzONPF and DBCO-biotin to a mixed solution of dimethyl sulfoxide and water, reacting the mixture to obtain ONPF-biotin. The preparation method of AzONPF is as follows: 1) Dissolve 2-azidoethanol in pyridine, add chloroformyl-(4-nitro)-phenol ester and stir until homogeneous. After reaction and purification, compound 1 is obtained. The structural formula of compound 1 is as follows: ; 2) Compound 1 was dissolved in dichloromethane, and N-tertiary oxycarbonyl-1,2-ethylenediamine and triethylamine were added to the system. The reaction was carried out overnight at room temperature. After the reaction was completed, the product was further extracted and purified to obtain compound 2. The structural formula of compound 2 is as follows: ; 3) Compound 2 was dissolved in dichloromethane, and trifluoroacetic acid was added. The reaction was carried out at room temperature to obtain compound 3. The structural formula of compound 3 is as follows: ; 4) Methyl 2-hydroxy-5-formylbenzoate was dissolved in dimethylformamide, cesium carbonate was added, and a dimethylformamide solution of o-nitrobenzyl bromide was added under ice bath conditions. The system was then brought to room temperature and reacted overnight. After the reaction was completed, the product was further extracted and purified to obtain compound 4, the structural formula of which is [insert structural formula here]. ; 5) Compound 4 was dissolved in methanol / tetrahydrofuran, and sodium borohydride was slowly added under ice bath conditions. The reaction was continued with stirring. After the reaction was completed, the product was further extracted and purified to obtain compound 5. The structural formula of compound 5 is as follows: ; 6) Compound 5 was dissolved in tetrahydrofuran, and an aqueous solution of lithium hydroxide was added. The mixture was reacted overnight at room temperature. After the reaction, the product was further extracted and purified. The resulting white crude product was redissolved in dimethylformamide, and compound 3, N,N-diisopropylethylamine, hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The mixture was reacted overnight at room temperature. After the reaction, the product was further extracted and purified to obtain a white solid, AzONPOH. The structural formula of AzONPOH is [insert structural formula here]. ; 7) Dissolve AzONPOH in dichloromethane, and slowly add a dichloromethane solution of bis(2-methoxyethyl)aminosulfur trifluoride under ice bath conditions. Raise the system to room temperature and react overnight. After the reaction is complete, further extract and purify the product to obtain a white solid, AzONPF. The structural formula of AzONPF is [insert structural formula here]. .

4. The preparation method according to claim 3, characterized in that, The molar ratio of AzONPF to DBCO-biotin was 1:1; the reaction conditions were room temperature and 2 hours.

5. A spatial single-cell transcriptome sequencing method based on light-controlled cell labeling, characterized in that: Includes the following steps: 1) Labeling the cells to be sequenced using the ONPF-biotin as described in claim 2; 2) Sorting and single-cell sequencing of ONPF-biotin-labeled cells.

6. The method according to claim 5, characterized in that, The marking method in step 1) includes: 11) Incubate the cells to be tested with HBSS solution containing ONPF-biotin; 12) Remove the solution from the cells incubated in step 11) and irradiate them with laser; 13) After washing the irradiated cells twice, add culture medium containing streptavidin-fluorescent molecular conjugate and incubate at room temperature; after incubation, wash the cells twice more with HBSS solution.

7. The method according to claim 6, characterized in that, The labeling in step 1) is repeated multiple times. After the last HBSS solution wash in step 13), steps 11)-13) are repeated.

8. The method according to claim 5, characterized in that, Step 2) specifically includes the following steps: 21) Add trypsin to the labeled cells, incubate, remove the trypsin, blow the cells with PBS containing 5% fetal bovine serum and then perform flow cytometry sorting. Select and separate the positive colonies that can be clearly observed in the flow cytometry signal. 22) The positive cells were sorted into 96-well plates pre-filled with lysis buffer, one cell per well, and the cells were lysed. 23) Subsequently, the RNA obtained from each well was reverse transcribed to obtain cDNA; 24) Perform PCR amplification on the cDNA products obtained in step 23); 25) The PCR products from each well were purified using magnetic beads and then used to construct next-generation sequencing libraries. 26) The completed library was quality controlled using 5200 Fragment Analyzer; 27) After merging the quality-controlled libraries, next-generation sequencing is performed. Approximately 1.4 million reads are generated per sample for subsequent analysis to obtain the spatial single-cell transcriptome sequencing results.

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