Detection method for dynamic change of metal ions based on nucleic acid aptamer single cell sequencing

Abstract

The invention discloses a single cell ion dynamic change detection method (scIon-seq) based on a nucleic acid aptamer, which comprises the following steps: anchoring a hydrophobic lipid modified capture probe on the surface of a cell membrane, and when metal ions are released out of the cell from the cell, detecting the dynamic change of the single cell ions. The detection probe with the sequencing joint and the capture probe on the membrane form a secondary structure through inter-chain folding in the presence of the metal ions; and then in a single cell suspension system, carrying out high-throughput sequencing on the detection probe with the sequencing joint in the secondary structure to obtain the dynamic change information of the metal ions on the single cell level. According to the invention, a research means is provided for researching the physiological function of the metal ions at the single cell level on the technical aspect by utilizing the characteristics that the nucleic acid aptamers can recognize the metal ions in a high-specificity manner and can be compatible with high-throughput sequencing; the development of multiple omics such as metabonomics, proteomics and transcriptomics on the single cell level of the nucleic acid aptamer can be promoted.

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 protein 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 important potential to supplement single-cell ionomics and provide a more comprehensive description and characterization of different cell 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 signals obtained only reflect the average state of complex biological samples (such as tissues or blood), obscuring the individual and specific dynamics of single cells.

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

[0005] The purpose of the present invention is to provide a method for detecting the dynamic changes of metal ions based on nucleic acid aptamer single-cell sequencing, called scIon-seq. The detection method of the present invention can overcome the limitations of antibodies in recognizing ions and can effectively detect the changes of ions 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, wherein the split nucleic acid aptamer comprises a first part and a second part, wherein the first part is a capture probe and the second part is 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 detection probe to combine with the capture probe to form a stable complex; then in a single-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 the 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 dialkyl 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 a single cell, which comprises 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 a single cell.

[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 in samples from colorectal cancer patients.

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

[0014] The present invention utilizes the specific recognition ability of split nucleic acid aptamers for metal ions. When the target ion is used as a cofactor, the two sequences of the split nucleic acid aptamer bind to the ion to form a complex, and the characteristics of being compatible with high-throughput sequencing, thereby determining the ion changes in the microenvironment of a single cell. This method breaks through the bottleneck that traditional single-cell high-throughput sequencing technology cannot detect ions in biological samples, provides a technical means for understanding the physiological functions of ions at the single-cell level, and also lays a foundation for promoting the development of nucleic acid aptamers in the study of signal transduction, transcriptomics and metabolomics at the single-cell level in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without 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 are used to obtain the gradient K + Response (a) and experimental results of its specificity (b);

[0018] Figure 3 The synthetic route of the hydrophobic lipid and the 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 on the cell membrane monitors K in Experimental Example 3 + Characterization results; (a) Working principle: Cy3-labeled capture probe is anchored on the cell membrane. + With the increase of 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); the statistical data are from three independent experiments and are expressed as mean ± SD; (d) K at different concentrations from 0 mM to 100 mM + 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 nucleic acid aptamer probe, the K + The generated K +Detection schematic diagram; (b) Flow cytometry 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 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 In Example 1, in a cell line, nucleic acid aptamers can be qualitatively and quantitatively identified by single-cell high-throughput sequencing; (a) violin plots show the quality control results of single-cell sequencing; (b) correlation of gene expression after nucleic acid aptamer treatment; (c) violin plots show the analysis of gene expression in cells that are not treated, treated with a random control sequence, or treated with nucleic acid aptamers; (d) box plots show the sequence counts between nucleic acid aptamer sgc8c-treated or control sequence-treated groups; (e) box plots show the sequence counts between split nucleic acid aptamer sgc8c-treated, random control sequence-treated, or split nucleic acid aptamer-treated groups;

[0023] Figure 8 The ion dynamic changes of cells in colorectal cancer samples were detected using scIon-seq in Example 2; (a) scIon-seq process analysis diagram; (b) transcriptome-based monocyte expression profile clustering reveals different cell populations; (c) marker gene expression in each cluster; (d) feature map shows the number of split nucleic acid aptamers in each cluster; (e) box plot shows the sequence counts of nucleic acid aptamer sgc8c, random control sequences and split nucleic acid aptamers between 3 drug treatment groups, 4 drug treatment groups or no drug treatment groups. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all 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, usually based on antibody-recognized proteomics and DNA barcode-based genomics and transcriptomics, and then analyze macromolecules such as nucleic acids and proteins at the single-cell level. However, the above methods cannot detect changes in ions at the single-cell level.

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

[0027] Split aptamers are a type of aptamer, which generally splits a complete aptamer into two or more fragments, and then in the presence of target molecules, these fragments can recombine and form a complex with high affinity and specificity. This split aptamer composed of two or more short nucleic acid chains has become a new tool in biosensor devices.

[0028] Based on this, the present invention proposes a detection method for the dynamic changes of single-cell ions based on split nucleic acid aptamers by modifying nucleic acid aptamers, and utilizes the characteristics of split nucleic acid aptamers that they 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 on the membrane surface of living cells, when metal ions are released from the living cells to the outside of the cells, 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 identify ions, and selects a split nucleic acid aptamer having a first part and a second part for ion detection. When the target ion exists in the detection system, the first part and the second part undergo structural folding in the presence of metal ions to form a split nucleic acid aptamer with a sequencing barcode. Then, the complex is detected on a machine to obtain the situation 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, cell volume, cell proliferation and apoptosis, nerve signal hyperpolarization, myocardial cell homeostasis, cell osmotic pressure and other physiological processes. It also has the function of 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 a single cell based on nucleic acid aptamers is further provided. The method is based on the above method to screen nucleic acid aptamers that can specifically bind to K + A split nucleic acid aptamer, the nucleotide sequence of the split nucleic acid aptamer is 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. + 、Na + NH4 + The present invention utilizes this property of G-quadruplex to detect potassium ions, wherein the split nucleic acid aptamer used is the split G-quadruplex below, and 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 , it forms a complete G-quadruplex structure with the capture probe. By performing single-cell sequencing on the detection probe, the changes in potassium concentration at the single-cell level can be obtained.

[0034] Figure b) shows the use of scIon-seq to monitor K + The process is as follows. After isolating cells from colorectal cancer patients, the capture probe is anchored on the cell membrane. Then the cells are stimulated by drugs and the detection probe is added. Finally, scIon-seq is performed to show the ion changes of cells isolated from the blood of 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 by the detection method of the present invention, it is only necessary to select a suitable nucleic acid aptamer according to the binding mechanism between the ion and the nucleic acid aptamer.

[0036] For the above-mentioned split nucleic acid aptamers, those skilled in the art can select them according to the target ions to be detected, and the present invention does not limit this, as long as the nucleic acid aptamers can specifically bind to the target ions, any one can be used.

[0037] In the present invention, the capture probe of the first part is composed of two parts, namely, hydrophobic lipid and nucleic acid aptamer, and the specific structure is: a covalent coupling body of hydrophobic lipid and nucleic acid aptamer. During detection, the hydrophobic lipid plays the role of anchoring to the cell membrane; the nucleic acid aptamer in the capture probe is used to specifically bind to the nucleic acid aptamer of the other part.

[0038] The hydrophobic lipid is a molecule containing two relatively long carbon chains, usually straight or branched alkane groups, and the two carbon chains can be the same or different. The present invention does not limit the number of carbon atoms in the 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 adaptor, specifically: a sequencing adapter sequence is extended at both ends of the nucleic acid adaptor, and the sequencing adapter here can be a sequencing adapter conventionally used in the art, and the present invention does not limit this.

[0040] In some embodiments, the two ends of the detection probe are connected to sequencing adapters, specifically, the 5' end is connected to the capture sequence (SEQ ID NO: 3-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaaa), and the 3' end is connected to Ploy A. By connecting the above sequencing adapters, it is more convenient to detect the complex formed by the split nucleic acid aptamer and the target ion on the machine.

[0041] Furthermore, the present invention uses nucleated cells separated from the peripheral blood of colorectal cancer patients as samples, and performs potassium ion analysis on them. The experimental results show that the detection method of the present invention can identify the potassium ion changes of each cell type in the colorectal cancer PBMC sample. 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, and may provide 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 belongs to the protection scope of the present invention.

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

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

[0045] Calculation of fluorescence resonance energy transfer (FRET) efficiency involved in the following experiment: 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 split aptamer dependent on potassium ion concentration. A segment of Poly A and a capture sequence (see Table 1) are added to the two ends of the second part of the split aptamer. The fluorescence spectrophotometer is used to examine the K concentration of the Cy3-labeled capture probe and the Cy5-labeled detection probe. + 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 increases, proving that the above selected sequence has + Split aptamers were formed in the presence of β-catenin and the activity was concentration-dependent.

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

[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 synthesis of lipids 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, the mixture was stirred at 110 ° C, and the reaction was monitored by thin layer chromatography (TLC). After 2 h of reaction, the mixture was cooled 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 with anhydrous Na2SO4. The solvent was removed by a rotary evaporator and then separated by a flash chromatography column. Finally, 3.19 g of compound (2) was obtained as a light brown solid product, which was directly used in the next step of the experiment 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-diisopropyl chlorophosphoramidite (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 by 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 by solid phase synthesis. The oligonucleotide was then cut from the corresponding controlled pore glass, and the cut DNA product was mixed with 1 mL of ice-cold ethanol and 40 μL of 3M NaCl and precipitated in cold ethanol at -20°C. The DNA product was then centrifuged at 12,000 rpm for 20 minutes at 4°C. Finally, 4,4'-dimethoxytrityl was removed from DNA by adding 80% acetic acid aqueous solution and precipitated in cold ethanol. The precipitate was dissolved in 400 μL 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 in 30 min. The isolated and purified product was lyophilized using a freeze centrifugal dryer, dissolved in ultrapure water, desalted using a DNA desalting column, lyophilized again, dissolved in sterile water and set aside.

[0060] Synthesis route: The phosphoramidite monomer is modified at the 5' end of DNA by solid phase synthesis. After modification, sterile water is added to dilute the reaction system to make the organic phase ratio less than 15%, filter, HPLC purification, collect chromatographic peaks in different time periods and perform mass spectrometry characterization.

[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 product capture probe is 11004.1, and the actual molecular weight measured is 11001.7 ( Figure 4 b), meet the requirement that the molecular weight error is within 0.03%.

[0062] Experimental Example 3

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

[0064] S1. After the LoVo cells grow to a suitable proportion 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 on 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 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, so that the FRET signal can be observed. Figure 5 As shown in b and 5c, 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 nucleic acid 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 validation assay for efflux that promotes 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 combination of added drugs included: 3 drug groups (amphotericin B + nigericin sodium salt + bumetanide) and 4 drug groups (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 in the figure. Figure 6 As shown in a, the steps are as follows:

[0072] S1. After the LoVo cells grow to a suitable 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 of the 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, 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 stimulated by drugs was quantitatively investigated. The results showed that in the absence of K + In the buffer solution, the K released into the environment was treated by adding the above three drug groups and four drug groups. + The concentrations reached 0.6mM and 0.9mM ( Figure 6 f and 6g).

[0077] Example 1

[0078] This embodiment is a single cell K based on nucleic acid aptamers + The dynamic change detection method, the specific operation method is as follows:

[0079] 1. Wash the cells twice with 1×DPBS, collect the digested cells into a 15mL centrifuge tube, centrifuge at 500g for 5min, discard the supernatant, wash once with 1×DPBS and collect the cells at the bottom of the tube, add an appropriate amount of BindingBuffer according to the cell density and resuspend, take 1 million cells in 400μL system, add 200nM nucleic acid aptamer, incubate on ice for 30min, centrifuge at 500g for 5min 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 22,000 cell suspensions of the above cells (filled to 24 μL with Binding Buffer) into a 0.2 mL low-adsorption PCR tube, mix well and place on ice for later use.

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

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

[0083] 5. After the droplet collection stops, let it stand at room temperature for 20 minutes before demulsification: rinse the C4 filter with 6×SSC, pour the droplet generation liquid into the C4 filter, and wash the magnetic beads in the C4 filter with 6×SSC 2-3 times, transfer the C4 filter to a 50mL centrifuge tube, add 700μL Collection Buffer, and gently blow the magnetic beads on the surface of the collection filter membrane to a 1.5mL centrifuge tube. Add 700μL Collection Buffer and gently blow the magnetic beads on the surface of the collection filter membrane to the above 1.5mL centrifuge tube, place it on the magnetic stand and let it stand for 3-5 minutes, discard the supernatant, remove the centrifuge tube from the magnetic stand, add 200μL 6×SSC and mix it with a pipette, and evenly divide it into 2 0.2mL PCR tubes, about 100μL in each tube. Repeat adding 200 μL of 6×SSC to the above 1.5 mL centrifuge tube and mix well by pipetting, divide evenly into the above two 0.2 mL PCR tubes, then place on the magnetic rack, let stand for 3 to 5 minutes, and discard the supernatant.

[0084] 6. Mix 87.5μL of RT Buffer, 5μL of RT Primer-V2, 5μL of RT Enzyme, and 2.5μL of RNaseInhibitor in a PCR tube, and incubate at 42℃ for 90min in a PCR instrument. Incubate at 50℃ for 2min, incubate at 42℃ for 2min for 10 times, incubate at 65℃ for 10min, and then maintain at 4℃.

[0085] 7. After the reaction is finished, 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, 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 200μL Stop Buffer and mix well to terminate the reaction. Centrifuge briefly and place on a magnetic rack. Let stand for 3-5 minutes and discard the supernatant (hereinafter referred to as "instantly discard and discard the supernatant"). Add 200μL Wash Buffer to wash once, instantly discard the supernatant, add 200μL 1× Denaturation Buffer and mix well. Incubate at room temperature for 5 minutes, instantly discard the supernatant, keep the centrifuge tube on the magnetic rack, add 200μL Wash Buffer and let stand for 2 minutes and discard the supernatant. Add 100μL of Second Strand Buffer-V2 67.5μL, Second Strand Primer-V2 7.5μL, and Second Strand Enzyme-V2 25μL to the above PCR tube and mix well. Centrifuge briefly and place in a PCR instrument to incubate at 25℃ for 10 minutes, incubate at 37℃ for 30 minutes, and then keep at 4℃.

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

[0088] 10. After the reaction, centrifuge the two tubes of PCR products and place them on a magnetic rack for 3 to 5 minutes. Take 95 μL of supernatant from each tube, combine the two tubes into a new 1.5 mL centrifuge tube, totaling 190 μL, add 114 μL of DNA Clean Beads to mix, incubate at room temperature for 10 minutes, centrifuge briefly and place on a magnetic rack, let stand for 2 to 5 minutes (hereinafter referred to as "instant centrifuge and stand"), transfer the supernatant to a new 1.5 mL centrifuge tube, mark it as Oligo product 1, and further purify the above-adsorbed magnetic beads, keep the centrifuge tube on the magnetic rack, add 500 μL of freshly prepared 80% ethanol, let stand for 30 seconds, discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant, open the tube cap, dry at room temperature, add 32 μL of NF Water and mix, incubate at room temperature for 5 minutes, centrifuge briefly and let stand, transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube, mark it as cDNA product, and take 1 μL The concentration of cDNA products was detected using QubitTM dsDNAHS Assay Kit.

[0089] 11. According to the concentration of cDNA product, take 150ng product into 0.2mL PCR tube, put the PCR tube on ice box, aspirate 10μL of Frag Buffer-V2 and 5μL of Frag Enzyme-V2, a total of 15μL, add to the above PCR tube and mix well. After centrifugation, place the PCR tube in a PCR instrument precooled to 4℃, then incubate at 30℃ for 9min, incubate at 65℃ for 20min, and then keep at 4℃.

[0090] 12. Add 20μL of Ligation Buffer-V2, 5μL of DNALigase-V2, 5μL of scRNA Adapter-V2, and 10μL of NFWater (a total of 40μL) to the above PCR tube and mix well. Centrifuge briefly and place in a PCR instrument with the hot lid closed, incubate at 20℃ for 15min, and then keep at 4℃.

[0091] 13. Add 100 μL DNAClean Beads to the adapter ligation product and mix well. Incubate at room temperature for 5 minutes, discard the supernatant instantly, keep the PCR tube on the magnetic stand, add 200 μL freshly prepared 80% ethanol, rinse the magnetic beads and tube wall, let stand for 30 seconds and discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant, dry at room temperature, and add 102 μL NF Water and mix well. 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, discard the supernatant instantly, add 15 μL of DNAClean Beads and mix well. Incubate at room temperature for 5 minutes, discard the supernatant instantly, keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, rinse the magnetic beads and the 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, 58°C for 20 s, and 72°C for 30 s for 12 cycles. Incubate at 72°C for 5 min and then maintain at 4°C.

[0092] 14. Add 55μL DNA Clean Beads to the PCR product and mix well. After magnetic separation, transfer the supernatant to a new 0.2mL centrifuge tube, add 15μL DNA Clean Beads and mix well. Incubate at room temperature for 5min, discard the supernatant instantly, keep the PCR tube on the magnetic rack, add 200μL freshly prepared 80% ethanol, rinse the magnetic beads and the tube wall, let stand for 30s and discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant, dry at room temperature, add 32μL TE Buffer and mix well. Transfer 30μL supernatant to a new 1.5mL centrifuge tube, mark it, and take 1μL of the product after fragment screening for concentration determination, waiting for cyclization.

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

[0094] 16. Pipette 28μL of Oligo product, 4μL of scRNA Barcode Primer II-1~scRNA Barcode PrimerII-16, 25μL of PCR Amp Enzyme, and 13μL of NF Water to a total of 50μL, add them to a new 0.2mL PCR tube and mix well. Centrifuge for a moment and incubate in a PCR instrument at 95℃ for 3min. Incubate at 98℃ for 20s, 62℃ for 30s, and 72℃ for 10s for 10 cycles. Incubate at 72℃ for 1min and keep at 4℃.

[0095] 17. Add 35μL DNA Clean Beads to the PCR product and mix well, incubate at room temperature for 5 minutes, centrifuge and let stand for a moment, transfer the supernatant to a new 0.2mL centrifuge tube, add 35μL DNA Clean Beads and mix well, incubate at room temperature for 5 minutes, centrifuge and discard the supernatant, keep the PCR tube on the magnetic rack, add 200μL freshly prepared 80% ethanol, let stand for 30s and discard the supernatant, repeat the 80% ethanol wash and completely discard the supernatant, dry at room temperature, add 32μL TE Buffer and mix well. Place the PCR tube on the magnetic rack, let stand for 2 to 5 minutes, transfer 30μL of supernatant to a new 1.5mL centrifuge tube, mark it, and measure the concentration.

[0096] 18. According to the concentration of PCR library product, take 400ng product into PCR tube, use TE Buffer to make the volume to 45μL, put the PCR tube on ice, add 5μL 20nM Ad153 splint oligo and mix well, centrifuge and incubate in PCR instrument at 95℃ for 3min, transfer the PCR tube to ice immediately after the reaction and let it stand for 5min, add 10×TA Buffer 6μL, 100nMATP 0.6μL, T4 DNA ligase (600U / μL) 0.4μL, TE Buffer 3μL, a total of 10μL, to the above PCR tube, mix well, centrifuge and incubate in PCR instrument at 75℃ and 37℃ for 60min, transfer the PCR tube to ice immediately after the reaction, add 10×TA Buffer 0.4μL, ExoⅠ (20U / μL), ExoⅢ (100U / μL) 0.65μL, TE Buffer 1μL of 4μL was added to the above PCR tube and mixed. After centrifugation, it was placed in a PCR instrument and incubated at 37℃ for 30min. After the reaction, 90μL of PEG32Beads was added and mixed and incubated at room temperature for 10min. After magnetic separation, 200μL of freshly prepared 80% ethanol was added to rinse the magnetic beads and the tube wall. After standing for 30s, the supernatant was discarded. Repeat the 80% ethanol washing and completely discard the supernatant. 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 by ssDNAHS Assay Kit. Finally, the product entered the sequencing platform process 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 of a shows that the quality control results of this method meet the analysis requirements, with an average of 3000 genes and 4500 UMI values ​​detected in each cell. Figure 7 b shows that gene expression still has a high correlation after nucleic acid aptamer treatment (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 cells is consistent. Due to the specific recognition ability of nucleic acid aptamer Sgc8c to its membrane protein target, such as Figure 7 As shown in Figure d, the value of the aptamer Sgc8c is higher than the count of its control sequence, which verifies the universality of the aptamer sequencing strategy. Finally, we used scIon-seq to investigate the effect of external drug stimulation on the promotion of 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 is a method for using scIon-seq to detect single-cell K + The dynamic change detection method uses peripheral blood mononuclear cells (PBMCs) collected and separated 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, add DPBS containing 2% FBS to 20 mL, gently invert to mix, leave at room temperature, and then separate PBMC as soon as possible;

[0103] 3. Use a pipette to pass the density gradient centrifuge (Ficoll) through SepMate TM Carefully add SepMate to the small hole in the center 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 test tube. This layer is rich in PBMCs. Wash the PBMCs with 10 mL of DPBS containing 2% FBS, centrifuge at 300 g for 8 min 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 blow and resuspend, add 3 mL of red blood cell lysis buffer (Solabo), gently blow and mix the cells, let it stand on ice for 15 minutes, gently invert 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 to resuspend the cells according to the cell density, and use a cell counting plate to count the live and dead cells to determine the cell survival rate 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. Then, PBMCs were 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, a nucleic acid aptamer sgc8c, and a split nucleic acid aptamer shown in Table 1, single-cell sequencing was performed to identify the dynamic changes of the transcriptome and ions. The above-mentioned flowchart 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, 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), erythrocytes (HBA1, HBD), and platelets (PPBP, PF4); four cell populations were annotated as T cells, including CD4+ T cells ( CD3D, TCF7, LTB), NKT cells (NKG7, GZMB, GZMA), mucosa-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 distributed indiscriminately 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 above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the dynamic changes of metal ions in a single cell based on split 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: after 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 structure; then in a single cell suspension system, by performing high-throughput sequencing on the detection probe in the complex structure, the dynamic change information of the metal ions on the cell membrane surface can be obtained.

2. The detection method according to claim 1, characterized in that: The capture probe consists of a hydrophobic lipid-coupled nucleic acid aptamer.

3. 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; Preferably, the sequencing adapter is a double-ended adapter, comprising Poly A at the 5' end and a capture sequence 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; Preferably, the metal ions include K + 、Na + , Ca 2+ and Mg 2+ .

5. 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 Poly A at the 5' end and a capture sequence at the 3' end.

6. A single cell metal ion dynamic change detection kit, characterized in that: Comprising the split nucleic acid aptamer according to claim 5.

7. A detection kit for dynamic changes of single-cell potassium ions, characterized in that: It comprises the split nucleic acid aptamer according to claim 6, 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.

8. Use of the detection kit as claimed in claim 6 or 7 in the preparation of a product for detecting the dynamic changes of metal ions in single cells.

9. Use of the detection kit as claimed in claim 6 or 7 in the preparation of a product for analyzing potassium ion changes in the microenvironment of colorectal cancer patient samples.

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