Droplet single-cell secreted RNA detection method based on reagent combination in combination with proximity to signal amplification reagent and application thereof

Abstract

This invention discloses a reagent combination for detecting and amplifying the proximity of secreted RNA, a droplet-based single-cell secreted RNA detection method based on the reagent combination, and its applications, belonging to the fields of single-cell analysis and nucleic acid detection technology. The reagent combination for detecting and amplifying the proximity of secreted RNA (a splitting recognition probe, a self-sealing PAM hairpin probe, a CRISPR / Cas12a system, a fluorescent reporter probe, and an amplification probe) is mixed with a cell suspension to form an aqueous system. This aqueous system is then mixed with an oil phase to form an oil-in-water droplet, encapsulating cells within the droplet to form single-cell droplets. After incubation, the cells secrete target RNA molecules within the droplet. The reagent combination recognizes and amplifies the secreted RNA, generating a fluorescent signal. Microscopic imaging of the droplet combined with image analysis enables single-cell level detection and analysis of secreted RNA. This method can achieve single-cell spacerization and secreted RNA enrichment within the droplet microenvironment and can be used for the detection of single-cell secreted RNA.

Description

Technical Field

[0001] This invention relates to the fields of single-cell analysis and nucleic acid detection technology, specifically to reagent combinations for detecting secreted RNA by identifying proximity and signal amplification, droplet-based single-cell secreted RNA detection methods based on reagent combinations, and their applications. Background Technology

[0002] Cellular secretion is a crucial process for maintaining physiological homeostasis and intercellular communication. Cells participate in biological processes such as regulating immune responses, tissue development, and disease progression by secreting various signaling molecules. Traditional research has mainly focused on secreted proteins, cytokines, and metabolic molecules. However, recent studies have shown that nucleic acid molecules can also be released into the extracellular environment through cellular secretion and participate in intercellular signal transduction. MicroRNAs (miRNAs) are a class of endogenous non-coding RNA molecules with a length of approximately 19–25 nucleotides. They regulate gene expression at the posttranscriptional level and play an important role in various physiological and pathological processes, including tumorigenesis, immune regulation, and metabolic diseases. Studies have found that miRNAs not only exist within cells but can also be released into the extracellular environment through extracellular vesicles or RNA-binding proteins. These secreted miRNAs can transmit regulatory information between cells, playing a vital role in tumor microenvironment regulation, intercellular communication, and disease progression. In addition to miRNAs, researchers have also discovered a novel class of glycosylated RNA molecules in recent years. GlycoRNAs are a class of nucleic acid molecules that have undergone glycosylation modification on RNA molecules. Their molecular structure includes both the RNA backbone and glycosylation modification groups. Studies have shown that glycoRNAs can be localized on the cell membrane surface and participate in processes such as intercellular interactions and immune recognition. The discovery of glycoRNAs has expanded our understanding of RNA modification and intercellular signal transduction mechanisms, and also suggests that cell-secreted RNA molecules may have more complex structural features and biological functions.

[0003] Currently, detection methods for small RNA molecules such as miRNAs mainly include real-time quantitative PCR, sequencing technology, and various nucleic acid amplification detection methods. These methods typically require the complete extraction of cell culture supernatant or tissue samples before detection, thus only providing information at the population average level and failing to reflect the secretory differences between individual cells. In reality, even within the same cell population, the secretory capacity of different cells can exhibit significant heterogeneity. Population-level detection often masks these intercellular differences, thereby limiting in-depth research into cellular secretory behavior.

[0004] To study cellular secretion heterogeneity, various single-cell analysis techniques have been developed in recent years. For example, single-cell technologies based on microwell arrays, microfluidic chips, or droplet encapsulation can isolate individual cells in independent microenvironments, thereby enabling the study of single-cell behavior. Although these technologies have been widely used in single-cell protein secretion detection and single-cell gene expression analysis, the small size, low secretion volume, and easy dilution of miRNAs and related RNA molecules in conventional systems limit their effectiveness. High-efficiency detection is difficult to achieve directly in traditional detection systems. For instance, single-cell miRNA secretion detection (Vortexing-generated high throughput single-cell droplets for facile analysis of multiplexed microRNA dynamic secretion) requires 4–8 hours to obtain a significant signal, which is time-consuming. Therefore, developing a method for efficiently detecting cellular secreted RNA molecules at the single-cell level is of great significance for studying cellular secretion heterogeneity and intercellular signaling communication. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a reagent combination for detecting secreted RNA by recognizing proximity and signal amplification, a droplet single-cell secreted RNA detection method based on the reagent combination, and its application, so as to solve the problem that existing single-cell level detection methods for cell secreted RNA molecules cannot achieve high-efficiency detection.

[0006] This invention is specifically achieved through the following technical solutions: A first aspect of the present invention discloses a reagent combination for detecting and identifying proximity and signal amplification in secreted RNA, comprising: The splitting recognition probes include recognition probe 1 and recognition probe 2, which can work together to recognize the target secreted RNA and trigger a neighboring hybridization reaction. Self-sealing PAM hairpin probes can undergo strand substitution reactions triggered by adjacent hybridization reactions to form CRISPR / Cas12a recognition sequences; The CRISPR / Cas12a system includes the Cas12a protein and its bound gRNA. The gRNA includes amplification gRNA and recognition gRNA. The Cas12a protein can recognize and cleave fluorescent probes and amplification probes. The amplification gRNA can recognize the single-stranded DNA released after the amplification probe is cleaved, and the recognition gRNA can recognize the double-stranded DNA structure formed after the self-closing PAM hairpin probe strand is replaced. Fluorescent reporter probes are nucleic acid probes containing both fluorescent and quenching groups; The amplification probe can participate in the signal amplification reaction after activation by the CRISPR / Cas12a system.

[0007] Preferably, it also includes a buffer solution.

[0008] Preferably, when the target secretory RNA is modified RNA, recognition probe 1 can specifically recognize the modified structure of the target secretory RNA molecule, and recognition probe 2 can specifically recognize the RNA sequence region of the target secretory RNA molecule; when the target secretory RNA is unmodified RNA, recognition probe 1 can specifically recognize a conserved specific nucleic acid sequence region of the target secretory RNA molecule, and recognition probe 2 can specifically recognize another conserved specific nucleic acid sequence region on the target secretory RNA molecule that does not overlap with the binding region of recognition probe 1 and is adjacent to it.

[0009] Preferably, when the target secreted RNA is glycoRNA, the nucleotide sequence of recognition probe 1 is as shown in SEQ ID NO.3, and the nucleotide sequence of recognition probe 2 is as shown in SEQ ID NO.4; when the target secreted RNA is miRNA, the nucleotide sequence of recognition probe 1 is as shown in SEQ ID NO.1, and the nucleotide sequence of recognition probe 2 is as shown in SEQ ID NO.2.

[0010] Preferably, in the fluorescent reporter probe, the fluorescent group and the quenching group are FAM and BHQ-1, or Cy3 and BHQ-2, or Cy5 and BHQ-3.

[0011] Preferably, the nucleotide sequence of the self-sealing PAM hairpin probe is shown in SEQ ID NO.5, the nucleotide sequence of the amplification gRNA is shown in SEQ ID NO.6, the nucleotide sequence of the recognition gRNA is shown in SEQ ID NO.7, the nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO.8, and the nucleotide sequence of the amplification probe is shown in SEQ ID NO.9.

[0012] In a second aspect, the present invention discloses the application of the above-described reagent combination for detecting secretory RNA by recognizing proximity and signal amplification in the detection of secretory RNA in single cells.

[0013] A third aspect of the present invention discloses a method for detecting droplet-secreted RNA based on a combination of reagents for recognizing proximity and signal amplification, comprising the following steps: 1) Mix the above-mentioned reagent combination for detecting and amplifying neighboring cells and signals for secretory RNA with the cell suspension to form an aqueous system; 2) Mix the aqueous phase system with the oil phase to form an oil-in-water droplet, which randomly encapsulates cells in the droplet to form a single-cell droplet; 3) Incubate single-cell droplets to induce the cells to secrete target RNA molecules within the droplets. A combination of reagents for detecting and amplifying the secreted RNA then performs a recognition reaction and amplifies the signal, generating a fluorescent signal. 4) Perform microscopic imaging of droplets and combine it with image analysis to achieve single-cell level detection and analysis of secreted RNA.

[0014] Preferably, in step 1), the preparation method of the reagent combination for detecting proximity and signal amplification of secreted RNA is as follows: Cas12a protein is incubated with amplification gRNA and recognition gRNA at room temperature in the presence of 5 mM MgCl2 to form a Cas12a-gRNA complex; the amplification probe solution is placed in a buffer solution containing 20 mM Tris-HCl, 50 mM NaCl and 10 mM MgCl2 and pre-incubated at room temperature; recognition probe 1, recognition probe 2, self-blocking PAM hairpin probe, Cas12a-gRNA complex, fluorescent reporter probe and incubated amplification probe are dissolved in the buffer solution to obtain the reagent combination for detecting proximity and signal amplification of secreted RNA.

[0015] Preferably, in step 1), in the reagent combination for detecting and amplifying the secreted RNA, the final concentrations of recognition probe 1 and recognition probe 2 are both 30-50 nM, the final concentration of the self-blocking PAM hairpin probe is 1-100 nM, the final concentration of Cas12a protein is 100 nM, the final concentration of the amplification gRNA is 50 nM, the final concentration of the recognition gRNA is 50 nM, the final concentration of the fluorescent reporter probe is 500 nM, and the final concentration of the amplification probe is 100 nM.

[0016] Preferably, in step 2), the oil phase is QX200™ Droplet Generation Oil for EvaGreen.

[0017] Preferably, in step 2), the mixing method is through vortex oscillation or microfluidic chip droplet generation.

[0018] Preferably, in step 2), the single-cell droplets are formed by random encapsulation, so that the droplets mainly contain single cells.

[0019] Preferably, in step 3), the incubation temperature of the single-cell droplet is 37°C, and the incubation time is 0.5 to 4 hours.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a reagent combination for detecting and amplifying the proximity of secreted RNA. When the target secreted RNA is present, the splitting recognition probes (recognition probe 1 and recognition probe 2) can bind to different regions of the target secreted RNA and undergo proximity hybridization, thereby triggering a strand displacement reaction of the self-closing PAM hairpin probe to form a CRISPR / Cas12a recognition sequence. After the formation of the CRISPR / Cas12a recognition sequence, the double-stranded DNA structure formed after strand displacement by the self-closing PAM hairpin probe is recognized by gRNA, and the CRISPR / Cas12a system is activated. After recognizing the fluorescent reporter probe and the amplification probe respectively, the fluorescent reporter probe is cleaved (releasing a fluorescent signal) and the amplification probe (achieving signal amplification). The amplification probe releases single-stranded DNA, which is amplified and recognized by gRNA, further activating the CRISPR / Cas12a system. The CRISPR / Cas12a system further cleaves the fluorescent reporter probe (releasing a fluorescent signal) and the amplification probe (achieving signal amplification), forming a positive feedback loop that continuously releases fluorescent signals and amplifies signals, thereby achieving the detection of the target secreted RNA molecule. This reagent combination can be used for the detection of secreted RNA in single cells, enabling effective identification and signal amplification of low-abundance nucleic acid molecules in a microscale space, accelerating the detection efficiency of the reaction, and keeping the detection time within 1 hour. It is suitable for high-efficiency analysis of secreted RNA molecules in single cells.

[0021] This invention provides a droplet-based single-cell secreted RNA detection method based on reagent combinations that recognize proximity and amplify signals. The method encapsulates single cells through droplet spacing, enriching secreted RNA within the droplet microenvironment. By combining a division recognition probe, a self-sealing PAM hairpin probe, a CRISPR / Cas12a system, a fluorescent reporter probe, and an amplification probe, it enables the detection of low-abundance RNA molecules. This method offers advantages such as rapid detection, ease of operation, wide applicability, and strong single-cell analysis capabilities. It can detect and analyze secreted RNA at the single-cell level and can detect different types of secreted RNA molecules by changing the division recognition probe, particularly applicable to the simultaneous detection of different types of secreted RNA molecules such as miRNA and glycoRNA. Attached Figure Description

[0022] Figure 1 This invention presents the principle and droplet encapsulation flowchart of a droplet-based single-cell secreted RNA detection method based on reagent combinations for recognizing proximity and signal amplification; wherein, A is the principle diagram, B is the mixing method using vortex oscillation, and C is the mixing method using microfluidic chip droplet generation. Figure 2 Gel electrophoresis verification of the structural changes of the self-closing PAM hairpin probe triggered by the splitting recognition probe; Figure 3 This is a graph showing the fluorescence detection results of target RNA molecules using a reagent combination for detecting and amplifying adjacent RNA molecules. The red bars represent the mixture of cell culture supernatant and the reagent combination for detecting and amplifying adjacent RNA molecules (miRNA). The gray bars on the left represent the mixture of culture medium and the reagent combination for detecting and amplifying adjacent RNA molecules (miRNA). The blue bars represent the mixture of cell culture supernatant and the reagent combination for detecting and amplifying adjacent RNA molecules (glycoRNA). The gray bars on the right represent the mixture of culture medium and the reagent combination for detecting and amplifying adjacent RNA molecules (glycoRNA). Figure 4 This is a microscopic image of single-cell secreted miRNA detection; the blue fluorescent signal is Hoechst dye, and the green fluorescent signal is the RNA detection signal. Figure 5 CAD design drawings of microfluidic chips and images of droplet formation in channels under a bright-field microscope; Figure 6 Microscopic images of single-cell secreted glycoRNAs; Figure 7 Statistical graph of RNA secretion results from single cells of different cell lines; Figure 8 This is a statistical graph showing the results of single-cell RNA secretion detection after cells were treated under different conditions. Detailed Implementation

[0023] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0024] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0025] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0026] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0027] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0028] In this article, "room temperature" refers to a temperature of approximately 20°C to 35°C, or approximately 23°C to 28°C, or approximately 25°C. It can be 20°C, 25°C, 30°C, or 33°C.

[0029] This invention provides a reagent combination for detecting secreted RNA, identifying proximity and amplifying signals, comprising: The splitting recognition probe includes recognition probe 1 and recognition probe 2, which can specifically bind to different regions of the target secreted RNA, respectively. Recognition probe 1 and recognition probe 2 can synergistically recognize the target secreted RNA and undergo proximity hybridization in the presence of the target secreted RNA. Specifically, when the target secreted RNA is modified RNA (e.g., glycoRNA), recognition probe 1 can specifically recognize the modified structure of the target secreted RNA molecule, and recognition probe 2 can specifically recognize the RNA sequence region of the target secreted RNA molecule. When the target secreted RNA is unmodified RNA (e.g., miRNA), recognition probe 1 can specifically recognize a conserved specific nucleic acid sequence region of the target secreted RNA molecule, and recognition probe 2 can specifically recognize another conserved specific nucleic acid sequence region of the target secreted RNA molecule that does not overlap with the binding region of recognition probe 1 and is adjacent to it. More specifically, when the target secreted RNA is g... When targeting lycoRNA, recognition probe 1 is a glycoaptor probe that specifically recognizes the glycosyl structure of the glycoRNA molecule, and recognition probe 2 is an RNA sequence recognition probe that specifically recognizes the RNA sequence region of the glycoRNA molecule. After recognition probes 1 and 2 bind to the glycoRNA, they can trigger a subsequent strand displacement reaction and generate a detectable fluorescent signal. When the target secreted RNA is miRNA, recognition probe 1 can specifically recognize a conserved specific nucleic acid sequence at the 5' end of the miRNA molecule, and recognition probe 2 can specifically recognize a conserved specific nucleic acid sequence at the 3' end of the miRNA molecule that does not overlap with the binding region of recognition probe 1 and is closely adjacent to it. After recognition probes 1 and 2 bind to the miRNA, they completely hybridize, causing the free ends of the two probes to approach each other and form a partially complementary binding, thereby triggering a subsequent strand displacement reaction and generating a detectable fluorescent signal. Self-sealing PAM hairpin probes are nucleic acid probes with a self-hybridizing hairpin structure. They undergo a strand substitution reaction triggered by a neighboring hybridization reaction to form a CRISPR / Cas12a recognition sequence. The CRISPR / Cas12a system is used to recognize target sequences and trigger the cleavage reaction of fluorescent reporter probes and amplification probes. It includes the Cas12a protein and the gRNA bound to it. The gRNA includes amplification gRNA (gRNA-A) and recognition gRNA (gRNA-T). The Cas12a protein can form a Cas12a-gRNA complex with the amplification gRNA and recognition gRNA, which is used to accurately recognize and cleave fluorescent probes and amplification probes. The amplification gRNA can recognize the single-stranded DNA released after the amplification probe is cleaved, and the recognition gRNA can recognize the double-stranded DNA structure formed after the self-closing PAM hairpin probe strand is replaced. A fluorescent reporter probe is a nucleic acid probe with a fluorescent group and a quencher group that releases a fluorescent signal when cleaved by a CRISPR / Cas12a system. The fluorescent group and quencher group include, but are not limited to, FAM (fluorescein) and BHQ-1, Cy3 and BHQ-2, or Cy5 and BHQ-3.

[0030] Amplification probes are nucleic acid probes used for signal amplification. They participate in the signal amplification reaction after being activated by the CRISPR / Cas12a system. Buffer solutions are used to provide the necessary ionic environment, maintain pH stability and osmotic pressure balance in the reaction system, and reduce non-specific reactions.

[0031] This invention provides a method for detecting droplet-based single-cell secreted RNA based on a combination of reagents that identify proximity and amplify signals, such as... Figure 1 As shown, it includes the following steps: 1. Mix the reagent combination used for detecting and identifying proximity and signal amplification of secretory RNA with the cell suspension to form an aqueous system; 2. Mix the aqueous phase system with the oil phase to form an oil-in-water droplet, and randomly encapsulate the cells in the droplet to form a single-cell droplet; The oil phase includes, but is not limited to, QX200. TM Droplet Generation Oil for EvaGreen, HFE 7500 fluorinated oil or FC40 fluorinated oil; mixing methods include, but are not limited to, vortex oscillation ( Figure 1 (B) or microfluidic chip droplet generation ( Figure 1 (C) method; 3. Incubate single-cell droplets to induce the cells to secrete RNA molecules within the droplets; 4. The incubated droplets were imaged using a fluorescence microscope, and the fluorescence intensity of the droplets was statistically analyzed using image analysis software to achieve single-cell level detection of secreted RNA.

[0032] The principle of the droplet single-cell secreted RNA detection method provided by this invention, based on the identification of proximity and signal amplification of reagent combinations, is as follows: Figure 1As shown in Figure A, when the target secreted RNA is present, recognition probe 1 and recognition probe 2 bind to different regions of the target secreted RNA and undergo proximity hybridization, thereby triggering a strand displacement reaction of the self-closing PAM hairpin probe to form a CRISPR / Cas12a recognition sequence. After the formation of the CRISPR / Cas12a recognition sequence, the double-stranded DNA structure formed after strand displacement of the self-closing PAM hairpin probe is recognized by gRNA, activating the CRISPR / Cas12a system. This system recognizes and cleaves the fluorescent reporter probe (releasing a fluorescent signal) and the amplification probe (achieving signal amplification). The amplification probe releases single-stranded DNA, which is then amplified and recognized by gRNA, reactivating the CRISPR / Cas12a system. The CRISPR / Cas12a system further cleaves the fluorescent reporter probe (releasing a fluorescent signal) and the amplification probe (achieving signal amplification), forming a positive feedback loop to achieve the detection of the target secreted RNA molecule.

[0033] To enable those skilled in the art to better understand the present invention, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0034] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Information on some reagents used in the following examples is as follows: Cas12a and NEB buffer r2.1 were both purchased from NEB, M0653S EnGen® Lba Cas12a (Cpf1), B6002V NEBuffer. TM r2.1; Tris-HCl, purchased from Shanghai Sangon Biotech Co., Ltd., B648003-500 mL; Magnesium chloride (MgCl2) solution, purchased from Shanghai Yuanye Biotechnology Co., Ltd., R22727-500 mL; Sodium chloride solution, purchased from Shanghai Sangon Biotech Co., Ltd., B548120-100 mL; Cell information used is as follows: PANC-1 cells, HeLa cells, and MCF-7 cells were all purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; Nucleotide sequence information of probes and gRNA used is shown in Table 1, synthesized by Sangon Biotech (Shanghai) Co., Ltd.; DMEM high glucose medium (phenol red-free), purchased from Wuhan Pronosei Life Sciences Co., Ltd., PM150223-500 mL; All other raw materials used, unless otherwise specified, are conventional commercial products with specifications in the field.

[0035] Table 1 Sequence List

[0036] Note: In Table 1, 6-FAM is modified with a fluorescent group, and BHQ1 is modified with a quenching group.

[0037] I. Experiment on the structural change of a self-sealing PAM hairpin probe triggered by a splitting recognition probe 1. Divide the experiment into the following six groups: Self-blocking PAM hairpin probe set: Add 100 nM of self-blocking PAM hairpin probes (nucleotide sequences are shown in Table 1 as SEQ ID NO.5); miR-21-free group: Add mixed reagent 2, which is prepared by mixing miR-21 recognition probe 1 (nucleotide sequence as shown in SEQ ID NO.1 in Table 1), miR-21 recognition probe 2 (nucleotide sequence as shown in SEQ ID NO.2 in Table 1), and self-blocking PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO.5 in Table 1) to obtain mixed reagent 2; wherein, the final concentration of miR-21 recognition probe 1, miR-21 recognition probe 2 and self-blocking PAM hairpin probe is 100 nM. Group 1 without recognition probes: Mixed reagent 3 was added. The mixed reagent 3 was prepared by mixing miR-21 (nucleotide sequence as shown in SEQ ID NO.10 in Table 1), miR-21 recognition probe 2 (nucleotide sequence as shown in SEQ ID NO.2 in Table 1), and self-blocking PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO.5 in Table 1) to obtain mixed reagent 3; wherein, the final concentration of miR-21 recognition probe 2 and self-blocking PAM hairpin probe is 100 nM, and the final concentration of miR-21 is 200 nM; Group 2 without recognition probes: Mixed reagent 4 was added. The mixed reagent 4 was prepared by mixing miR-21 (nucleotide sequence as shown in SEQ ID NO.10 in Table 1), miR-21 recognition probe 1 (nucleotide sequence as shown in SEQ ID NO.1 in Table 1), and self-blocking PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO.5 in Table 1) to obtain mixed reagent 4; wherein, the final concentration of miR-21 recognition probe 1 and self-blocking PAM hairpin probe is 100 nM, and the final concentration of miR-21 is 200 nM; Hairpin-free probe set: Add mixed reagent 5, which is prepared by mixing miR-21 recognition probe 1 (nucleotide sequence as shown in SEQ ID NO.1 in Table 1), miR-21 recognition probe 2 (nucleotide sequence as shown in SEQ ID NO.2 in Table 1), and miR-21 (nucleotide sequence as shown in SEQ ID NO.10 in Table 1) to obtain mixed reagent 5; wherein, the final concentration of miR-21 recognition probe 1 and miR-21 recognition probe 2 is 100 nM, and the final concentration of miR-21 is 200 nM; Reagent combination group: Add mixed reagent 6, which is prepared by mixing miR-21 (nucleotide sequence as shown in SEQ ID NO.10 in Table 1), miR-21 recognition probe 1 (nucleotide sequence as shown in SEQ ID NO.1 in Table 1), miR-21 recognition probe 2 (nucleotide sequence as shown in SEQ ID NO.2 in Table 1), and self-blocking PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO.5 in Table 1) to obtain mixed reagent 6; wherein, the final concentration of miR-21 recognition probe 1, miR-21 recognition probe 2 and self-blocking PAM hairpin probe is 100 nM, and the final concentration of miR-21 is 200 nM; 2. The reagents of each of the above experimental groups were placed at 37℃ for 1 h. The products after the reaction were subjected to polyacrylamide gel electrophoresis (180 V, 35 min). The gel imaging results are as follows: Figure 2 As shown, when the target RNA (miRNA) is present, the splitting recognition probes bind to different regions of the target RNA and undergo proximity hybridization, thereby triggering the self-closing PAM hairpin probes to undergo strand displacement reaction, forming the CRISPR / Cas12a recognition sequence.

[0038] II. Reagent combinations for detecting target RNA molecules by recognizing proximity and signal amplification in secretory RNA detection. 1. Human pancreatic cancer cells PANC-1 were seeded in DMEM high-glucose medium (phenol red-free) containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured in a 37°C, 5% CO2 incubator, maintaining stable humidity during the culture process. Cell growth was observed regularly, and the medium was changed as needed. When the cells reached approximately 80% confluence and were ready for passage, the cell culture supernatant was collected for subsequent secreted RNA detection; DMEM high-glucose medium (phenol red-free) served as a control. 2. Preparation of reagent combinations (miRNA) for the detection and recognition of neighboring RNAs and signal amplification. 1) Before the reaction, Cas12a protein was incubated with amplification gRNA (nucleotide sequence as shown in SEQ ID NO.6 in Table 1) and recognition gRNA (nucleotide sequence as shown in SEQ ID NO.7 in Table 1) at room temperature for 20 min in 5 mM MgCl2 to form Cas12a-gRNA complex. 2) The amplification probe (nucleotide sequence as shown in SEQ ID NO.9 in Table 1) should be annealed before use. Place the 2.5 μM amplification probe solution in a buffer containing 20 mM Tris-HCl, 50 mM NaCl and 10 mM MgCl2 and pre-incubate at room temperature for 4-5 h before use. 3) Mix miR-21 recognition probe 1 (nucleotide sequence as shown in SEQ ID NO.1 in Table 1), miR-21 recognition probe 2 (nucleotide sequence as shown in SEQ ID NO.2 in Table 1), self-blocking PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO.5 in Table 1), the Cas12a-gRNA complex obtained in step 1), the fluorescent reporter probe (nucleotide sequence as shown in SEQ ID NO.8 in Table 1), the amplification probe obtained in step 2), and 1×NEB buffer r2.1 to obtain a reagent combination (miRNA) for detecting secreted RNA, recognizing neighboring RNA, and amplifying signals. The final concentrations of miR-21 recognition probe 1 and miR-21 recognition probe 2 were both 30 nM, the final concentration of the self-blocking PAM hairpin probe was 1 nM, the final concentration of Cas12a protein was 100 nM, the final concentration of amplification gRNA was 50 nM, the final concentration of recognition gRNA was 50 nM, the final concentration of fluorescent reporter probe was 500 nM, and the final concentration of amplification probe was 100 nM. 3. Preparation of reagent combinations (glycoRNA) for detecting secreted RNA, identifying proximity and amplifying signals. The glycoRNA recognition probe 1 (nucleotide sequence as shown in SEQ ID NO. 3 in Table 1), glycoRNA recognition probe 2 (nucleotide sequence as shown in SEQ ID NO. 4 in Table 1), self-closing PAM hairpin probe (nucleotide sequence as shown in SEQ ID NO. 5 in Table 1), Cas12a-gRNA complex (prepared in the same way as described above), fluorescent reporter probe (nucleotide sequence as shown in SEQ ID NO. 8 in Table 1), incubated amplification probe (prepared in the same way as described above), and 1×NEB buffer 2.1 were mixed to obtain the reagent combination (glycoRNA) for detecting secreted RNA, recognizing neighboring RNA, and amplifying signals. The final concentrations of glycoRNA recognition probe 1 and glycoRNA recognition probe 2 were both 30 nM, the final concentration of the self-blocking PAM hairpin probe was 1 nM, the final concentration of Cas12a protein was 100 nM, the final concentration of amplification gRNA was 50 nM, the final concentration of recognition gRNA was 50 nM, the final concentration of fluorescent reporter probe was 500 nM, and the final concentration of amplification probe was 100 nM.

[0039] 4. Mix the cell culture supernatant (or control medium) collected in step 1 with either the reagent combination (miRNA) for detecting and recognizing neighboring RNA and amplifying signals obtained in step 2 or the reagent combination (glycoRNA) for detecting and recognizing neighboring RNA and amplifying signals obtained in step 3. The amount of cell culture supernatant (or control medium) added should be 1 / 10 of the total volume of the mixed solution. React at 37°C for 1 hour. Characterize the reaction product using an enzyme-linked immunosorbent assay (ELISA) instrument. Set the excitation wavelength to 488 nm and the emission wavelength to 520 nm, and detect the signal at 520 nm.

[0040] The results are as follows Figure 3 As shown, the signal in the control culture medium was significantly smaller than that in the experimental group. This result demonstrates that after the CRISPR / Cas12a recognition sequence was formed in the experimental group, the Cas12a system was activated and cleaved the fluorescent reporter probe and amplification probe, thereby releasing the fluorescent signal and amplifying the signal, and thus enabling the detection of miRNA / glycoRNA molecules.

[0041] III. Droplet-based single-cell secreted RNA detection method based on reagent combinations for proximity recognition and signal amplification Example 1: Detection of secreted miRNAs in single cells 1. Preparation of reagent combinations for detecting secreted RNA, including proximity recognition and signal amplification. The preparation method is the same as in step 2, to obtain a reagent combination (miRNA) for detecting and recognizing neighboring RNA and amplifying signals.

[0042] 2. Constructing a single-cell droplet system 1) After culturing PANC-1 cells to 80% confluence, collect the cells, wash them with PBS buffer, and then use Hoechst working solution (2 μg / mL, 1 Cell nuclei were stained with PBS buffer at 37°C for 15 min, and then resuspended in the reagent combination (miRNA) obtained in step 1 for detecting and amplifying neighboring secreted RNA, forming an aqueous system. The final cell concentration in the aqueous system was 2 × 10⁻⁶. 6 cells / mL; 2) Add 50 μL of the aqueous phase system obtained in step 1) and 250 μL of QX200™ Droplet Generation Oil for EvaGreen (as the oil phase) to a 15 mL centrifuge tube, and vortex at 3000 rpm for 30 s to form an oil-in-water droplet system, so that the cells are randomly encapsulated in the droplets to form single-cell droplets.

[0043] 3. Detection of secreted miRNAs by single cells The single-cell droplet system formed in step 2 was incubated at 37°C to allow the cells to secrete miRNA molecules in the droplet microenvironment for 0.5–4 h.

[0044] 4. Detection and analysis of secreted RNA at the single-cell level Fluorescence microscopy imaging was performed on the droplet system after incubation in step 3, and the results are as follows: Figure 4 As shown, individual cells do not interfere with each other within the droplet. After the cells secrete the target molecule (miRNA) within the droplet, the target molecule (miRNA) can be recognized by the detection system. This result proves that the method can achieve rapid detection of secreted miRNA at the single-cell level.

[0045] Example 2 Detection of GlycoRNA Secreted by Single Cells 1. Preparation of reagent combinations for detecting secreted RNA, including proximity recognition and signal amplification. The preparation method is the same as in step 2.3, to obtain a reagent combination (glycoRNA) for detecting and recognizing neighboring RNA and amplifying signals.

[0046] 2. Constructing a single-cell droplet system 1) After culturing PANC-1 cells to 80% confluence, collect the cells, wash them with PBS buffer, and then use DiD working solution (1 μM, 1 Cell membranes were stained with PBS buffer at 37°C for 15 min, and then resuspended in the reagent combination (glycoRNA) obtained in step 1 for detecting and amplifying neighboring cells and signals of secreted RNA, forming an aqueous system. The final cell concentration in the aqueous system was 2 × 10⁻⁶ cells / mL. 6 cells / mL; 2) Inject 50 μL of the aqueous phase system obtained in step 1) and 250 μL of QX200™ Droplet Generation Oil for EvaGreen (as the oil phase) into the microfluidic chip channel respectively. Droplets are generated under continuous phase shearing, achieving single-cell droplet spacing. Figure 5 ).

[0047] 3. Detection of secreted glycoRNAs by single cells The single-cell droplet system formed in step 2 was incubated at 37°C to allow the cells to secrete glycoRNA molecules in the droplet microenvironment for 0.5–4 h.

[0048] 4. Detection and analysis of secreted RNA at the single-cell level Fluorescence microscopy imaging of the droplet system yielded the following results: Figure 6 As shown, individual cells do not interfere with each other within the droplet. After the cells secrete the target molecule (glycoRNA) within the droplet, the target molecule (glycoRNA) can be recognized by the detection system. This result proves that the method can achieve rapid detection of secreted glycoRNA at the single-cell level.

[0049] Single-cell secreted RNA was detected in different cell lines, and the secretion levels of different cell lines were compared and analyzed by statistically analyzing the fluorescence signal intensity of droplets, as detailed below: Example 3 The difference from Example 2 is that the cells used in step 2, 1) are HeLa cells. The other steps are the same as in Example 2.

[0050] Example 4 The difference from Example 2 is that the cells used in step 2, 1) are MCF-7 cells. The other steps are the same as in Example 2.

[0051] Statistical analysis of fluorescence signals was performed on the droplet systems obtained in Examples 2 to 4, and the results are as follows: Figure 7 As shown, differences in fluorescence signal intensity can be observed between different cell lines, indicating that this method can be used to compare the secreted glycoRNA levels of different cell lines.

[0052] Single-cell glycoRNA secretion was detected after cell enzymatic or drug treatment to analyze the effect of different treatment conditions on cellular RNA secretion and further verify the applicability of the detection system. The specific steps are as follows: Example 5 The difference from Example 2 is that in step 2, after treating the cells with RNase Cocktail (including RNase A and RNase T1), the cells are resuspended in a reagent combination for detecting and amplifying the proximity of secreted RNA.

[0053] The specific steps are as follows: RNase A (500 U / mL) and RNase T1 (20000 U / mL) are diluted 50 times with 1×PBS and then the cells are treated at 37℃ for 30 min.

[0054] The other steps are the same as in Example 2.

[0055] Example 6 The difference from Example 2 is that in step 2, after treating the cells with PNGase F, they are resuspended in a reagent combination for detecting and amplifying the proximity of secreted RNA.

[0056] The specific steps are as follows: treat cells with PNGase F (10 U / 100 μL, dissolved in HBSS buffer) at 37℃ for 30 min.

[0057] The other steps are the same as in Example 2.

[0058] Example 7 The difference from Example 2 is that in step 2, 1) the cells are treated with the glycosylation inhibitor NGI-1 and then resuspended in a reagent combination for detecting and amplifying the proximity of secreted RNA.

[0059] The specific steps are as follows: Add 10 μM NGI-1 (dissolved in DMSO) to the culture medium and treat the cells for 24 h.

[0060] The other steps are the same as in Example 2.

[0061] Statistical analysis of fluorescence signals was performed on the droplet systems obtained in Examples 5-7, with the droplet system from Example 2 (without any enzyme or drug treatment) serving as a control. Results are as follows: Figure 8 As shown, there are significant differences in fluorescence signals in droplets under different treatment conditions, indicating that this method can be used to analyze the changes in glycoRNA secreted by cells under different treatment conditions.

[0062] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A reagent combination for detecting and identifying proximity and amplifying signals in secreted RNA, characterized in that, include: The splitting recognition probes include recognition probe 1 and recognition probe 2, which can work together to recognize the target secreted RNA and trigger a neighboring hybridization reaction. Self-sealing PAM hairpin probes can undergo strand substitution reactions triggered by adjacent hybridization reactions to form CRISPR / Cas12a recognition sequences; The CRISPR / Cas12a system includes the Cas12a protein and gRNA bound to it, wherein the gRNA includes amplification gRNA and recognition gRNA. The Cas12a protein can recognize and cleave fluorescent probes and amplification probes. The amplification gRNA can recognize the single-stranded DNA released after the amplification probe is cleaved. The recognition gRNA can recognize the double-stranded DNA structure formed after the self-closing PAM hairpin probe strand is replaced. Fluorescent reporter probes are nucleic acid probes containing both fluorescent and quenching groups; The amplification probe can participate in the signal amplification reaction after activation by the CRISPR / Cas12a system.

2. The reagent combination for detecting and amplifying neighboring signals in secreted RNA according to claim 1, characterized in that, It also includes buffer solutions.

3. The reagent combination for detecting and amplifying neighboring signals in secreted RNA according to claim 1, characterized in that, When the target secreted RNA is modified RNA, recognition probe 1 can specifically recognize the modified structure of the target secreted RNA molecule, and recognition probe 2 can specifically recognize the RNA sequence region of the target secreted RNA molecule. When the target secreted RNA is unmodified RNA, recognition probe 1 can specifically recognize a conserved specific nucleic acid sequence region of the target secreted RNA molecule, and recognition probe 2 can specifically recognize another conserved specific nucleic acid sequence region of the target secreted RNA molecule that does not overlap with the binding region of recognition probe 1 and is adjacent to it.

4. The reagent combination for detecting and identifying proximity and amplifying signals in secreted RNA according to claim 1, characterized in that, When the target secreted RNA is glycoRNA, the nucleotide sequence of recognition probe 1 is shown in SEQ ID NO.3, and the nucleotide sequence of recognition probe 2 is shown in SEQ ID NO.4; when the target secreted RNA is miRNA, the nucleotide sequence of recognition probe 1 is shown in SEQ ID NO.1, and the nucleotide sequence of recognition probe 2 is shown in SEQ ID NO.

2.

5. The reagent combination for detecting and identifying proximity and amplifying signals in secreted RNA according to claim 1, characterized in that, In the fluorescent reporter probe, the fluorescent group and quencher group are FAM and BHQ-1, or Cy3 and BHQ-2, or Cy5 and BHQ-3.

6. The reagent combination for detecting and amplifying neighboring signals in secreted RNA according to claim 1, characterized in that, The nucleotide sequence of the self-sealing PAM hairpin probe is shown in SEQ ID NO.5, the nucleotide sequence of the amplification gRNA is shown in SEQ ID NO.6, the nucleotide sequence of the recognition gRNA is shown in SEQ ID NO.7, the nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO.8, and the nucleotide sequence of the amplification probe is shown in SEQ ID NO.

9.

7. The application of the reagent combination for detecting secreted RNA by means of any one of claims 1 to 6 in the detection of secreted RNA in single cells.

8. A method for detecting secreted RNA from single cells in droplets based on reagent combinations that identify proximity and signal amplification, characterized in that, Includes the following steps: 1) The reagent combination for detecting and identifying proximity and amplifying signals for secretory RNA as described in any one of claims 1 to 6 is mixed with the cell suspension to form an aqueous system; 2) Mix the aqueous phase system with the oil phase to form an oil-in-water droplet, which randomly encapsulates cells in the droplet to form a single-cell droplet; 3) Incubate single-cell droplets to induce the cells to secrete target RNA molecules within the droplets. A combination of reagents for detecting and amplifying the secreted RNA, along with neighboring reagents, is used to identify the secreted RNA and amplify the signal, generating a fluorescent signal. 4) Perform microscopic imaging of droplets and combine it with image analysis to achieve single-cell level detection and analysis of secreted RNA.

9. The method for detecting droplet-based single-cell secreted RNA based on a reagent combination for recognizing proximity and signal amplification according to claim 8, characterized in that, In step 1), the reagent combination for detecting and amplifying the neighboring and signal of secreted RNA has the following final concentrations: recognition probe 1 and recognition probe 2 are both 30-50 nM; the self-blocking PAM hairpin probe has a final concentration of 1-100 nM; the Cas12a protein has a final concentration of 100 nM; the amplification gRNA has a final concentration of 50 nM; the recognition gRNA has a final concentration of 50 nM; the fluorescent reporter probe has a final concentration of 500 nM; and the amplification probe has a final concentration of 100 nM.

10. The method for detecting droplet-based single-cell secreted RNA based on a reagent combination of proximity recognition and signal amplification according to claim 8, characterized in that, In step 2), the mixing method is through vortex oscillation or microfluidic chip droplet generation.

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