A method for detecting dynamic changes of metal ions based on nucleic acid aptamer single-cell sequencing

Abstract

The invention discloses a detection method (scIon‑seq) for the dynamic changes of single-cell ions based on nucleic acid aptamers, comprising: anchoring a capture probe modified by hydrophobic lipids to the cell membrane surface, and when metal ions are released from the cell to the extracellular space, a detection probe with a sequencing adapter forms a secondary structure with the capture probe on the membrane by interchain folding in the presence of the metal ion; then in a single-cell cell suspension system, high-throughput sequencing is carried out on the detection probe with a sequencing adapter in the secondary structure, and the dynamic change information of metal ions on the single-cell level can be obtained. The present invention utilizes the ability of nucleic acid aptamers to identify metal ions with high specificity and the characteristics of compatible high-throughput sequencing, and provides a research method for studying the physiological functions of metal ions at the single-cell level at a technical level, which is conducive to promoting the development of multiple omics such as nucleic acid aptamers in studying metabolomics, proteomics and transcriptomics at the single-cell level.

Description

Technical Field

[0001] The present invention relates to the technical field of metal ion detection, and in particular to a method for detecting dynamic changes of metal ions based on nucleic acid aptamer single-cell sequencing. Background Art

[0002] The emergence of single-cell high-throughput sequencing technology has triggered a revolutionary shift in research methods in the fields of genomics and transcriptomics. Single-cell transcriptome sequencing can reveal unprecedented cellular diversity. Although RNA and proteins provide important insights into cell function, potassium ions, as one of the most abundant metal ions in cells, play an important role in regulating cell potential and volume, affecting cell proliferation and apoptosis, neural signal hyperpolarization, myocardial cell homeostasis, cell osmotic pressure and other physiological processes. In addition, potassium ions (K + ) also plays a role in maintaining normal muscle function, enzyme activity, and physiological pH. Therefore, unbiased quantitative analysis of the ionome has the potential to complement single-cell ionomics and provide a more comprehensive description and characterization of different cellular states.

[0003] However, current analytical methods have limited ability to monitor potassium ion homeostasis at the single-cell level, hindering a deeper understanding of potassium ion dynamics at the single-cell level. Currently commonly used ion-selective electrodes achieve direct detection of ion concentrations with high temporal and spatial resolution, but are susceptible to interference from other ions, require frequent calibration, and have a short service life. Although small molecule-based fluorescent probes have achieved non-invasive and easy-to-operate ion monitoring methods, their accuracy is limited by fluorescence signal interference and photobleaching. Due to multiple interferences in the biological microenvironment, only a few fluorescent probes have been used for potassium ion detection under physiological conditions. Importantly, although these methods provide K + However, the obtained signals only reflect the average state of complex biological samples (such as tissue or blood), masking the individual and specific dynamics of single cells.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a method for detecting dynamic changes in metal ions based on single-cell sequencing using nucleic acid aptamers, termed scIon-seq. This method overcomes the limitations of antibodies in ion recognition and can effectively detect ion changes at the single-cell level.

[0006] In a first aspect, the present invention provides a method for detecting the dynamic changes of metal ions in a single cell based on a split nucleic acid aptamer. The split nucleic acid aptamer comprises a first part and a second part, the first part being a capture probe and the second part being a detection probe;

[0007] The detection method includes: anchoring the capture probe on the membrane surface of living cells. When metal ions are released from the living cells to the outside of the cells, the metal ions act as cofactors to promote the combination of the detection probe and the capture probe to form a stable complex; then, in a single-cell cell suspension system, high-throughput sequencing of the detection probe in the complex structure can be performed to obtain the dynamic changes of metal ions on the cell membrane surface.

[0008] In a second aspect, the present invention provides a split nucleic acid aptamer, which includes a first part and a second part, the first part is a capture probe, and the second part is a detection probe; the capture probe is composed of a hydrophobic lipid based on a double alkyl chain coupled to a nucleic acid aptamer; the detection probe is composed of a nucleic acid aptamer with a sequencing adapter extended at both ends; the sequencing adapter is a double-ended adapter, including Poly A at the 5' end and a capture sequence at the 3' end.

[0009] In a third aspect, the present invention provides a detection kit for the dynamic changes of metal ions in single cells, which includes the above-mentioned split nucleic acid aptamer.

[0010] In a fourth aspect, the present invention provides a detection kit for the dynamic changes of potassium ions in a single cell, which comprises the above-mentioned split nucleic acid aptamer, wherein the nucleotide sequence of the capture probe is as shown in SEQ ID NO: 1, and the nucleotide sequence of the detection probe is as shown in SEQ ID NO: 2.

[0011] In a fifth aspect, the present invention provides the use of the above-mentioned detection kit in the preparation of a product for detecting the dynamic changes of metal ions in single cells.

[0012] In a sixth aspect, the present invention provides the use of the above-mentioned detection kit in the preparation of a product for analyzing potassium ion changes in the cellular microenvironment of samples from colorectal cancer patients.

[0013] The present invention has the following beneficial effects:

[0014] This method leverages the specific recognition ability of split-type aptamers for metal ions. With the target ion as a cofactor, the two sequences of the split-type aptamer bind to the ion to form a complex, and its compatibility with high-throughput sequencing allows it to determine ion changes in the microenvironment of a single cell. This method overcomes the bottleneck of traditional single-cell high-throughput sequencing technology, which is unable to detect ions in biological samples. It provides a technical means for understanding the physiological functions of ions at the single-cell level and lays the foundation for the future development of aptamers in the study of signal transduction, transcriptomics, and metabolomics at the single-cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of using scIon-seq to monitor the dynamic changes of potassium ions in the microenvironment of colorectal cancer cells;

[0017] Figure 2 The capture probe labeled with Cy3 and the detection probe labeled with Cy5 in Experimental Example 1 were used to generate the gradient K + Response (a) and experimental results of its specificity (b);

[0018] Figure 3 This is the synthetic route of the hydrophobic lipid and its modified capture probe in Experimental Example 2;

[0019] Figure 4 HPLC purification (a) and characterization results (b) of the hydrophobic lipid-modified capture probe in Experimental Example 2;

[0020] Figure 5 The split aptamer monitoring K on the cell membrane in Experimental Example 3 + Characterization results; (a) Working principle: Cy3-labeled capture probe is anchored on the cell membrane. + With the increase of the concentration, the free Cy5-labeled detection probe is captured by the capture probe to form a complex structure with higher stability; (b) Flow cytometry analysis shows that the free Cy5-labeled detection probe is captured by the capture probe at different K values from 0 mM to 100 mM. + K at concentration + dependent response; (c) Fluorescence intensity statistics corresponding to Figure (b); Statistical data were obtained from three independent experiments and expressed as mean ± SD; (d) K + CLSM imaging under ; scale bar, 20 μm;

[0021] Figure 6 The fluorescence characterization results of potassium ion efflux in living cells stimulated by drugs in Experimental Example 4; (a) Based on the split-type nucleic acid aptamer probe, the K + The generated K +Detection schematic diagram; (b) Flow cytometric analysis results of cells treated with three drug combinations (amphotericin B + nigericin sodium salt + bumetanide) and four drug combinations (strophanthin G + amphotericin B + nigericin sodium salt + bumetanide); (c) Fluorescence intensity statistics corresponding to Figure (b); (d) CLSM imaging of LoVo cells; scale bar, 10 μm; (e) Quantitative analysis based on CLSM imaging; (f) Commercial K + Detection principle of fluorescent probe; (g) Commercial K + Titration curves of fluorescent probes and K values after treatment of cells with 3 or 4 drugs + Concentration analysis; Statistical data were obtained from three independent experiments and expressed as mean ± standard deviation;

[0022] Figure 7 For the cell line described in Example 1, nucleic acid aptamers can be qualitatively and quantitatively identified by single-cell high-throughput sequencing; (a) Violin plot showing the quality control results of single-cell sequencing; (b) Correlation of gene expression after nucleic acid aptamer treatment; (c) Violin plot showing analysis of gene expression in cells that were untreated, treated with a random control sequence, or treated with nucleic acid aptamers; (d) Box plot showing sequence counts between nucleic acid aptamer sgc8c-treated or control sequence-treated groups; (e) Box plot showing sequence counts between split nucleic acid aptamer sgc8c-treated, random control sequence-treated, or split nucleic acid aptamer-treated groups;

[0023] Figure 8 Figure 2 shows the detection of dynamic changes in ions in cells of colorectal cancer samples using scIon-seq; (a) scIon-seq process analysis diagram; (b) transcriptome-based clustering of monocyte expression profiles reveals different cell populations; (c) marker gene expression in each cluster; (d) feature map showing the number of split-type nucleic acid aptamers in each cluster; (e) box plot showing sequence counts of nucleic acid aptamer sgc8c, random control sequences, and split-type nucleic acid aptamers between the three-drug treatment group, the four-drug treatment group, or the no-drug treatment group. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0025] Current single-cell sequencing technologies are able to detect the phenotype of each cell in a complex population, typically through antibody-based proteomics and DNA barcode-based genomics and transcriptomics, to analyze macromolecules such as nucleic acids and proteins at the single-cell level. However, these methods are unable to detect changes in ions at the single-cell level.

[0026] Aptamers, produced through exponential enrichment ligand system evolution, can be screened in a short period of time. They are DNA or RNA oligonucleotides and have become powerful tools for biomarker discovery, bioimaging, molecular diagnostics, and targeted therapy. As identification tools, the principle of aptamer detection is as follows: Aptamers bind to their targets based on the diversity of single-stranded nucleic acid structures and spatial conformations. Through pairing, electrostatic interactions, hydrogen bonding, and van der Waals forces between complementary bases within the chain, adaptive folding occurs, forming stable structures such as hairpins, pseudoknots, and convex loops. These structures bind to the target molecule, exhibiting high affinity for the target.

[0027] Split aptamers are a type of aptamer that typically splits a complete aptamer into two or more fragments. These fragments then recombine in the presence of a target molecule to form a complex with high affinity and specificity. These split aptamers, composed of two or more short nucleic acid chains, have become a new tool in biosensor devices.

[0028] Based on this, the present invention proposes a method for detecting the dynamic changes of single-cell ions based on split-type nucleic acid aptamers by modifying nucleic acid aptamers. The method uses the characteristic that split-type nucleic acid aptamers will bind to specific ions to form complexes in the presence of specific targets, thereby determining the specific ion changes in the microenvironment of a single cell. This method combines single-cell sequencing with ion sensing methods based on nucleic acid aptamers for the first time, providing new ideas for understanding the physiological functions of ions at the single-cell level and enabling a deeper understanding of the potential mechanisms of therapeutic intervention. At the same time, this technology is compatible with commercial single-cell RNA sequencing platforms and can achieve mRNA, protein and ion detection in the same sample.

[0029] Specifically, the present invention provides a method for detecting the dynamic changes of single-cell ions based on nucleic acid aptamers (scIon-seq), which includes: the split nucleic acid aptamer includes a first part and a second part, the first part is a capture probe, and the second part is a detection probe; the detection method includes: anchoring the capture probe to the membrane surface of a living cell, and when metal ions are released from the living cell to the outside of the cell, under the action of the metal ions, the detection probe will combine with the capture probe to form a stable complex structure; then, in a single-cell cell suspension system, by performing high-throughput sequencing on the detection probe in the complex structure, the dynamic changes of metal ions on the cell membrane surface can be obtained.

[0030] The present invention utilizes the above-mentioned characteristics of split nucleic acid aptamers that can specifically recognize ions, and selects a split nucleic acid aptamer having a first part and a second part for ion detection. When the target ion is present in the detection system, the first part and the second part undergo structural folding in the presence of the metal ion to form a split nucleic acid aptamer with a sequencing barcode. The complex is then tested on a machine to obtain the status of the metal ions in the cell, wherein the on-machine detection is an existing commercial single-cell RNA sequencing detection method.

[0031] Potassium ion is one of the most abundant metal ions in cells. It plays an important role in regulating cell potential, regulating cell volume, affecting cell proliferation and apoptosis, nerve signal hyperpolarization, myocardial cell homeostasis, cell osmotic pressure and other physiological processes. It also plays a role in maintaining normal muscle function, enzyme activity and physiological pH. In order to verify the effect of the detection method of the present invention, K + As an example, a method for detecting the dynamic changes of potassium ions in single cells based on nucleic acid aptamers is further provided. This method is based on the above method to screen nucleic acid aptamers that can specifically bind to K + The split nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 1-2.

[0032] K + The mechanism of action of nucleic acid aptamers is related to the formation of the G-quadruplex structure. G-quadruplex: four guanines in the same plane are bonded by Hoogsteen hydrogen bonds; G-tetrad has a large π surface, and due to π-π stacking, G-tetrads also tend to stack with each other to form a G-quadruplex structure. The G-quadruplex structure is in K + 、Na + NH4 + The present invention utilizes this characteristic of G-quadruplex to detect potassium ions. The split nucleic acid aptamer used is the split G-quadruplex below. The two parts of the split G-quadruplex probe are represented by G4-a and G4-b. Figure 1 Demonstrate the detection principle of the present invention:

[0033] Figure a) shows the use of split nucleic acid aptamers to detect cell microenvironment K + Schematic diagram of the process. In which the lipid-labeled capture probe is anchored on the cell membrane. + After eluting from the cell, the free detection probe is activated by the cofactor K + In the presence of , a complete G-quadruplex structure is formed with the capture probe. By performing single-cell sequencing on the detection probe, changes in potassium concentration at the single-cell level can be obtained.

[0034] Figure b) is the use of scIon-seq to monitor K + The process follows. After isolating cells from colorectal cancer patients, capture probes are anchored to the cell membrane. The cells are then stimulated with drugs and detection probes are added. Finally, scIon-seq is performed to visualize ion changes in cells isolated from blood from colorectal cancer patients.

[0035] The above detection methods are not limited to single cell K + The detection of dynamic changes can also be used for other common metal ions, such as Na + , Ca 2+ and Mg 2+ When other metal ions are detected using the detection method of the present invention, it is only necessary to select a suitable nucleic acid aptamer based on the binding mechanism between the ion and the nucleic acid aptamer.

[0036] For the above-mentioned split-type nucleic acid aptamer, those skilled in the art can select it according to the target ion to be detected. The present invention does not limit this. As long as it is a nucleic acid aptamer that can specifically bind to the target ion, it can be used.

[0037] In this invention, the first capture probe consists of two components: a hydrophobic lipid and an aptamer. Specifically, the capture probe is a covalent conjugate of the hydrophobic lipid and the aptamer. During detection, the hydrophobic lipid anchors the cell membrane; the aptamer in the capture probe specifically binds to the aptamer in the other component.

[0038] The hydrophobic lipid is a molecule containing two relatively long carbon chains, typically linear or branched alkane groups. These two carbon chains can be the same or different. The present invention does not limit the number of carbon atoms in these two carbon chains, as long as they can be anchored to the cell membrane during detection. In some embodiments, the capture probe uses a C18 dialkyl chain.

[0039] In the present invention, the second part of the detection probe is composed of a sequencing adapter and a nucleic acid aptamer. Specifically, a sequencing adapter sequence is extended at both ends of the nucleic acid aptamer. The sequencing adapter here can be a sequencing adapter commonly used in the art, and the present invention does not limit this.

[0040] In some embodiments, the detection probe is connected to sequencing adapters at both ends, specifically the 5' end is connected to the capture sequence (SEQ ID NO: 3-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaaa), and the 3' end is connected to PloyA. By connecting these sequencing adapters, the complex formed by the split-type nucleic acid aptamer and the target ion can be more conveniently detected on a machine.

[0041] Furthermore, the present invention uses nucleated cells isolated from the peripheral blood of colorectal cancer patients as samples for potassium ion analysis. The experimental results show that the detection method of the present invention can identify changes in potassium ions in each cell type in colorectal cancer PBMC samples. The detection method of the present invention may not only play a key role in understanding the biological processes of signal transduction and metabolomics, but also help to reveal the pathological mechanisms of these diseases, potentially providing new diagnostic and therapeutic targets.

[0042] Based on the above detection method, a single cell metal ion dynamic change detection kit can be obtained, which contains a split nucleic acid aptamer. The split nucleic acid aptamer contained in the kit can be a split nucleic acid aptamer that can specifically bind to Na + , Ca 2+ Mg 2+ or K + It can also be a nucleic acid aptamer that binds to other ions. As long as it is a split nucleic acid aptamer that can detect ion changes at the single-cell level, it falls within the scope of protection of the present invention.

[0043] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0044] The nucleic acid sequences purified by HPLC used in the following experiments were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0045] The following experiments involved calculation of fluorescence resonance energy transfer (FRET) efficiency: The ratio of the fluorescence intensity of Cy3 (FD, donor) to Cy5 (FA, acceptor) was defined as the FRET ratio (FA / FD) to evaluate the energy transfer efficiency.

[0046] The nucleic acid aptamers and their sequences used in the above experiments are shown in Table 1:

[0047] Table 1 Nucleic acid aptamers and their sequence information

[0048]

[0049] Experimental Example 1

[0050] This experiment is a fluorescence spectrum test of the potassium ion concentration-dependent split aptamer. A Poly A segment and a capture sequence (see Table 1) were added to both ends of the second part of the split aptamer. The K ion concentration of the Cy3-labeled capture probe and the Cy5-labeled detection probe were examined using a fluorescence spectrophotometer. + dependent complex formation to assess K + Concentration dependence and specificity of the probe.

[0051] like Figure 2As shown in a, with K + As the concentration increases, the FRET value becomes larger and larger, which proves that the above selected sequences have + Split aptamers were formed in the presence of β-catenin and the activity was concentration-dependent.

[0052] The specificity of the split nucleic acid aptamer to different ions was then verified by measuring the fluorescence intensity of different samples containing 20mM concentrations such as NaCl, NH4Cl, CaCl2, and MgCl2.

[0053] The experimental results are as follows Figure 2 As shown in b, the selected Cy3-labeled capture probe and Cy5-labeled detection probe are K + It has good specificity and provides a basis for subsequent single-cell sequencing-based K + Testing provides strong protection.

[0054] Experimental Example 2

[0055] This experiment is based on the lipid synthesis of phosphoramidite monomers and the synthesis route and characterization of lipid-modified capture probes. Figure 3 As shown, the details are as follows:

[0056] 1-iodooctadecane (5 g, 13.1 mmol), N,N-diisopropylethylamine (DIPEA) (3.40 g, 26.2 mmol) and 4-aminophenol (0.72 g, 6.6 mmol) were dissolved in 50 mL of anhydrous DMF and the mixture was stirred at 110 ° C. The reaction was monitored by thin layer chromatography (TLC). After 2 h of reaction, the mixture was allowed to cool to room temperature, then washed with saturated NaHCO (200 mL) and extracted three times with 150 mL of dichloromethane. The organic layer was collected and dried over anhydrous Na2SO4. The solvent was removed by rotary evaporation and then separated by flash chromatography. Finally, 3.19 g of compound (2) was obtained as a light brown solid product, which was directly used in the next step after purification.

[0057] Synthesis of compound lipid phosphoramidite (3). Under nitrogen protection, 0.7g of compound 2 (1.1mM) was dissolved in 20mL of anhydrous dichloromethane and DIPEA (0.3g, 2.3mmol) was added. The bottle was placed in an ice bath and 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (0.4g, 1.7mmol) was added dropwise. The reaction was then slowly warmed to room temperature and stirred for another 1h. The solvent was then removed using a rotary evaporator and finally purified by silica gel chromatography to obtain 0.7g of colorless solid phosphoramidite lipid monomer (3) with a yield of 75.4%.

[0058] The parameters of the H NMR spectrum are: 1H NMR (400MHz, Chloroform-d): δ6.89 (d, J=9.0, 1.3Hz, 2H), 6.55 (d, J=9.1Hz, 2H), 3.91 (dq, J=8.3, 6.6Hz, 2H), 3.78-3.60 (m, 2H), 3. 25-3.11(m,4H),2.64(td,J=6.6,2.0Hz,1H),2.56-2.39(m,1H),1.52(td,J=6.9,3.3Hz,2H),1.41-1.15(m,72H),0.97-0.78(m,6H). 13 C NMR (400MHz, Chloroform-d): δ144.56,144.25,144.17,120.94,120.86,117.57,113.23,58.95,58.76,51.63,48.54,4 3.64,43.51,39.13,31.97,29.76,29.72,29.68,29.61,29.42,27.28,24.71,24.64,24.46,24.39,22.73,20.66,14.15. 31 P NMR (400MHz, Chloroform-d): δ146.902ppm.

[0059] The phosphoramidite monomer synthesized above was dissolved in ultra-dry dichloromethane and coupled to the 5' end of the oligonucleotide via solid-phase synthesis. The oligonucleotide was then cleaved from the corresponding controlled-pore glass. The cleaved DNA product was mixed with 1 mL of ice-cold ethanol and 40 μL of 3 M NaCl and precipitated in -20°C ethanol. The DNA product was then centrifuged at 12,000 rpm for 20 minutes at 4°C. Finally, the 4,4'-dimethoxytrityl group was removed from the DNA by adding 80% aqueous acetic acid and precipitated in cold ethanol. The precipitate was dissolved in 400 μL of 0.1 M triethylamine acetate (TEAA) and purified by HPLC under the following conditions: C4 column (BioBasic-4, 200 mm × 4.6 mm, Thermo Scientific), 25°C; flow rate: 3.0 mL / min; eluent A: 0.1 M TEAA (pH 7.0); eluent B: acetonitrile; linear gradient, 5%-65% B over 30 min. The purified product was lyophilized using a freeze-drying centrifuge, dissolved in ultrapure water, desalted using a DNA desalting column, lyophilized again, dissolved in sterile water, and used.

[0060] The synthetic route is to modify the phosphoramidite monomer at the 5' end of DNA through solid phase synthesis. After modification, sterile water is added to dilute the reaction system to reduce the organic phase ratio to less than 15%. The reaction is filtered and purified by HPLC. The chromatographic peaks at different time periods are collected and characterized by mass spectrometry.

[0061] The results are as follows Figure 4 As shown in a, the peak with a retention time of about 25 min is the product peak of the capture probe. The theoretical molecular weight of the obtained capture probe is 11004.1, and the actual molecular weight obtained is 11001.7 ( Figure 4 b) The molecular weight error is within 0.03%.

[0062] Experimental Example 3

[0063] In this study, fluorescence was used to characterize the effect of added K + To promote the formation of split aptamers on the cell membrane, the operation process is as follows:

[0064] S1. After LoVo cells grow to a suitable ratio in DMEM medium, use PBS (without K + ) Wash the cells three times to reduce the K + interference;

[0065] S2. Add 500 nM capture probe to serum-free DMEM medium and incubate with cells for 30 min to anchor the capture probe to the cell surface;

[0066] S3. After washing away the unanchored probes with PBS, the cells were incubated with K + 500 nM detection probe was added to PBS and incubated at room temperature for 1 h;

[0067] S4. Wash the cells once with PBS and perform flow cytometry and confocal imaging, respectively.

[0068] Split-type nucleic acid aptamers on cell membranes monitor K + The working principle is as follows Figure 5 As shown in a. + In this case, the Cy3-labeled capture probe and the Cy5-labeled detection probe form a complex structure, and thus a FRET signal can be observed. Figure 5 As shown in b and 5c, the flow cytometry results showed that: + As the concentration increases, the FRET signal becomes stronger, indicating that more and more detection probes are captured. + Promotes the formation of complex structure. Figure 5As shown in d, the confocal imaging results also confirmed the flow cytometry results, verifying that the probe has the ability to inhibit K + Among them, the Dio channel (cell membrane) and Cy5 can overlap well, indicating that the split aptamer is located on the cell membrane surface.

[0069] Experimental Example 4

[0070] In this experiment, the addition of drugs stimulated the intracellular K + Fluorescence verification test of efflux to promote complex structure formation.

[0071] In the presence of external drug stimulation, cell K + In the case of efflux, the FRET signal generated by the Cy3-labeled capture probe and the Cy5-labeled detection probe was observed for verification. The drug combinations included: a 3-drug group (amphotericin B + nigericin sodium salt + bumetanide) and a 4-drug group (strophanthin G + amphotericin B + nigericin sodium salt + bumetanide). The experimental roadmap for the dynamic detection of K+ in living cells stimulated by drugs is shown below. Figure 6 As shown in a, the steps are as follows:

[0072] S1. After the LoVo cells grow to an appropriate proportion in DMEM medium, wash the cells three times with PBS to reduce the K + interference.

[0073] S2. Add 500 nM capture probe to serum-free DMEM medium and incubate with cells for 30 min to anchor the capture probe on the cell surface.

[0074] S3. After washing away the unanchored probes with PBS, 500 nM detection probes were added to DMEM buffer containing 10% fetal bovine serum, and the cells were pretreated with 20 μM of different drug combinations for 3 h.

[0075] S4. Wash the cells once with PBS and perform flow cytometry and confocal imaging, respectively.

[0076] like Figure 6 As shown in b and 6c, the flow cytometry results showed that: + As the concentration increases, the FRET signal becomes stronger, indicating that more and more detection probes are captured, and at the same time, the K + Promotes the formation of complex structure. Figure 6 As shown in Figures 6d and 6e, the confocal imaging results also confirmed the flow cytometry results. Among them, the Dio channel (cell membrane) and Cy5 can overlap well, indicating that the addition of drugs does not affect the localization of the probe on the cell membrane surface. Subsequently, we used the commercial probe IPG-4TMA +The concentration of K+ excreted from cells by drugs was quantitatively investigated. The results showed that: + The K released into the environment was detected by adding the above three drug groups and four drug groups into the buffer solution. + The concentrations reached 0.6mM and 0.9mM ( Figure 6 f and 6g).

[0077] Example 1

[0078] This embodiment is a single cell K + The specific operation method of dynamic change detection method is as follows:

[0079] 1. Wash the cells twice with 1×DPBS, collect the digested cells into a 15 mL centrifuge tube, centrifuge at 500 g for 5 min, discard the supernatant, wash once with 1×DPBS and collect the cells at the bottom of the tube. Add an appropriate amount of Binding Buffer according to the cell density and resuspend. Take 1 million cells in 400 μL system, add 200 nM nucleic acid aptamer, incubate on ice for 30 min, and centrifuge at 500 g for 5 min at room temperature. Discard the supernatant and wash the cells 1-2 times with washing buffer solution.

[0080] 2. Prepare the cell suspension system: Add 24 μL of mixed Cell Solution, 28 μL of mixed Index Carrier, 4 μL of mixed RNase Inhibitor, and the cell suspension of 22,000 cells (make up to 24 μL with Binding Buffer) to a 0.2 mL low-adhesion PCR tube, mix well, and place on ice until use.

[0081] 3. Prepare the magnetic bead suspension system: add 100 μL of mixed Cell Beads-V2 to a 0.2 mL low-adsorption PCR tube, place it on a magnetic stand and let it stand for 3-5 minutes. Discard the supernatant and add 72 μL Lysis Buffer and 8 μL DIR Reagent-V2. Mix well and place on ice until ready to use.

[0082] 4. 80 μL of cell suspension, 800 μL of P50 Oil, and 80 μL of magnetic bead suspension were sequentially added to the slide, and then placed in a single-cell droplet generator (C4, manufactured by BGI) for droplet generation.

[0083] 5. After droplet collection stops, let it stand at room temperature for 20 minutes before performing the demulsification procedure: Rinse the C4 filter with 6×SSC, pour the droplet formation solution onto the C4 filter, and wash the magnetic beads in the C4 filter with 6×SSC two to three times. Transfer the C4 filter to a 50mL centrifuge tube, add 700μL of Collection Buffer, and gently pipette the beads off the surface of the filter membrane into a 1.5mL centrifuge tube. Add another 700μL of Collection Buffer and gently pipette the beads off the surface of the filter membrane into the 1.5mL centrifuge tube. Place the tube on a magnetic stand and let it stand for 3 to 5 minutes. Discard the supernatant, remove the centrifuge tube from the magnetic stand, add 200μL of 6×SSC, and mix thoroughly by pipetting. Divide the tube equally into two 0.2mL PCR tubes, approximately 100μL per tube. Repeat adding 200 μL of 6×SSC to the above 1.5 mL centrifuge tube and mix by pipetting. Divide the mixture evenly into the two 0.2 mL PCR tubes mentioned above. Then place the tubes on a magnetic stand and let them stand for 3-5 minutes, then discard the supernatant.

[0084] 6. In a PCR tube, mix 87.5 μL of RT Buffer, 5 μL of RT Primer-V2, 5 μL of RT Enzyme, and 2.5 μL of RNase Inhibitor. Place the tube in a PCR instrument and incubate at 42°C for 90 min. Follow this with 10 cycles of incubation at 50°C for 2 min, incubation at 42°C for 2 min, incubation at 65°C for 10 min, and then hold at 4°C.

[0085] 7. After the reaction is complete, discard the supernatant, add 200μL Wash Buffer and mix well. Let it stand on the magnetic rack for 3-5 minutes and discard the supernatant. Add 10μL D Buffer, 5μL D Enzyme, and 85μL NF Water (a total of 100μL) to the above PCR tube and mix well. Centrifuge briefly and incubate in a PCR instrument at 37℃ for 15 minutes.

[0086] 8. After discarding the supernatant, add 200 μL Stop Buffer and mix to terminate the reaction. Centrifuge briefly, place on a magnetic rack, let stand for 3-5 minutes, and discard the supernatant (hereinafter referred to as "instant supernatant"). Add 200 μL Wash Buffer, wash once, and discard the supernatant. Add 200 μL 1× Denaturation Buffer and mix. Incubate at room temperature for 5 minutes, and discard the supernatant briefly. Keep the centrifuge tube on the magnetic rack, add 200 μL Wash Buffer, let stand for 2 minutes, and discard the supernatant. Add 100 μL of Second Strand Buffer-V2 (67.5 μL), Second Strand Primer-V2 (7.5 μL), and Second Strand Enzyme-V2 (25 μL) to the above PCR tube, mix, centrifuge briefly, and incubate in a PCR instrument at 25°C for 10 minutes, incubate at 37°C for 30 minutes, and then hold at 4°C.

[0087] 9. After discarding the supernatant, add 200μL Stop Buffer to each tube and mix to terminate the reaction. Discard the supernatant briefly and add 200μL Wash Buffer to wash once. Discard the supernatant briefly and add 100μL of cDNA Amp Enzyme 50μL, cDNA Amp Primer-V2 4μL, Suspension Reagent-V2 10μL, and NF Water 35μL to the above PCR tubes and mix. After centrifugation, place the tubes in a PCR instrument with a heated cover at 105℃ and incubate at 95℃ for 3min. Incubate at 98℃ for 20s, 60℃ for 30s, and 72℃ for 3min for 18 cycles. Incubate at 72℃ for 5min and then hold at 4℃.

[0088] 10. After the reaction, centrifuge the two tubes of PCR products and place them on a magnetic stand for 3-5 minutes. Take 95 μL of supernatant from each tube and combine the two tubes into a new 1.5 mL centrifuge tube, totaling 190 μL. Add 114 μL of DNA Clean Beads and mix well. Incubate at room temperature for 10 minutes. Centrifuge briefly and place on a magnetic stand for 2-5 minutes (hereinafter referred to as "snap and stand"). Transfer the supernatant to a new 1.5 mL centrifuge tube and mark it as Oligo Product 1. Further purify the above-adsorbed magnetic beads. Keep the centrifuge tube on the magnetic stand and add 500 μL of freshly prepared 80% ethanol. Let it stand for 30 seconds. Discard the supernatant. Repeat the 80% ethanol wash and completely discard the supernatant. Open the tube cap and dry it at room temperature. Add 32 μL of NF Water and mix well. Incubate at room temperature for 5 minutes. Centrifuge briefly and let it stand. Transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube and mark it as cDNA product. Take 1 μL The concentration of cDNA products was determined using the Qubit™ dsDNA HS Assay Kit.

[0089] 11. Based on the cDNA product concentration, transfer 150 ng of product to a 0.2 mL PCR tube. Place the PCR tube on ice. Pipette 10 μL of Frag Buffer-V2 and 5 μL of Frag Enzyme-V2 (a total of 15 μL) into the tube and mix thoroughly. Centrifuge briefly and place the tube in a PCR instrument precooled to 4°C. Incubate at 30°C for 9 minutes, then at 65°C for 20 minutes, and then maintain at 4°C.

[0090] 12. Add 20 μL of Ligation Buffer-V2, 5 μL of DNALigase-V2, 5 μL of scRNA Adapter-V2, and 10 μL of NFWater (a total of 40 μL) to the above PCR tube, mix well, centrifuge briefly, and incubate at 20°C for 15 minutes in a PCR instrument with the heated lid closed, then maintain at 4°C.

[0091] 13. Add 100 μL DNAClean Beads to the adapter-ligated product and mix thoroughly. Incubate at room temperature for 5 minutes. Discard the supernatant quickly. Keep the PCR tube on the magnetic stand and add 200 μL freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. Let it stand for 30 seconds and discard the supernatant. Repeat the 80% ethanol wash and completely discard the supernatant. After drying at room temperature, add 102 μL NF Water and mix thoroughly. Place the PCR tube on the magnetic rack and let it stand for 2 to 5 minutes. Transfer 30 μL of supernatant to a new 0.2 mL centrifuge tube, add 55 μL of DNA CleanBeads and mix well. Incubate at room temperature for 5 minutes, quickly discard the supernatant, add 15 μL of DNAClean Beads and mix well. Incubate at room temperature for 5 minutes, quickly discard the supernatant, keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, rinse the magnetic beads and tube wall, let it stand for 30 seconds and discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant. After drying at room temperature, add 48 μL of NF Water and mix well. Transfer 46 μL of supernatant to a new 0.2 mL centrifuge tube, add 50 μL of PCR Amp Enzyme and mix well. Centrifuge briefly and incubate in a PCR instrument at 95°C for 3 min. Incubate at 98°C for 20 s, incubate at 58°C for 20 s, and incubate at 72°C for 30 s for 12 cycles. Incubate at 72°C for 5 min and then maintain at 4°C.

[0092] 14. Add 55 μL of DNA Clean Beads to the PCR product and mix thoroughly. After magnetic separation, transfer the supernatant to a new 0.2 mL centrifuge tube and add 15 μL of DNA Clean Beads, mix thoroughly, and incubate at room temperature for 5 minutes. Discard the supernatant quickly. Keeping the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. Let stand for 30 seconds and discard the supernatant. Repeat the 80% ethanol wash and completely discard the supernatant. After drying at room temperature, add 32 μL of TE Buffer and mix thoroughly. Transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube, label it, and aspirate 1 μL of the fragment screening product for concentration determination. Wait for circularization.

[0093] 15. Add 152 μL DNA Clean Beads to the previously retained Oligo Product 1 and mix well. Incubate at room temperature for 5 minutes, discard the supernatant, keep the centrifuge tube on the magnetic rack, add 500 μL freshly prepared 80% ethanol, let it stand for 30 seconds, discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant. After drying at room temperature, add 32 μL NF Water and mix well. Transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube, mark it as Oligo Product 2, and use Qubit TM The product concentration was detected using the dsDNA HS Assay Kit.

[0094] 16. Pipette 28 μL of oligo product, 4 μL of scRNA Barcode Primer II-1 to scRNA Barcode Primer II-16, 25 μL of PCR Amp Enzyme, and 13 μL of NF Water (a total of 50 μL) into a new 0.2 mL PCR tube and mix well. After a quick centrifugation, place the tube in a PCR instrument and incubate at 95°C for 3 minutes. The tube was then incubated at 98°C for 20 seconds, 62°C for 30 seconds, and 72°C for 10 seconds for 10 cycles. After incubation at 72°C for 1 minute, the tube was maintained at 4°C.

[0095] 17. Add 35 μL of DNA Clean Beads to the PCR product and mix thoroughly. Incubate at room temperature for 5 minutes. Centrifuge briefly and let stand. Transfer the supernatant to a new 0.2 mL centrifuge tube. Add another 35 μL of DNA Clean Beads and mix thoroughly. Incubate at room temperature for 5 minutes. Centrifuge briefly and discard the supernatant. Keeping the PCR tube on a magnetic rack, add 200 μL of freshly prepared 80% ethanol and let stand for 30 seconds before discarding the supernatant. Repeat the 80% ethanol wash and completely discard the supernatant. After drying at room temperature, add 32 μL of TE Buffer and mix thoroughly. Place the PCR tube on a magnetic rack and let stand for 2-5 minutes. Transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube, label it, and determine the concentration.

[0096] 18. According to the concentration of PCR library product, take 400ng product into PCR tube, add TE Buffer to the volume of 45μL, put the PCR tube on ice, add 5μL 20nM Ad153 splint oligo and mix well. Centrifuge briefly and incubate in PCR instrument at 95℃ for 3min. After the reaction, transfer the PCR tube to ice and let it stand for 5min. Add 10μL of 10×TA Buffer 6μL, 100nM ATP 0.6μL, T4 DNA ligase (600U / μL) 0.4μL, TE Buffer 3μL to the above PCR tube, mix well, centrifuge briefly and incubate in PCR instrument at 37℃ with heated cover at 75℃ for 60min. After the reaction, transfer the PCR tube to ice immediately. 1μL of the total 4μL was added to the above PCR tube and mixed. After a brief centrifugation, the tube was placed in a PCR instrument and incubated at 37°C for 30 minutes. After the reaction, 90μL of PEG32Beads was added and mixed and incubated at room temperature for 10 minutes. After magnetic separation, 200μL of freshly prepared 80% ethanol was added to rinse the magnetic beads and the tube wall. The tube was allowed to stand for 30 seconds and the supernatant was discarded. The 80% ethanol was repeated and the supernatant was completely discarded. After drying at room temperature, 32μL of TE Buffer was added and mixed. 30μL of supernatant was transferred to a new 1.5mL centrifuge tube, marked, and used Qubit TM The product concentration was detected using the ssDNAHS Assay Kit. Finally, the cells were processed on the sequencing platform for single-cell high-throughput sequencing.

[0097] In order to demonstrate the versatility of single-cell sequencing technology based on nucleic acid aptamers, this experiment added nucleic acid aptamer Sgc8c (whose target is the membrane protein PTK7 overexpressed in LoVo cells) and its random control sequence to perform the above-mentioned single-cell high-throughput sequencing. Figure 7 As shown, group 1 is a buffer solution without nucleic acid aptamer, group 2 is nucleic acid aptamer Sgc8c, and group 3 is a random control sequence.

[0098] Through bioinformatics analysis, Figure 7 The violin plot in a shows that the quality control results of this method meet the analysis requirements, with an average of 3,000 genes and 4,500 UMI values detected in each cell. Figure 7 b shows that gene expression still has a high correlation after treatment with nucleic acid aptamers (r=0.95, p<2.2e-16), which once again proves that the addition of nucleic acid aptamers does not affect the normal expression of genes. Figure 7The violin plot of c shows that the distribution of low-expression and high-expression genes in the cells is consistent. Due to the specific recognition ability of the nucleic acid aptamer Sgc8c to its membrane protein target, such as Figure 7 As shown in Figure d, the number of nucleic acid aptamer Sgc8c is higher than that of its control sequence, which verifies the universality of the nucleic acid aptamer sequencing strategy. Finally, we used scIon-seq to investigate the effect of exogenous drug stimulation on the K + Egress. Figure 7 As shown in Figure 4, Group 4 was not treated with drugs, Group 5 was treated with three drugs in Experimental Example 4, and Group 6 was treated with four drugs in Experimental Example 4. The analysis results showed that the number of detection probes in the drug-treated group was higher than that in the drug-untreated group, proving that scIon-seq can monitor the K of living cells at the single-cell level. + External discharge.

[0099] Example 2

[0100] This example uses scIon-seq to analyze single-cell K + The dynamic change detection method uses peripheral blood mononuclear cells (PBMCs) collected and isolated from colorectal cancer patients, and the processing method is as follows:

[0101] 1. Take out fresh whole blood from the anticoagulant tube and transfer it to a 50mL centrifuge tube. Centrifuge at 3000rpm / min for 10min.

[0102] 2. Discard the upper plasma layer and add DPBS containing 2% FBS to 20 mL. Gently invert to mix, and place at room temperature. Then proceed with PBMC isolation as soon as possible.

[0103] 3. Use a pipette to pass the density gradient centrifuge (Ficoll) through SepMate TM Carefully insert SepMate into the central hole of the plug TM In the test tube, the top of the density gradient centrifugation solution should just submerge the insert;

[0104] 4. Maintain SepMate TM Hold the test tube vertically and use a pipette to add the diluted sample along the wall of the test tube (the sample can be poured directly along the wall of the tube, but be careful not to pour the diluted sample directly onto the central hole);

[0105] 5. Centrifuge at 1200g for 10 minutes at room temperature without turning off the brake (for samples stored for more than 24 hours, the recommended centrifugation time is 20 minutes);

[0106] 6. Quickly transfer the buffy coat layer to a new tube. This layer is rich in PBMCs. Wash the PBMCs with 10 mL of DPBS containing 2% FBS, centrifuge at 300 g for 8 minutes at room temperature, and discard the supernatant.

[0107] 7. If the precipitate is light pink, it does not need to be lysed. If it is dark red, it needs to be lysed: add 1 mL of 1× DPBS, use a pipette to gently pipette to resuspend, add 3 mL of red blood cell lysis buffer (Solabo), gently pipette to mix the cells, let it stand on ice for 15 minutes, gently inverting it from time to time to mix, centrifuge at 500g for 5 minutes at 4°C, and remove the supernatant;

[0108] 8. Add an appropriate amount of Binding Buffer according to the cell density to resuspend the cells, and use a cell counting plate to count the live and dead cells to determine the cell viability and cell concentration.

[0109] The specific detection method is the same as in Example 1.

[0110] In this example, peripheral blood was collected from patients with colorectal cancer and nucleated cells were isolated. PBMCs were then treated with the aforementioned three drug combinations and four drug combinations, respectively, and the untreated group was used as a control. After each group was incubated with a random control sequence, the nucleic acid aptamer sgc8c, and the split nucleic acid aptamer shown in Table 1, single-cell sequencing was performed to identify the dynamic changes in the transcriptome and ions. The above-mentioned flow chart for analyzing cells in colorectal cancer samples by single-cell high-throughput sequencing is shown in FIG. Figure 8 As shown in a.

[0111] Fourteen cell populations were obtained by unsupervised clustering ( Figure 8 b) Based on the expression of classic cell type markers, immune cells such as T cells, B cells, monocytes, dendritic cells and red blood cells can be identified ( Figure 8 c), three cell populations were annotated as monocytes using this method, including CD14+ monocytes (CD14, S100A12, LYZ), CD16+ monocytes (FCGR3A, CDKN1C, MS4A7), and intermediate monocytes (FCN1, CD14, FCGR3A); one cell population was labeled as dendritic cells (CD1C, LYZ), red blood cells (HBA1, HBD), and platelets (PPBP, PF4); and four cell populations were annotated as T cells, including CD4+ T cells ( CD3D, TCF7, LTB), NKT cells (NKG7, GZMB, GZMA), mucosal-associated invariant T cells (MAIT cells; CD3D, MAGI2), and circulating T cells (CD3D, MKI67, STMN1); four cell populations were annotated as B cells, including plasma-1 cells (MZB1, JCHAIN), plasma-2 cells (MZB1, IGLC3), plasma-3 cells (MZB1, IGHG1), circulating B cells (MKI67, MZB1, STMN1) ( Figure 8c). We then investigated the distribution of split-aptamers in specific subpopulations. The UMAP plots and violin plots based on the split-aptamer count analysis showed that the split-aptamers were uniformly distributed in each cluster ( Figure 8 d). Figure 8 As shown in e, the split-aptamer counts in the drug-treated group were higher than those in the non-drug-treated group. In addition, the number of split-aptamers in the group treated with four drugs was higher than that in the group treated with three drugs, indicating that scIon-seq can identify the K of each cell type in colorectal cancer PBMC samples. + change.

[0112] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for detecting dynamic changes of metal ions in single cells based on split-type nucleic acid aptamers, characterized in that: The split nucleic acid aptamer comprises a first part and a second part, the first part is a capture probe, and the second part is a detection probe; The detection method comprises: anchoring the capture probe to the membrane surface of a living cell; when metal ions are released from the living cell to the outside of the cell, the metal ions act as cofactors to promote the binding of the detection probe with the capture probe to form a stable complex structure; then, in a single-cell suspension system, high-throughput sequencing is performed on the detection probe in the complex structure to obtain dynamic changes in metal ions on the cell membrane surface; The capture probe is composed of a hydrophobic lipid-coupled nucleic acid aptamer; the nucleotide sequence of the capture probe is shown in SEQ ID NO: 1, and the nucleotide sequence of the detection probe is shown in SEQ ID NO:

2.

2. The detection method according to claim 1, characterized in that The detection probe is composed of a nucleic acid adaptor with sequencing adapters extended at both ends.

3. The detection method according to claim 2, characterized in that The sequencing adapter is a double-ended adapter, including a capture sequence at the 5' end and Poly A at the 3' end.

4. The detection method according to any one of claims 1 to 3, characterized in that The metal ions include monovalent and divalent metal ions.

5. The detection method according to claim 4, characterized in that The metal ions include K + 、Na + , Ca 2+ and Mg 2+ .

6. A split nucleic acid aptamer, characterized in that: The split nucleic acid aptamer comprises a first part and a second part, the first part is a capture probe, and the second part is a detection probe; The capture probe is composed of a hydrophobic lipid based on a dialkyl long chain coupled to a nucleic acid aptamer; the detection probe is composed of a nucleic acid aptamer with sequencing adapters extended at both ends; The sequencing adapter is a double-ended adapter, including a capture sequence at the 5' end and Poly A at the 3' end; The nucleotide sequence of the capture probe is shown in SEQ ID NO: 1, and the nucleotide sequence of the detection probe is shown in SEQ ID NO:

2.

7. A kit for detecting the dynamic changes of metal ions in single cells, characterized in that: Including the split nucleic acid aptamer according to claim 6.

8. Use of the detection kit according to claim 7 in preparing a product for detecting the dynamic changes of metal ions in single cells.

9. Use of the detection kit according to claim 7 in preparing a product for analyzing potassium ion changes in the microenvironment of colorectal cancer patient samples.

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