Method for detecting cell surface RNA glycosylation based on flow cytometry

Abstract

The invention provides a method for detecting cell surface RNA glycosylation based on flow cytometry, and belongs to the field of RNA glycosylation detection. The method comprises the following steps: carrying out azido sugar labeling on RNA on the surface of a cell by utilizing a metabolic pathway, coupling an azido group with a DBCO-fluorescent probe based on a click chemistry reaction, and detecting a fluorescent signal by utilizing flow cytometry, so as to realize efficient and sensitive detection on cell RNA glycosylation. The method is easy and convenient to operate and high in flux, has good repeatability and quantification capacity, provides powerful technical support for functional research of cell surface RNA glycosylation, is suitable for researching the effect of cell surface RNA glycosylation in immune regulation and control, and provides a new technical means for further revealing a cell communication mechanism related to RNA glycosylation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of RNA glycosylation detection, and particularly relates to a method for detecting cell surface RNA glycosylation based on flow cytometry. BACKGROUND

[0002] Glycosylation is a post-translational modification that plays a crucial role in molecular metabolism and cellular function. Especially on the cell surface, glycosylated proteins and lipids not only serve as mediators of signal transduction, but also as markers for cell recognition and interfaces for interaction with the external environment. Glycosylation is a ubiquitous cellular modification that encompasses all life forms studied, including prokaryotes and eukaryotes. Interestingly, recent studies have shown that RNA can also be glycosylated and distributed on the cell membrane. These glycosylated small RNAs not only play a role inside the cell, but are also localized on the cell surface, where glycosylated binding receptors recognize them. In 2023, in situ imaging of cell membrane surface RNA glycosylation was successfully achieved using ARPLA technology, revealing the spatial distribution of glycosylated RNA at the subcellular level and observing its migration process within the cell. In addition, other research groups further studied and elucidated the role of glycosylated RNA in immune responses. Most glycosylated RNAs exist in a stable form on the outer surface of neutrophils, and these surface RNAs are essential for recruiting neutrophils in mouse inflammatory tissues, revealing important mechanisms for regulating the expression level of glycosylated RNA. Therefore, the discovery of RNA glycosylation on the cell membrane not only deepens researchers' understanding of glycosylation modification, but also provides a new perspective for studying cell-cell interactions, mechanisms of disease occurrence, and future drug development.

[0003] However, there is currently a lack of efficient, sensitive and reproducible technical means to detect cell surface RNA glycosylation. The commonly used detection means is Northern blot (NB), but this method has many limitations. First, the operation process of NB is complicated, involving gel preparation, sample denaturation, electrophoresis, membrane transfer and probe hybridization, etc. The overall experimental period is long and the efficiency is low. Second, the membrane transfer and hybridization process is sensitive to operating conditions, and is prone to human error, affecting the stability and reproducibility of the results. In addition, formaldehyde, MOPS buffer and other commonly used in NB experiments are toxic, which is not conducive to laboratory safety management. Patent "Glycosylated RNA imaging method based on RNA and sugar double metabolic co-labeling" (Application number: 202411817683.X). The present application takes metabolic markers Ac4ManNAz and 5-EU as an example, labels azide and alkyne on the cell surface sugar and RNA respectively, and then connects DBCO-AF647 and N3-AF488 to azide and alkyne through click chemistry. The two fluorescent groups label the same glycosylated RNA to produce FRET signal. Laser confocal microscope detection can observe green cell images in the fluorescence donor channel, red cell images in the fluorescence acceptor channel, and yellow cell images in the FRET channel, thereby realizing the precise positioning of cell surface RNA, sugar and glycosylated RNA. However, this method is complex and requires the same glycosylated RNA to be labeled with 2 metabolic markers to be detected, which may result in a low detection result. Moreover, the distribution and specific conformation of RNA on the cell membrane are not clear, and the distance between adjacent RNAs may be too close, resulting in mutual activation between adjacent RNAs instead of fluorescence polarization due to glycosylation of RNA molecules. Similarly, the azide label and alkyne label within the RNA molecule may be far apart in space or blocked by proteins on the cell membrane, resulting in no yellow fluorescence signal. These factors will lead to inaccurate detection results of the above method. More importantly, this method can only be semi-quantitative.

[0004] In contrast, flow cytometry has significant advantages such as simple operation, high detection throughput, strong sensitivity, and the ability to obtain single-cell level information. It does not require gel transfer process, and can complete quantitative detection of large-scale samples in a short time, significantly improving experimental efficiency and data reliability. Therefore, establishing a flow cytometry-based RNA glycosylation detection method will provide more efficient and reproducible technical support for studying whether cells exist and their potential functional mechanisms. SUMMARY

[0005] The existing detection methods such as Northern blot are complicated in operation and limited in sensitivity, and it is difficult to meet the requirements of high-throughput and single-cell analysis. Based on this, the present application provides a method for detecting RNA glycosylation on the surface of cells based on flow cytometry, which belongs to the field of RNA glycosylation detection. The method refers to using metabolic pathways to label the RNA on the surface of cells with azido sugar, and based on the click chemistry reaction, the azido group is coupled with the DBCO-fluorescent probe, and then the fluorescence signal is detected by flow cytometry, thereby realizing efficient and sensitive detection of RNA glycosylation on the surface of cells. The method is simple in operation, high in throughput, has good repeatability and quantitative capacity, provides strong technical support for the functional research of RNA glycosylation on the surface of cells, is suitable for studying the role of RNA glycosylation on the surface of cells in immunoregulation, and provides a new technical means for further revealing the cell communication mechanism related to RNA glycosylation.

[0006] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0007] On the one hand, the present application provides a method for detecting RNA glycosylation on the surface of cells, which comprises the following steps:

[0008] (1) labeling RNA with a metabolic marker;

[0009] (2) coupling reaction of fluorescent probe and labeled group on RNA molecule;

[0010] (3) detecting fluorescence intensity by flow cytometry.

[0011] It should be understood that existing researches have shown that glycosylated RNA (glycoRNA) is mainly located on the surface of cells (yan A. Flynn, Kayvon Pedram, Stacy A. Malaker, Pedro J. Batista, Benjamin A. H. Smith, Alex G. Johnson, Benson M. George, Karim Majzoub, Peter W. Villalta, Jan E. Carette, Carolyn R. Bertozzi, Small RNAs are modified with N-glycans and display on the surface of living cells, Cell), and the present application also verifies this conclusion by Northern blot technology and flow cytometry, so the detection method provided by the present application is mainly used for detecting glycosylated RNA on the surface of cells.

[0012] Further, the metabolic marker is any one or more of Ac4GalNAz, Ac4GlcNAz, Ac4ManNAz, Ac4GlcNAl, Ac4ManNAl, Ac4GalNAl. The metabolic marker is used to label the glycosylated RNA after incubation with the cell, and the glycosylated RNA is labeled with a tag group after being metabolized by the cell. It can be understood that the type of the tag group is determined by the metabolic marker, and specifically, Ac4GalNAz, Ac4GlcNAz, Ac4ManNAz enable the target RNA molecule to be labeled with an azido group after being metabolized by the cell, and Ac4GlcNAl, Ac4ManNAl, Ac4GalNAl enable the glycosylated RNA to be labeled with an alkyne group. In some ways, the metabolic marker is Ac4ManNAz, and the labeled glycosylated RNA is labeled with an azido group.

[0013] Further, the concentration of the fluorescent probe is 1-100 μM; preferably 5-15 μM; more preferably 5 μM. Further, the fluorescent probe is composed of a reaction group and a fluorescent group; the fluorescent group is a hydrophobic fluorescent group or a hydrophilic fluorescent group.

[0014] The present application uses a hydrophilic fluorescent group and a hydrophobic fluorescent group to label the target RNA respectively, and it is found that in the case of detecting the same target, the hydrophilic fluorescent group (AF647) produces a higher fluorescence signal value, which is closer to the result of Northern blot, and will not be endocytosed by the cell, so the hydrophilic group is preferred. Further, the fluorescent group is a hydrophilic fluorescent group; the hydrophilic fluorescent group is any one or more of FITC, TRITC, Cy3, Cy5.5, Cy7, PE, APC, AF647, AF680, AF750.

[0015] In some ways, the hydrophilic fluorescent group is AF647. The excitation wavelength and emission wavelength of the AF647 (Alexa Fluor 647) are 650 nm and 668 nm respectively, which belongs to the dye in the Far-Red / NIR region. AF647 has a very high molar extinction coefficient, good fluorescence quantum yield, and excellent light stability. The AF647 dye molecule is usually modified with a sulfonate group, which makes AF647 have excellent hydrophilicity and water solubility.

[0016] Further, the reaction group is any one or more of -DBCO, -N3, -C≡C-H, DIBO, -SH, -CH=CH2, a tetrazine group, a tetrazole group, -SO2F.

[0017] It needs to be understood that the role of the reaction group is to carry out high-efficiency chemical reaction with the labeling group on the RNA molecule, and therefore, a suitable reaction group needs to be selected according to the type of the labeling group, such as when the labeling group is an azide group, a DBCO group is used as the reaction group, and when the labeling group is an alkyne group (-C≡C-H) or an alkene group (-CH=CH2), a thiol group (-SH) is used as the reaction group. Based on this, the coupling reaction is also determined by the types of the labeling group and the reaction group.

[0018] Further, the labeling group is generated by a metabolic marker through an intracellular metabolic pathway; the labeling group is any one or more of -DBCO, -N3, -C≡C-H, DIBO, -SH, -CH=CH2, a tetrazine group, a tetrazole group, and -SO2F. In other words, the metabolic marker directly or indirectly determines the types of the labeling group, the reaction group, and the coupling reaction.

[0019] Further, after the coupling reaction is completed, the RNA needs to be captured.

[0020] Further, the RNA is captured by using magnetic beads; the amount of the magnetic beads is 1-20 μL, preferably 10-12 μL, and more preferably 10 μL. Further, the time for capturing the RNA is 5 min-1 h, preferably 15-20 min, and more preferably 20 min.

[0021] Preferably, the metabolic marker is Ac4ManNAz; the fluorescent probe is composed of a DBCO group and an AF647 fluorescent group; the concentration of the fluorescent probe is 5 μM; and the labeling group is -N3.

[0022] In another aspect, the present application provides a use of a hydrophilic fluorescent group for improving the accuracy of the detection result of RNA glycosylation, characterized in that the hydrophilic fluorescent group is any one or more of FITC, TRITC, Cy3, Cy5.5, Cy7, PE, APC, AF647, AF680, and AF750.

[0023] In summary, glycosylation, as an important post-translational modification, not only exists on proteins and lipids, but also exists on RNA, and plays a function on the cell membrane surface according to recent research. The present application establishes a cell surface RNA glycosylation detection method based on flow cytometry, and lays a foundation for the research in the field of RNA glycosylation.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. By introducing azido sugar metabolic markers in cell culture, combined with the click chemistry reaction of DBCO-fluorescent probe, this method realizes the specific labeling and high sensitivity detection of glycosylated RNA on the cell membrane surface.

[0026] 2. Using flow cytometry, the expression level of RNA glycosylation in different cell types can be quickly quantitatively analyzed, which has the advantages of simple operation, good repeatability and suitability for large-scale sample detection.

[0027] 3. This method provides a reliable technical platform for the functional study of cell surface glycosylated RNA, and has important significance for revealing the role of RNA glycosylation in cell communication, immune response and disease mechanism. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0029] Figure 1 : Flow chart of flow cytometry for detecting cell surface RNA glycosylation;

[0030] Figure 2 : Confocal imaging of N3 labeling on the membrane surface of HeLa cells using DBCO-AF647;

[0031] Figure 3 : Confocal imaging of N3 labeling on the membrane surface of LOVO cells using DBCO-AF647;

[0032] Figure 4 : Confocal imaging of N3 labeling on the membrane surface of TH-29 cells using DBCO-AF647;

[0033] Figure 5 : Quantitative analysis of cell membrane surface N3 using flow cytometry, wherein the first row is a flow displacement map, and the second row is a fluorescence statistical map; data represent mean ± standard deviation, n = 3;

[0034] Figure 6 : Quality detection of RNA extracted from cells using Qsep100 automatic nucleic acid analysis system;

[0035] Figure 7RNA imaging of HeLa cells, wherein, lane 1 (L1): RNA marker; lane 2 (L2): HeLa cell group treated with 100 mM Ac4ManNAz for 48 h (experimental group); lane 3 (L3): HeLa cell group without any treatment (negative control group); lane 4 (L4, experimental group) and lane 5 (L5, negative control group) correspond to the RNA samples in L2 and L3 lanes, respectively, which are transferred to membrane, labeled with DBCO-biotin, and combined with streptavidin-IR800, and the red arrow in the figure points to the region where glycosylated RNA exists;

[0036] Figure 8 RNA imaging analysis of HeLa cell-derived RNA based on Northern blot technology, which is sequentially labeled with Ac4ManNAz and treated with RNase Cocktail, wherein, lane 1 (L1): RNA marker; lane 2 (L2): HeLa cell group treated with 100 mM Ac4ManNAz for 48 h and digested with RNase Cocktail (experimental group); lane 3 (L3): HeLa cell group treated with 100 mM Ac4ManNAz for 48 h but not digested with RNase Cocktail (control group); lane 4 (L4, experimental group) and lane 5 (L5, negative control group) correspond to the RNA samples in L2 and L3 lanes, respectively, which are transferred to membrane, labeled with DBCO-biotin, and combined with streptavidin-IR800;

[0037] Figure 9 Detection results of RNA treated with RNase Cocktail before extraction based on Northern blot technology;

[0038] Figure 10 Influence of different types of dyes on detection results of N3-labeled glycosylated RNA; wherein, hydrophilic type refers to AF647-DBCO, and hydrophobic type refers to Cy5-DBCO;

[0039] Figure 11 Influence of different concentrations of DBCO-AF647 dye on detection results of N3-labeled glycosylated RNA; 5 μM, 10 μM and 15 μM concentrations of DBCO-AF647 probe were used to label N3-modified RNA, and then RNA-specific magnetic beads were used for capture and purification, and the fluorescence signal intensity was detected by flow cytometry, wherein, Fig. a is a flow cytometry fluorescence shift diagram, and Fig. b is a corresponding fluorescence intensity statistical analysis diagram;

[0040] Figure 12 Influence of magnetic bead dosage on detection results of N3-labeled glycosylated RNA;

[0041] Figure 13 :The effect of capture time on the detection results of glycosylated RNA with N3 labeling;

[0042] Figure 14 :Based on flow cytometry, RNA detection results after coupling and RNase Cocktail treatment;

[0043] Figure 15 : Based on flow cytometry, RNA detection results were obtained after RNase Cocktail treatment before RNA extraction. DETAILED DESCRIPTION

[0044] The present invention is further elaborated below in conjunction with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0046] Example 1: Experimental method

[0047] 1.1 Cellular glucose metabolism markers

[0048] HeLa (human cervical cancer cells) and HT-29 (human colon adenocarcinoma cells) were obtained from the Shanghai Cell Bank, and LOVO (human colorectal cancer cells) were obtained from the American Type Culture Collection (ATCC). HeLa cells were cultured in DMEM medium (Basal Media), and other cell lines were cultured in RPMI-1640 medium (Basal Media). All cell culture media were supplemented with 10% (volume ratio) fetal bovine serum (FBS, Gibco). 1-1.2×10 6 (preferably 1.2×10 6 HeLa, HT-29, and LOVO cells (100 μg each) were seeded in 100 mm culture dishes containing the corresponding culture medium and cultured overnight at 5% CO₂, 37°C, and 95% humidity. To promote the formation of azide groups (N₃) on the cell surface, the cells were incubated with 100 μM tetraacetylazidemannoamine (Ac₄ManNAz, Aladdin, T303546-250 mg) in a 37°C CO₂ incubator for 48 h.

[0049] Unless otherwise specified, the present invention uses Ac4ManNAz to label the sugar metabolism of HeLa cells.

[0050] 1.2 Cell fixation and staining

[0051] For adherent cells, first remove the cells that have been successfully labeled with azide (N3) groups from the incubator. After removing the culture medium, carefully add 4% paraformaldehyde fixing solution (Biological Industries) to ensure complete coverage of the entire cell sample, and fix at room temperature for 10 to 20 min (preferably 15 min). After fixation is complete, to thoroughly remove residual paraformaldehyde, place the cells on a shaker and add an appropriate amount of DPBS buffer (Biosharp) to rinse the cells, rinse 3 times, 5 min each time, to ensure thorough washing. Next, add DAPI working solution (Biological Industries) to the fixed cells and stain at room temperature for 5 to 20 min (preferably 15 min). DAPI is a fluorescent dye commonly used for visualization of cell nuclei. After staining is complete, to remove unbound DAPI dye, rinse the cells again using DPBS buffer, with the cells placed on a shaker, rinse 3 times, 5 min each time.

[0052] 1.3 Confocal laser scanning microscopy imaging

[0053] To observe the distribution of N3 groups in cells, first seed cells (total number 1-1.2 x 10 6 cells in a confocal dish and incubate the dish at 5% CO2, 37°C, 95% humidity overnight to promote cell attachment and growth; then, treat the cells with 0 or 100 μM Ac4ManNAz for 48 h to introduce N3 groups into the cells; after treatment, wash the cells twice with DPBS buffer to remove unreacted Ac4ManNAz; then, incubate the cells at 37°C in the corresponding fresh culture medium containing 20 nM DBCO-Cy5 (a fluorescent probe that can specifically label azide groups through click chemistry) (CONFLUORE) for 1 h to achieve fluorescent labeling; after incubation, wash the cells twice with DPBS buffer in the dark to remove unbound fluorescent probes; then, fix the cells with 4% paraformaldehyde fixing solution for 10 min to preserve the cell structure and the position of the fluorescent label; after fixation, wash the cells again twice with DPBS buffer, then use 4,6-diamidino-2-phenylindole (DAPI) for cell nucleus staining to provide staining contrast for subsequent cell nucleus observation; finally, use a confocal laser scanning microscope (Nikon A1 HD25, made in Japan) for observation, specifically, use 405 nm and 633 nm lasers to excite DAPI and Cy5 fluorescent dyes, respectively, to observe and record the distribution of N3 groups on the cells.

[0054] 1.4 Flow cytometry characterization of cell membrane azido labeling

[0055] To verify whether the N3 group is successfully labeled on the cell membrane using flow cytometry, the method of N3 labeling cells described above (step 1.1) was adopted. Specifically, the cells were first treated with 0 or 100 mM Ac4ManNAz for 48 h to introduce azido groups onto the cell membrane (as described in step 1.1); after treatment, the cells were washed twice with DPBS buffer to remove unreacted substances; then, the cells were incubated with fresh culture medium containing 20 nM DBCO-AF647 at 37°C for 1 h. DBCO-AF647 can specifically bind to N3 through click chemistry to achieve fluorescent labeling of the cell membrane with N3 groups; after incubation, the cells were washed twice with DPBS in the dark to ensure the removal of all unbound fluorescent probes; then, the cells were resuspended in 1 mL of CTS buffer pre-cooled at 4°C, and the fluorescence signal intensity was detected using a flow cytometer (Beckman Coulter) to quantitatively evaluate the labeling efficiency of N3 on the cell membrane. The present application collected 10,000 cells and analyzed the data using FlowJo (v10.8.1) software. TM TrypLE TM Select enzyme (Gibco) was used to digest adherent cells to separate them from the surface of the culture dish; after centrifugation (1000 rpm, 3 min) to collect the cells, the cells were washed twice with DPBS buffer, and finally 1 x 10 5 cells were resuspended in DPBS buffer for flow cytometry (Beckman Coulter) analysis to detect the intensity of the fluorescence signal and quantitatively evaluate the labeling efficiency of N3 on the cell membrane. The present application collected 10,000 cells and analyzed the data using FlowJo (v10.8.1) software.

[0056] 1.5 RNA extraction and purification

[0057] 1.5.1 For adherent cells, the culture medium was removed and washed with 1 x DPBS buffer; then, 1 mL of RNA lysis solution TRIzol (Invitrogen) was added to 5 x 10 6 to 1 x 10 7 cells.

[0058] 1.5.2 Transfer the lysate to a 1.5 mL centrifuge tube, mix by vortexing or repeatedly pipetting to ensure complete lysis of the cells, then place on ice for 5 min; then add 20% by volume of chloroform (Yonghua, CAS: 67-66-3) to the solution, after high-speed shaking with a vortex for 15 s, a milky emulsion is formed; place the emulsion at 4°C for 5 min, then centrifuge at 4°C at 12,000 x g for 15 min, at this time, the solution will have a clear layering phenomenon, and will be divided into three layers, namely the colorless aqueous phase on top, a white middle layer, and a red organic phase on the bottom, carefully transfer the aqueous phase to another new enzyme-free centrifuge tube with a pipette.

[0059] 1.5.3 After transfer, add an equal volume of 4°C pre-cooled isopropanol (Shu Shi, 40064360 CAS: 67-63-0), mix the solution slowly by inverting, then place at 4°C for 10 min; again centrifuge at 4°C at 12,000 x g for 10 min, at this time, a white precipitate can be observed in the centrifuge tube, which is the RNA.

[0060] 1.5.4 After carefully removing the supernatant from the centrifuge tube with a pipette, add 1 mL of 75% ethanol prepared with enzyme-free water, resuspend the precipitate by tapping the tube bottom, slowly invert several times, then place at room temperature for 5 min; centrifuge at 4°C at 12,000 x g for 5 min, and as cleanly as possible discard the supernatant to minimize impurity residues; in a clean environment, leave the centrifuge tube open to dry for a few minutes, avoiding over-drying of the RNA, which would make it difficult to dissolve.

[0061] 1.5.5 Add an appropriate amount (preferably 20 μL) of enzyme-free water to dissolve the RNA, use a pipette to gently blow to speed up the dissolution of the RNA; after complete dissolution, take a small amount for quality testing, and store the rest in a -65 to -85°C environment.

[0062] 1.5.6 For protein digestion, mix 25 μg of RNA obtained in step 1.5.5 with 1 μg of proteinase K (Sigma, P2308-25MG), incubate at 37°C for 45 min, then repeat the RNA extraction steps of 1.5.2-1.5.5 for separation.

[0063] The purified column (Shengong) is used to purify the separated RNA as follows:

[0064] 1.5.7 Add 9 volumes of Buffer A to the RNA solution, mix well, then add 1 / 2 volume of anhydrous ethanol (Aladdin, E111991-500 ml), mix again; place the adsorption column in a collection tube, transfer the above mixture to the adsorption column, centrifuge at 12,000 rpm at room temperature for 30 s, and discard the waste liquid in the collection tube.

[0065] 1.5.8Subsequently, the adsorption column was placed back into the collection tube, and sequentially washed with 700 μL and 300 μL of RPE solution, each time centrifuged at 12,000 rpm for 30 s, and the waste liquid in the collection tube was removed; this step was repeated once.

[0066] 1.5.9Centrifuged at 10,000 rpm for 2 min to remove residual RPE solution.

[0067] 1.5.10The adsorption column was placed into a new RNase-free 1.5 mL centrifuge tube, 30 μL of DEPC-treated H2O was added to the center of the membrane, and after standing at room temperature for 2 min, centrifuged at 12,000 rpm for 1 min, and the obtained RNA solution was used for subsequent experiments or stored at -80°C.

[0068] 1.6 Treatment of cell surface RNA with RNase

[0069] 1.6.1First, the culture medium of adherent cells was removed, and washed twice with 1×DPBS.

[0070] 1.6.2To 5×10 6 to 1×10 7 cells, 1 mL of CTS TM TrypLE TM Select enzyme (Gibco) was added, and digested at 37°C for 10 min.

[0071] 1.6.3After digestion, the digested cells were transferred to a 1.5 mL centrifuge tube, centrifuged briefly to remove the supernatant, 1 mL of 1×DPBS buffer was added, and the cells were uniformly blown with a pipette gun, centrifuged briefly to remove the supernatant, and the operation was repeated once.

[0072] 1.6.4The RNase Cocktail (the components are RNase A 500 U / mL and RNase T1 20,000 U / mL) was diluted 50 times with the corresponding cell culture medium; after removing the supernatant in step 1.6.3, 500 μL of RNase Cocktail diluted with the culture medium was added (Thermo Fisher), the cells were uniformly blown with a pipette gun, and incubated at 37°C for 15 min.

[0073] 1.6.5After centrifugation, the supernatant was removed, 1 mL of 1×DPBS buffer was added, and the cells were uniformly blown with a pipette gun, centrifuged briefly to remove the supernatant, and the operation was repeated once.

[0074] 1.6.6After removing the supernatant, 1 mL of RNA lysis solution was added to the centrifuge tube. Subsequent steps for RNA extraction were performed according to the steps described in “1.5”.

[0075] It should be noted that this experimental step refers to the use of RNase Cocktail to treat the cell surface before extracting the RNA in the cell, in order to pre-clean the glycosylation-modified RNA on the cell membrane surface, and then prove that the glycosylation-modified RNA is mainly located on the cell membrane surface.

[0076] 1.7 Biotin labeling of RNA

[0077] 20 μg of RNA with N3 modification was mixed with DBCO-biotin (Biotin) at a final concentration of 500 μM at 37°C for 2 h. The specific operation is as follows: take 9 μL of RNA solution, add 1 μL of 10 mM DBCO-biotin solution, and then add 10 μL of enzyme-free water to ensure that the total reaction volume is 20 μL; after mixing, incubate at 37°C for 2 h to ensure complete reaction; after the reaction is completed, heat the reaction mixture at 60°C for 10 min to denature the possible secondary structure; then, quickly place the reaction tube on ice for 3 min to stop the reaction; finally, add this reaction system to the pre-prepared RNA purification column, and purify the RNA according to the standard process (as described in step “1.5”).

[0078] 1.8 RNA gel electrophoresis, blotting and imaging

[0079] Before performing RNA gel electrophoresis, the RNA needs to be denatured first. The specific operation steps are as follows: incubate the RNA sample at 55°C for 10 min to destroy its secondary structure; then, quickly cool the sample on ice for 3 min; add 1 x SybrGold (Thermo Fisher Scientific, S11494) and 1 x loading buffer (Shanghai Biotech, B548640-0001) to the denatured RNA sample, and dilute with sterile water to volume, mix well, and store for later use. If it is necessary to verify whether the N3 modification is RNA, add 1 μL RNase Cocktail (RNase A 500 U / mL and RNase T120,000 U / mL) to the sample, digest at 37°C for 30 min, then add 1 x SybrGold and 1 x loading buffer, dilute with sterile water to volume, mix well, and store for later use. The steps of gel electrophoresis are as follows: first, clean the gel preparation tools with 70% ethanol and dry them; prepare a 1% agarose-formaldehyde denaturing gel, i.e., add 0.55 g of agarose (Shanghai Biotech, CAS: 9012-36-6) to 36 mL of DEPC water, heat in a microwave oven until the agarose is completely dissolved, and cool the solution to 50-60°C; then, add 9 mL of 37% formaldehyde (Macklin, F809702-500 mL) and 5 mL of 10 x MOPS electrophoresis buffer (Shanghai Biotech, C516042-0001) to prepare the gel and insert the comb, and let it stand for at least 30 min to ensure that the gel is solidified; after the gel is solidified, place it in the electrophoresis tank and add 1 x MOPS electrophoresis buffer; carefully remove the comb, and add 20-40 μL of the prepared sample to each gel well; cover the electrophoresis tank cover, set the voltage to 110 V, and perform electrophoresis for 45 min; stop electrophoresis when the bromophenol blue dye migrates to 2 / 3 or 3 / 4 of the gel; and use a UV gel imager (GE, InmageQuant 800) to observe the distribution of total RNA in the gel.

[0080] The membrane transfer operation is as follows: place a glass plate horizontally on a plastic box, cut a piece of filter paper with the same width as the box but twice the length of the box, and place it horizontally on the glass plate with both ends dipping into 20xSSC solution (Shenguo, B548110-0200); trim the edges of the gel with a knife and cut a small piece at the top left corner as a marker; place the filter paper, gel, PVDF membrane, and more filter paper in order, and finally place a weight on top to flatten; the membrane transfer process is carried out at 4°C for 22-24h; after the membrane transfer is complete, wash the membrane with 0.05mol / L NaOH solution for 1-2min, and then with 2xSSC for 1min. After the membrane is dried, cross-link the RNA to the PVDF membrane with UV-C light; the blocking and staining steps are as follows: block the PVDF membrane with PBS (Odyssey Blocking Buffer) at 25°C for 45min; dilute streptavidin-IR800 (Li-Cor Biosciences, 926-32230) to 1:10,000 and stain the PVDF membrane at 25°C for 30min; then, wash the PVDF membrane with 1xPBS containing 0.1% Tween-20 for 3 times, 5min each time, to remove excess streptavidin; finally, scan the PVDF membrane on a Typhoon multifunctional biomolecular imager and use Image J software for image quantitative analysis.

[0081] 1.9 Flow cytometry detection of glycosylated RNA

[0082] Take 20μg of N3-labeled RNA (the steps for labeling RNA with N3 are described in step 1.1) or RNA without N3 labeling and mix with DBCO-AF647 dye (Click Chemistry Tools, CAT#1302) at 37°C for 2h. The specific operation is as follows: take 20μg of RNA, add DBCO-AF647 dye with a final concentration of 5μM, and supplement with sterile enzyme-free water to a total reaction volume of 15μL; after mixing, incubate at 37°C for 2h to ensure complete reaction; after the reaction is complete, heat the reaction mixture at 60°C for 10min to denature the possible secondary structure; quickly place the reaction tube on ice for 3min to stop the reaction; add 10μL of purified magnetic beads (Biyun Tian) to capture the RNA, incubate for 20min, place it on the magnetic stand to remove the supernatant, and wash it with 80% ethanol for 3 times to ensure that all unbound fluorescent probes are removed; finally, add 400μL of sterile enzyme-free water, mix well with a pipette gun, and then detect the fluorescence signal intensity of DBCO-AF647 by flow cytometry (Beckman Coulter), and analyze the data with FlowJo (v10.8.1) software.

[0083] It should be noted that the above experimental method is the method for detecting cell surface RNA glycosylation based on flow cytometry provided by the present application.

[0084] Example 2: Principle and process of flow cytometry for detecting cell surface RNA glycosylation

[0085] RNA glycosylation refers to a post-translational modification process in which a sugar molecule is covalently linked to an RNA molecule (usually a nucleotide base) through a glycosidic bond. It is similar to protein glycosylation but occurs on RNA and is one of the hotspots in recent RNA epigenetic (epitranscriptomic) research, and is considered an important mechanism for regulating RNA function. The types of research that have been more thoroughly studied mainly involve two types of glucose modification: first, β-D-glucose is connected to the N4 nitrogen atom of 5-methylcytosine (m 5 C) through a β-glycosidic bond to form N4-glycosyl-5-methylcytidine (glc 4 m 5 C); second, β-D-glucose is connected to the N6 nitrogen atom of N6-methyladenine (m 6 A) through a β-glycosidic bond to form N6-glycosyl-N6-methyladenosine (glc 6 m 6 A). In general, RNA glycosylation is a rapidly developing research field, and its role in gene expression regulation, cell physiology and disease is the focus of current research.

[0086] Due to the many limitations of traditional Northern blot technology in terms of operational complexity, repeatability and safety, there is an urgent need to develop a new method that is efficient, sensitive and can be used to detect cell surface RNA glycosylation. The present application develops a detection strategy based on flow cytometry for analyzing the glycosylation modification of cell surface RNA. The principle of the detection method is as follows: After Ac4ManNAz (full name: 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine) enters the cell, it is metabolically converted into CMP-Sia5Az, which is used as a substrate by sialyltransferases in the Golgi apparatus to transfer Sia5Az at the end of the glycan chain to newly synthesized glycoproteins or glycolipids, so that the cell surface and secreted glycoconjugates containing terminal sialic acid (mainly glycoproteins) have a biorthogonal azido group (-N3); then, using the biorthogonality of the azido group, a reporter group (such as a fluorescent dye, biotin, etc., the present application selects a fluorescent group) is covalently linked to the molecule with the azido group through a click chemistry reaction (CuAAC or SPAAC, the present application selects CuAAC), and then the fluorescence of the cells is detected by flow cytometry, thereby achieving analysis of the glycosylated RNA of the cells.

[0087] The experimental procedure of the above strategy is as follows: first, the cell is treated with Ac4ManNAz for 48 hours to introduce azido group on the cell membrane; then total RNA is extracted by Trizol method, and the RNA with N3 modification is subjected to click reaction with a fluorescent probe (preferably DBCO-AF647) with DBCO group; then, the RNA molecules are enriched by magnetic beads, and the fluorescence signal is detected by flow cytometry. Figure 1 ).

[0088] It should be understood that N3 modification is the most universal, sensitive and biocompatible labeling method in the study of glycosylated RNA at present, and can realize efficient, visualized and enriched analysis in combination with click chemistry, and is the first choice for glycoRNA (glycosylated RNA) detection; DBCO probe is one of the most commonly used and effective probes for N3 modified molecules (such as glycosylated RNA) at present. As the core reaction group in copper-free click chemistry, it has a series of significant advantages, especially suitable for live cell labeling, sugar chain metabolism labeling and glycoRNA research.

[0089] According to actual needs and experimental purposes, those skilled in the art can modify glycosylated RNA by cell metabolism using different sugars, and then select a fluorescently labeled labeling molecule (DBCO probe in the present application) according to the group (N3 group in the present application) modified on the glycosylated RNA.

[0090] Example 3: N3 labeling of cell surface using Ac4ManNAz molecules

[0091] Ac4ManNAz will be metabolized into sialic acid with azido group, which will be connected to the sugar chain structure of RNA under the action of glycosylase; these azido-containing sugar metabolites (such as SiaNAz) are finally modified on specific RNA, thereby realizing azido labeling of glycoRNA (glycosylated RNA). Therefore, theoretically, after 48h of Ac4ManNAz treatment of cells and the sugar metabolism process, the cell membrane surface will be labeled with N3. In order to verify the effectiveness of this labeling, HeLa cells were co-incubated with DBCO-AF647 in this embodiment, and after the incubation was completed, the cells were fixed and stained with DAPI, and imaging analysis was performed (as described in Example 1). This embodiment sets up three groups of experiments: 1) untreated HeLa cell group (negative control); 2) HeLa cell group without sugar treatment but co-incubated with BDCO-AF647 dye; 3) HeLa cell group treated with sugar (Ac4ManNAz) and co-incubated with BDCO-AF647 dye.

[0092] As Figure 2As shown in FIG. 1A, no fluorescence signal was detected in the Cy5.5 channel for untreated HeLa cells; while a weak fluorescence signal was observed for HeLa cells that were not treated with sugar but co-incubated with BDCO-AF647 dye, which might be due to non-specific adsorption or endocytosis; in contrast, strong fluorescence signal was observed on the cell membrane surface for HeLa cells that were treated with sugar and co-incubated with BDCO-AF647 dye, indicating that N3 groups were successfully labeled on the surface of HeLa cells, which is consistent with the theory. Meanwhile, the above conclusion was also verified in colorectal cancer cell lines LOVO and TH-29 cells (specific steps are as described above), as shown in FIG. 1B and FIG. 1C, respectively. Figure 3 and Figure 4 As shown in FIG. 1B and FIG. 1C, the imaging results of the two cell lines were similar to those of HeLa cells, which preliminarily proved that N3 labeling on the cell surface could be achieved using Ac4ManNAz.

[0093] Further, flow cytometry was also used in this embodiment to quantitatively analyze the N3 labeling on the cell membrane of HeLa, HT-29 and LOVO cells. The experimental design was consistent with the previous imaging analysis, i.e., first using Ac4ManNAz to label the sugar molecules on the cell membrane of HeLa / HT-29 / LOVO cells, and then using DBCO-AF647 to label the N3 groups for subsequent analysis. The specific experimental steps are as described in Embodiment 1, and the experimental results are shown in FIG. 2. Figure 5

[0094] The results showed that regardless of the type of cells, only after the cells were treated with Ac4ManNAz, a significant shift was observed, which not only indicated the successful labeling of N3 again, but also further verified the universality of Ac4ManNAz for labeling sugar molecules, i.e., it could label sugar molecules on the surface of various cells. In order to more accurately evaluate the change of fluorescence signal, this embodiment quantitatively analyzed the fluorescence value obtained by flow cytometry. The results showed that Figure 5 ), compared with the negative control, only after the N3 was successfully labeled on the cell membrane, the intensity of the fluorescence signal was significantly improved, which was consistent with the microscope imaging data.

[0095] In summary, this embodiment fully confirmed that Ac4ManNAz could label N3 on the sugar molecules on the cell membrane surface of various cells, and these experimental results laid a foundation for subsequent experiments and analysis.

[0096] Embodiment 4: Detection of glycosylated RNA in cells using Northern blot technology

[0097] ​Example 3 successfully labeled the sugar molecules on the cell surface through the cell glycometabolism process, but the modification object of the sugar molecules on the cell surface is uncertain because the sugar molecules can be modified on RNA, protein and lipid. Based on this, it is assumed that the RNA on the cell surface is glycosylated, and then the sugar chain of the RNA will be labeled with N3 group after the cell glycometabolism. To further verify the assumption, the present application extracts the RNA of the cells treated with Ac4ManNAz, and uses protease K to digest the RNA to process the protein on the sugar chain of the RNA that may be labeled with N3 (the specific steps are the same as described in Example 1); then, the Qsep100 automatic nucleic acid analysis system is used to detect the quality of the processed RNA, that is, the quality of the RNA is evaluated by the RNA quality value (RQN), and the score range is 1-10, the higher the score, the better the RNA quality. In addition, the DV200 value is also used for evaluation in this example, and generally, when the DV200 value is above 75%-80%, it indicates that the RNA quality is good.

[0098] As shown in Figure 6 , the RNA quality test results extracted from three different cell lines (HeLa, HT-29 and LOVO cells) show that the RQN value of the RNA of HeLa and LOVO cells is as high as 9.7, indicating that the RNA quality is excellent; in contrast, the RQN value of the RNA extracted from HT-29 cells is 7.5, which is slightly lower than that of the other two cells, indicating that the RNA quality is slightly worse; however, the DV200 value of the RNA extracted from the three cells is above 90%. Overall, the quality of the RNA extracted in this example is very good in general and can be used for subsequent experiments.

[0099] Next, the RNA of the HeLa cells treated with Ac4ManNAz and passed the quality inspection was coupled with DBCO-biotin in solution. After the coupling reaction, the total RNA was analyzed by RNA gel electrophoresis and imaged under UV irradiation to evaluate the quality and integrity of the sample. Then, the RNA in the agarose-formaldehyde denaturing gel was transferred to a PVDF membrane and fixed on the membrane by UV crosslinking to ensure the stability of the RNA sample in the subsequent processing. In order to detect and image the glycosylated RNA band, the RNA modified with N3 group was subjected to click chemistry reaction with DBCO-biotin (i.e. N3-DBCO reaction), and the product was purified to remove unreacted DBCO-biotin. In theory, the RNA extracted from the cells without Ac4ManNAz treatment does not have N3 label, so it cannot be modified by biotinylation. Then, the membrane was stained with streptavidin labeled with fluorescent marker (IR-800). The high affinity between streptavidin and biotin enables specific binding, so that the target RNA can be accurately imaged by Northern blot analysis through fluorescence detection. The experiment also set a negative control, i.e. the HeLa cell group without Ac4ManNAz treatment (the specific steps are the same as described in Example 1). The specific results are shown in Figure 7 .

[0100] As can be seen from the results, under UV light, the total RNA of both the control group (lane 3 in Figure 7 ) and the experimental group (lane 2 in Figure 7 ) can be successfully imaged, and the bands are clear. This result is consistent with the RNA quality inspection result. The Northern blot detection result shows that (lanes 4-5 in Figure 7 ) only lane 4 has a clear band, which preliminarily proves that Ac4ManNAz can label RNA with N3 group in cells, and confirms the existence of RNA glycosylation. It should be understood that there is also a faint background band in lane 5, which may be because the RNA modified with N3 group is also produced in normal cells.

[0101] To further prove that the object modified with N3 group is RNA, the RNA modified with azido group was digested by RNAase cocktail (the specific steps are the same as described in Example 1), and then the quality of the RNA was preliminarily detected by RNA gel electrophoresis, and the RNA imaging analysis was performed by Northern blot technique. The experiment used the RNA sample group without RNAase cocktail treatment as a control, and the experimental steps were the same as described in Example 1. The specific results are shown in Figure 8 .

[0102] The results show that the RNase cocktail can effectively degrade the total RNA extracted from the Ac4ManNAz-labeled HeLa cells Figure 8 Correspondingly, no band appears in the sample after RNase cocktail digestion, while a clear band appears in the control, which means that the Ac4ManNAz successfully labels the glycosylated RNA in the cells, and also verifies the existence of RNA glycosylation.

[0103] Although the above results fully prove the existence of glycosylated RNA in cells, they cannot directly prove that RNA glycosylation mainly occurs on the cell surface, in other words, the localization of glycosylated RNA detected by Northern blot technology cannot be confirmed. In order to further verify that glycosylated RNA exists on the cell membrane surface, the present embodiment uses RNase cocktail enzyme (Thermo) to specifically digest the RNA on the surface of HeLa cells after N3 labeling, extracts the RNA of the cells, and uses RNA gel electrophoresis and Northern blot technology to analyze the imaging of the RNA (the specific experimental steps are the same as those described in Example 1), and the results are shown in Figure 9 .

[0104] Under ultraviolet light, the total RNA can be successfully imaged in both the control group (lane 1 in Figure 9 ) and the experimental group (lane 2 in Figure 9 ), and the bands are clear, indicating that the cell RNA is successfully extracted and has high quality, which is suitable for subsequent detection. The Northern blot results show that after the cell surface is treated with RNase cocktail enzyme, the glycosylated RNA band obviously disappears (lane 4 in Figure 9 ), and correspondingly, the glycosylated RNA band exists in the normal experimental group (lane 3 in Figure 9 ), which further proves that the glycosylated modified RNA mainly exists on the cell membrane surface, which is consistent with the previous research; and it also proves that the Northern blot technology detects the glycosylated RNA on the cell surface.

[0105] Example 5: Optimization of metabolic markers in flow cytometry

[0106] Although Northern blot analysis can be used to detect the glycosylation modification of cell surface RNA, this method has the disadvantages of complicated operation, long time consumption and low sensitivity. Therefore, after confirming that Ac4ManNAz can be used to label glycosylated RNA, the present application develops a new method based on fluorescent labeling and flow cytometry, which can quickly and sensitively detect the glycosylation modification of cell surface RNA. This method has the advantages of simple operation, high detection throughput and strong sensitivity by fluorescent labeling of specific RNA and combining magnetic bead purification and flow cytometry analysis. The specific steps are shown in "1.9" of Example 1. At the same time, the method is also optimized, including sugar metabolism markers, fluorescent dyes and their concentrations, and the way of capturing RNA. The results obtained by flow cytometry in the optimization process are compared with the results of Northern blot-based experiments to evaluate the accuracy of the flow cytometry detection results, wherein the same RNA sample is detected by the two experimental techniques, which is described in detail in Examples 5-8.

[0107] Since the metabolic markers largely determine the types of labeling groups on the glycosylated RNA molecules and the labeling efficiency, they are the key factors to improve the accuracy of the results of flow cytometry detection. Therefore, the types of the markers were optimized in this embodiment, i.e. Ac4GalNAz, Ac4GlcNAz, Ac4ManNAz, Ac4GlcNAl, Ac4ManNAl and Ac4GalNAl were respectively used to label the glycosylated RNA of HeLa cells. Since the types of labeling groups generated by different sugar metabolites are different, the probes used are also different. Specifically, the labeling groups generated by Ac4GalNAz, Ac4GlcNAz and Ac4ManNAz are azido groups, and the corresponding probe is DBCO-AF647 probe; the labeling groups generated by Ac4GlcNAl, Ac4ManNAl and Ac4GalNAl are alkyne groups, and the corresponding probe is SH-AF647 probe. The specific experimental steps are described in Embodiment 1. The results show that glycosylated RNA signals are not detected when labeled by Ac4GlcNAz, and the other five molecules can successfully label glycosylated RNA. Further, in terms of labeling groups, compared with alkyne groups, the fluorescence signal generated by azido groups is stronger and closer to the results of Northern blot, which may be because azido groups are relatively stable, while alkyne and thio groups are more easily oxidized, which in turn leads to the inability of the probe to efficiently label the target, or the need to increase the amount of probe to achieve the same effect as the azido-DBCO group. Further, among the two sugar metabolites that can generate azido labeling groups (Ac4GalNAz and Ac4ManNAz), the signal-to-noise ratio of the Ac4ManNAz group is the highest and closest to the results of Northern blot, i.e. Ac4ManNAz is preferred to label the target molecules.

[0108] Embodiment 6: Optimization of fluorescent dyes in flow cytometry

[0109] In this embodiment, the fluorescent dyes were optimized, i.e. the hydrophilic dye AFDye 647 (AF647)-DBCO (Click Chemistry Tools, CAT#1302) and the hydrophobic dye Cy5-DBCO (CONFLUORE) were coupled with azido (N3) modified RNA, then the labeled RNA was captured and purified by RNA specific magnetic beads, and the fluorescence signal intensity was detected by flow cytometry (the specific experimental steps are described in Embodiment 1).

[0110] The results are as follows: Figure 10As shown, the fluorescence signal value of the hydrophilic dye (AF647) detected by flow cytometry is higher than that of the hydrophobic dye (Cy5), which is closer to the experimental results of Northern blot, and it can be seen that the hydrophilic dye reacts more fully and efficiently. In addition, the luminescence of the above two dyes was observed under a confocal microscope, and it was found that HeLa cells showed significant endocytosis of Cy5 fluorescent groups, which would increase the background fluorescence signal and interfere with the interpretation of specific signals on the membrane surface, while AF647 fluorescent genes would not have such phenomenon. Therefore, the hydrophilic dye is preferred in subsequent experiments, specifically, DBCO-AF647 dye.

[0111] Example 7: Optimization of the concentration of fluorescent dyes in flow cytometry

[0112] Example 6 proves that using AF647 as a fluorescent group is more conducive to the detection of glycosylated RNA. Based on this, this example optimizes the use concentration of DBCO-modified fluorescent probes (DBCO-AF647) with 6 μg of azido (N3)-modified RNA as the labeled object, and the concentration is set to 5 μM, 10 μM and 15 μM. Then, the labeled RNA is captured and purified by RNA-specific magnetic beads, and the fluorescence signal intensity is detected by flow cytometry. For specific steps, refer to “1.9” in Example 1, and the specific detection results are shown in Figure 11 .

[0113] The results show that Figure 11 ) DBCO-AF647 dyes with a concentration of 5 μM to 15 μM are suitable for flow cytometry detection of glycosylated RNA. It is worth noting that the higher the concentration of the dye, the more sensitive the detection result, that is, the higher the concentration of the dye, the lower the signal-to-noise ratio. This may be because the higher the concentration of the dye, the stronger the non-specific adsorption, resulting in a decrease in the signal-to-noise ratio. In other words, when the concentration of DBCO-AF647 is 5 μM, the best signal-to-noise ratio is obtained. Therefore, in subsequent experiments, the concentration of DBCO-AF647 probe is preferably set to 5 μM for specific coupling labeling with N3-modified RNA.

[0114] Example 8: Optimization of captured RNA in flow cytometry

[0115] After the click reaction of the RNA with N3 modification with the fluorescent probe with DBCO group (preferably DBCO-AF647), the RNA molecules need to be enriched. Specifically, the present application uses magnetic beads to enrich the target RNA. If the RNA enrichment efficiency is low, the subsequent detection value will be low, therefore, in order to improve the efficiency of enriching RNA, the amount of magnetic beads (5 μL, 8 μL, 10 μL) and the capture time were optimized respectively, wherein 6 μg of RNA with N3 group derived from HeLa cells was used for the following optimization experiments (the specific experimental steps are the same as those described in Example 1). First, the amount of magnetic beads was optimized, the amount of magnetic beads was set to 5 μL, 8 μL and 10 μL respectively, and the capture time was 10 min, and the capture results are shown in Figure 12 The results show that, under the same capture time, with the increase of the amount of magnetic beads, the total amount of RNA enriched also increases, and when the amount of magnetic beads reaches 10 μL, the total amount of RNA enriched does not increase, which may be because excessive magnetic beads can cause aggregation or sedimentation between particles, resulting in a decrease in effective surface area. In addition, too many magnetic beads can introduce more background binding, interfering with effective capture, therefore, the amount of 10 μL of magnetic beads is preferred for capture.

[0116] Then, under the optimal amount of magnetic beads (10 μL), the capture time was optimized, and the capture time was set to 5 min, 10 min, 15 min and 20 min respectively, and the results are shown in Figure 13 The capture time is positively correlated with the total amount of RNA enriched, and the capture time is finally selected to be 20 min. In summary, in order to maximize the enrichment of RNA, 10 μL of magnetic beads is preferred for capturing RNA in the subsequent experiments, and the capture time is 20 min, at this time, the capture efficiency of RNA is more than 50%. The capture efficiency of RNA still has a large space for improvement, which may be because the RNA is firmly combined on the magnetic beads, and it is not completely released in the elution step, or the magnetic beads are not completely settled in the magnetic separation step, resulting in the magnetic beads being sucked away.

[0117] Example 9: Effect of flow cytometry detection of glycosylated RNA

[0118] Based on the exploration of Examples 5-8, an optimized method for detecting glycosylated RNA based on flow cytometry was obtained. In this example, the method was used to detect the glycosylation modification of RNA in HeLa cells. There were three experimental groups, respectively, RNA+DBCO-AF647 group for detecting RNA without N3 label using DBCO-AF647 probe, RNA-N3+DBCO-AF647 group for detecting RNA with N3 label using DBCO-AF647 probe, and RNA-N3+RNase+DBCO-AF647 group for detecting RNA with N3 label but treated with RNase using DBCO-AF647 probe. The RNA of HeLa cells without any treatment was used as a negative control group (RNA group). The specific experimental steps were the same as those described in Example 1, and the results were shown in Figure 14 .

[0119] As can be seen from Figure 14 , compared with the RNA group and the RNA+DBCO-AF647 group, the fluorescence value of the RNA-N3+DBCO-AF647 group was significantly increased. Further, after RNase treatment, the fluorescence signal of N3 labeled RNA (RNA-N3+RNase+DBCO-AF647 group) was significantly weakened and was comparable to that of the RNA+DBCO-AF647 group, which indicated that the flow cytometry method could effectively detect the glycosylation modification of RNA on the cell surface, which was consistent with the result of Northern blot technology Figures 7-8 .

[0120] In order to verify the existence of RNA glycosylation on the cell membrane surface, HeLa cells were labeled with N3 in this example, and then RNase enzyme was used to specifically digest the RNA on the cell surface. After treatment, the RNA of the cells was extracted, and the fluorescence signal was detected by flow cytometry. The specific experimental steps were the same as those described in Example 1, and the experimental results were shown in Figure 15 .

[0121] As can be seen from Figure 15 , compared with the RNA-N3+647 group without RNase digestion, the fluorescence signal of N3 labeled RNA (RNase+Cell-RNA-N3+647 group) in the cells treated with RNase was significantly reduced, further proving that there was glycosylation modified RNA on the surface of HeLa cell membrane, i.e., the flow cytometry detected was the glycosylated RNA located on the cell membrane (cell surface), which was consistent with the result of Northern blot technology Figure 9 .

[0122] The foregoing presents and describes the basic principles and main features of the present application and the advantages thereof, it being apparent that the present application is not limited to the details of the above-described exemplary embodiments, but that it can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application, and that it is therefore intended to cover all the changes and modifications which fall within the meaning and scope of the equivalent elements of the claims, the claims being intended to cover all the equivalents of the product and method and the scope of protection of the present application being defined by the claims, it being intended, therefore, that all the changes and modifications which fall within the meaning and scope of the equivalent elements of the claims be embraced therein, any reference signs in the claims not being regarded as limiting the claims concerned.

Claims

1. A method for detecting cell surface RNA glycosylation, characterized in that: The method comprises the following steps: (1) Labeling RNA with metabolic markers; (2) The fluorescent probe undergoes a coupling reaction with the labeling group on the RNA molecule; (3) Detect fluorescence intensity using flow cytometry.

2. The method according to claim 1, wherein The metabolic marker is any one or more of Ac4GalNAz, Ac4GlcNAz, Ac4ManNAz, Ac4GlcNAl, Ac4ManNAl, and Ac4GalNAl.

3. The method according to claim 1, wherein The concentration of the fluorescent probe is 1-100 μM.

4. The method according to claim 1, wherein The fluorescent probe consists of a reactive group and a fluorescent group; the fluorescent group is a hydrophobic fluorescent group or a hydrophilic fluorescent group, and the reactive group is any one or more of -DBCO, -N3, -C≡CH, DIBO, -SH, -CH=CH2, tetrazine group, tetrazole group, and -SO2F.

5. The method according to claim 1, wherein The labeling group is generated by a metabolic marker through an intracellular metabolic pathway; the labeling group is any one or more of -DBCO, -N3, -C≡CH, DIBO, -SH, -CH=CH2, tetrazine group, tetrazole group, and -SO2F.

6. The method according to claim 1, wherein After the coupling reaction is completed, the RNA needs to be captured.

7. The method according to claim 6, wherein Magnetic beads are used to capture RNA; the amount of the magnetic beads used is 1 to 20 μL.

8. The method according to claim 7, wherein The time for capturing RNA is 5 minutes to 1 hour.

9. The method according to claim 1, wherein The metabolic marker is Ac4ManNAz; the fluorescent probe consists of a DBCO group and an AF647 fluorescent group; the concentration of the fluorescent probe is 5 μM; and the labeling group is -N3.

10. Use of a hydrophilic fluorescent group to improve the accuracy of RNA glycosylation detection results, characterized in that: The hydrophilic fluorescent group is any one or more of FITC, TRITC, Cy3, Cy5.5, Cy7, PE, APC, AF647, AF680, and AF750.

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