A method, system and device for purifying and screening tumor glycoRNA

Abstract

The present invention discloses a method, system and equipment for purifying and screening tumor glycoRNA, belonging to the field of bioinformatics and RNA purification and screening. The present invention provides a probe group for specifically purifying tumor glycoRNA, the present invention provides a method for purifying and screening tumor glycoRNA and the purified and screened tumor glycoRNA, the present invention provides a method for purifying polysaccharides in tumor glycoRNA and the purified tumor glycoRNA polysaccharides, the present invention provides a system for glycoRNA glycan analysis and a system for predicting glycoRNA three-dimensional structure, and related equipment and computer-readable storage media. The present invention also provides the use of the aforementioned probe group and the aforementioned method in the preparation of tumor glycoRNA purification, tumor glycoRNA screening, and tumor glycoRNA glycan purification products.

Description

Technical Field

[0001] The present invention belongs to the field of bioinformatics and RNA purification and screening, and specifically relates to a method, system and equipment for purifying and screening tumor glycoRNA. Background Art

[0002] Glycosylated RNA (glycoRNA) is a new type of polysaccharide-modified biomolecule discovered by Flynn et al. in 2021. It is widely present in various types of cells, including human cervical cancer cell lines Hela, 293T, and human T lymphocyte line H9. It is believed that it may play an important role in signal transduction, immune response, and tumor development. Flynn et al. separated glycoRNA through a series of extraction and purification processes, and then detected and identified the two components of glycoRNA: sialic acid polysaccharide molecules and small RNAs by liquid chromatography-mass spectrometry and small RNA sequencing technology.

[0003] When detecting and identifying multiple glycoRNAs derived from cells, the two components of glycoRNA are detected separately and sorted according to the content of the multiple small RNAs and glycans detected. However, the correspondence between the multiple small RNAs and glycans is unknown, which hinders further structural and functional studies of glycoRNA. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides a probe group for specifically purifying tumor glycoRNA, wherein the probe group comprises: glycoRNA U2 probe SEQ ID NO: 1, and glycoRNA U4 probe SEQ ID NO: 2.

[0006] The present invention provides a method for purifying and screening tumor glycoRNA, the method comprising:

[0007] 1) Labeling glycoRNA in cells;

[0008] 2) extracting and purifying the total cellular RNA labeled with glycoRNA to obtain purified total RNA;

[0009] 3) Purifying small RNA from the purified total RNA to obtain purified small RNA;

[0010] 4) Purify the purified small RNA using streptavidin magnetic beads to obtain crude purified glycoRNA;

[0011] 5) Purify the crude purified glycoRNA using glycoRNA-specific magnetic beads and elute to obtain purified tumor glycoRNA;

[0012] The glycoRNA-specific magnetic beads are prepared by the following method:

[0013] The magnetic bead dispersion was thoroughly mixed, and then the carboxyl-derivatized microbeads were transferred, the magnetic beads were separated by magnetic attraction, and the supernatant was removed; the magnetic beads were washed 2-3 times with R Buffer, and the magnetic beads were separated by magnetic attraction, and the magnetic beads were retained; R Buffer was transferred to dissolve the amino oligo, and the amino oligo was added to the magnetic beads, mixed, and rotated at room temperature; EDC solution dissolved in R Buffer was added, mixed, R Buffer was added to make up the volume, and the reaction was rotated at room temperature, and the magnetic beads were separated by magnetic attraction, and the magnetic beads were washed 2-3 times with W Buffer to obtain glycoRNA-specific magnetic beads;

[0014] In step 5), the crude purified glycoRNA is purified by glycoRNA-specific magnetic beads, which specifically includes the following steps:

[0015] The glycoRNA sample purified by streptavidin magnetic beads was denatured by incubation at 65°C, and the probe set described above was added to the denatured sample, hybridized at 37°C for 30 min, denatured at 50°C for 5 min, and hybridized at 37°C for 90-180 min; the prepared glycoRNA-specific magnetic beads were added, mixed and incubated at room temperature for 30 min, the magnetic beads were collected and the supernatant was removed, WashBuffer was added repeatedly, and the magnetic beads were collected and the supernatant was removed after shaking and washing at 37°C.

[0016] Furthermore, the magnetic bead dispersion is a PBS aqueous solution containing MagnaBind™ carboxyl-derivatized microbeads, with a pH value of >4.0.

[0017] Furthermore, the amino oligo is an amino RNA probe specifically complementary to the aforementioned probe set for specific purification of tumor glycoRNA.

[0018] Furthermore, the R Buffer is a reaction buffer, the components of which are 100 mM MES, pH 4.5.

[0019] Furthermore, the W Buffer is a washing buffer, the components of which are 1×PBS, pH 7.4.

[0020] Furthermore, the glycoRNA-specific magnetic beads were stored in S Buffer at 4° C. and the concentration of the magnetic beads was 10 mg / mL.

[0021] Furthermore, the S Buffer is a storage buffer, and its components are 1×PBS, pH 7.4, and 0.05% (w / v) Proclin 300.

[0022] Furthermore, the ratio of the carboxyl-derivatized microbeads to the magnetic bead dispersion is 10 mg:200 μL.

[0023] Furthermore, the ratio of amino oligo, R Buffer for dissolving amino oligo, EDC solution, and R Buffer to make up the volume was 3OD: 300 μL: 150 μL: 50 μL.

[0024] Furthermore, the concentration of the EDC solution is 100 mg / mL.

[0025] Further, the tumor includes solid tumors, specifically including Hodgkin's lymphoma, non-Hodgkin's lymphoma, lung cancer, breast cancer, ovarian cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, kidney cancer, prostate cancer, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, Wilms' tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, neuroblastoma, ganglion cell carcinoma, Cell neuroblastoma, ganglioneuroma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, leiomyosarcoma, laryngeal cancer, paranasal sinus cancer, tongue cancer, gingival cancer, cheek cancer, thyroid tumor, oral cancer, head and neck malignancies, urinary system malignancies, skin squamous cell carcinoma, melanoma, gastrointestinal stromal tumors, thymoma, thymic carcinoma, glioma, meningioma, medulloblastoma, neurofibroma, hamartoma, glioma.

[0026] Furthermore, the prepared glycoRNA-specific magnetic beads are specific for U2 and U4.

[0027] Furthermore, the elution refers to eluting the glycoRNA carried on the glycoRNA-specific magnetic beads, specifically including: adding RNA Elution Buffer to the glycoRNA-specific magnetic beads and mixing, heating at 95°C for 2 min, collecting the magnetic beads and the supernatant, adding RNA Elution Buffer again and mixing, heating at 95°C for 2 min, collecting the magnetic beads and the supernatant; adding the supernatant to the RNA purification column, adding Binding Buffer, centrifuging at 10000g for 20s, discarding the waste liquid, adding prep Buffer to wash once, washing twice with Wash Buffer, evacuating once, adding RNA free water droplets, incubating for 5min, centrifuging at 10000g for 20s, repeating once, and collecting the eluate.

[0028] Furthermore, the labeling of glycoRNA in the cell includes the steps of: dissolving Ac4ManNAz (MedChemExpress, HY-W728531) with DMSO to 500 mM, and then diluting it with DMSO to 100 mM. GalNAc and Gal are dissolved with sterile water to 500 mM and 50 mM, respectively, and diluted with sterile water to 100 mM and 10 mM; treating U87, LN229 or Hela cells in a 10 cm culture dish with Ac4ManNAz (1:1000 dilution, 10 ml culture medium plus 10 μl 100 mM Ac4ManNAz) at a final concentration of 100 μM for 0 and 24 hours, and then harvesting the cells to extract RNA. Gal (Sigma, G0750) (10 μM, 1:1000 dilution) and GalNAc (Sigma, A2795) (100 μM, 1:1000 dilution) were used as medium supplements and added simultaneously with Ac4ManNAz.

[0029] Further, the extraction and purification of total RNA of cells labeled with glycoRNA includes the following steps: extracting total RNA by Trizol method. Collecting cells, lysing and mixing in TRIzol, incubating the sample at 37°C for 10 minutes to further denature non-covalent interactions. Then adding 0.2 times volume of chloroform to extract RNA. Vortex mixing, and finally centrifuging at 4°C, carefully aspirating the aqueous phase, transferring it to a new tube, and mixing it with 2 times volume of anhydrous ethanol; adding the above solution containing total RNA to Zymo RNA purification and concentration column (Zymo Research, 1017), centrifuging at 10,000 xg for 20 s, discarding the waste liquid, washing once with 400 μL RNA preparation buffer and washing twice with 400 μL RNA washing buffer to wash and purify the RNA, and then centrifuging at 10,000 xg for 20 s. Elute RNA with two volumes of pure water. Add 1 μg of proteinase K to 25 μg of purified RNA, incubate at 37°C for 45 min, and purify again using a Zymo column. Zymo Spin IC and IIICG columns can bind 50-350 μg of total RNA. Select the column in each experiment based on the amount of RNA to be purified.

[0030] Further, the purification of small RNA from the purified total RNA includes the following steps: adding 2 ml RNAzol to each 15 cm culture dish cell; DNA / protein precipitation: fully oscillating and mixing, adding 0.4 ml water for every 1 ml RNAzol used, inverting and mixing, standing for 15 minutes, and centrifuging at 12000 g for 15 minutes; mRNA precipitation: adding 0.4 times the volume of 75% ethanol to the supernatant, inverting and mixing, standing for 10 minutes, and centrifuging at 12000 g for 8 minutes; mRNA washing: collecting the supernatant into a new tube, adding 0.4 ml 75% ethanol to the precipitate for washing, centrifuging at 8000 g for 2 minutes and discarding the supernatant, washing twice; mRNA dissolution: using DEPC water to dissolve mRNA; miRNA precipitation: adding 0.8 times the volume of isopropanol to the supernatant obtained by mRNA precipitation, inverting and mixing, standing for 30 minutes, and centrifuging at 12000 g for 15 minutes. Centrifuge for 15 minutes; miRNA washing: discard the supernatant, add 0.4 ml, 70% isopropanol to the precipitate, 8000g, 3min, wash twice; miRNA dissolution: use DEPC water to dissolve miRNA and purify it through Zymo purification column.

[0031] Further, the streptavidin magnetic bead purification of the purified small RNA includes the following steps: streptavidin magnetic bead purification of glycoRNA: 10 μL MyOne C1 streptavidin magnetic beads, with 50 ng / mL glycogen in 500 μL Biotin WashBuffer (10 mM Tris HCl pH7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Tween-20), incubated at 25°C for 1 hour for blocking, centrifuged at 4°C, 3000rpm for 2 minutes after blocking, placed on a magnetic stand, and discarded the supernatant; 500 μL Biotin WashBuffer respin magnetic beads, added to biotinylated miRNA, sealed with sealing film, rotated and mixed at 4°C for 2 hours, centrifuged at 3000rpm for 2 minutes, placed on a magnetic stand, collected supernatant, and subsequently passed through a purification column for loading; washing beads to remove unbound RNA: use 1mL Biotin Wash Wash twice with buffer, 3 minutes each time; Elute glycoRNA: Incubate biotinylated RNA with magnetic beads in 60 μl 0.75M NaCl, 10 mM EDTA and 10 mM Tris-Cl (pH 7.4) (prepared in advance) at 22°C for 5 minutes. Elute RNA by resuspending the magnetic beads in 1× volume of 5% b-mercaptoethanol (BME) solution (do not discard the solution in the previous step) and incubate at 22°C for 5 minutes, centrifuge at 3000rpm for 2 minutes, collect the supernatant, and purify glycoRNA through Zymo concentration purification column.

[0032] The present invention provides a tumor glycoRNA obtained by the above-mentioned method for purifying and screening tumor glycoRNA.

[0033] The present invention provides a method for purifying polysaccharides in tumor glycoRNA, the method comprising the following steps:

[0034] The tumor glycoRNA described above was diluted with RNA free water, PNGaseF and 10×GlycoBuffer 2 were added, incubated at 37°C for 18 hours, cooled to room temperature, and purified twice using a pretreated PGC-SPE column. The column was then washed once with 5% acetonitrile / 0.1% TFA, and the glycans were eluted with 1.5 mL, 4× 50% acetonitrile / 0.1% TFA. The sample was immediately frozen on dry ice and then lyophilized to dryness. After lyophilization, it was dissolved in 1 ml of water and dried again to obtain the glycans in the purified tumor glycoRNA.

[0035] Furthermore, the volume ratio of the RNA free water, PNGaseF, and 10× GlycoBuffer 2 is 8:1:1.

[0036] Further, the pretreatment step of the pretreated PGC-SPE purification column includes: pretreating each PGC-SPE purification column with reagents in the following order: 15ml 1M NaOH, 15ml HPLC grade water, 15ml 30% acetic acid, 15ml HPLC grade water, 15ml 50% acetonitrile / 0.1% trifluoroacetic acid (TFA) washing the column 3 times, 15m 5% acetonitrile / 0.1% trifluoroacetic acid washing the column 2 times to obtain a pretreated PGC-SPE purification column.

[0037] The present invention provides a kit for purifying polysaccharides in tumor glycoRNA used in the above-mentioned purification method, wherein the kit comprises: RNA free water, PNGaseF, 10× GlycoBuffer 2, PGC-SPE purification column, 5% acetonitrile / 0.1% TFA, 4×50% acetonitrile / 0.1% TFA, 1M NaOH, and 30% acetic acid.

[0038] The present invention provides a tumor glycoRNA glycan obtained by the above-mentioned method for purifying glycan in tumor glycoRNA.

[0039] The present invention provides a system for glycoRNA glycan analysis, the system comprising: a data acquisition unit for obtaining data of glycans in glycoRNA, wherein the data is the liquid chromatography data of tumor glycoRNA glycans as described above; a data processing unit for performing library identification analysis based on the data in the data acquisition unit; and a data output unit for outputting the results of the data processing unit to obtain the analysis results of glycoRNA glycans.

[0040] In some embodiments, the system for glycan analysis in glycoRNA includes the steps of identifying and analyzing glycans in glycoRNA using liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the specific steps are: drying the polysaccharide solution in a vacuum concentrator and then dissolving it in pure water. CH3I and DMSO are added to the solution, and NaOH micro-spiral columns are used for full methylation. The methylated glycans are extracted with chloroform and rinsed with water several times. The chloroform layer is then collected and concentrated to dryness in vacuum. The LC separation parameters were set as follows: analytical column: 360 μm OD × 75 μm ID, 75 cm; filler: Phenomenex Jupiter C18, 5 μm, 300 Å; isolation column: 360 μm OD × 200 μm ID, 5 cm; filler: Phenomenex Jupiter C18, 5μm, 300 Å; mobile phase: buffer A is a mixture of 99.9% H2O and 0.1% FA, and buffer B is a mixture of 99.9% ACN and 0.1% FA. The parameters of the tandem mass spectrometer were: mass resolution 22.5 k, data-dependent acquisition top 20, automatic gain control target value 1×10 5 , maximum ion injection time 200 ms, precursor ion isolation window 3 m / z, dynamic exclusion 50.0 s, step NCE 10%. Based on LC-MS / MS data, library identification was performed using Glyseeker software (FDR ≤ 1%).

[0041] The present invention provides a system for predicting the three-dimensional structure of glycoRNA, the system comprising: a data acquisition unit for obtaining data of glycoRNA, wherein the data is the sequencing data of the tumor glycoRNA described above; a data processing unit for performing three-dimensional structure prediction based on the data in the data acquisition unit; and a data output unit for outputting the result of the data processing unit to obtain the three-dimensional structure of the glycoRNA.

[0042] Furthermore, the three-dimensional structure prediction comprises the steps of: using RNAStructure to predict the secondary structure of glycoRNA, then importing the secondary structure into 3dRNA to construct the three-dimensional structure, and then using dynamics software for optimization.

[0043] Furthermore, the kinetic software used was Gromacs 5.0.

[0044] Furthermore, the simulation conditions of Gromacs 5.0 during optimization were as follows: force field Gomacs 54a7, water model SPC, temperature coupling of the simulation system was determined by the “Berendsen thermostat” method, pressure coupling was determined by the “manometer” method, and the reference pressure was 1.01×10 5 , periodic boundary conditions are adopted in all directions of the system.

[0045] Furthermore, the Gromacs 5.0 used the following steps during optimization: optimizing the side chains for 5000 steps in a simulated aqueous solution environment with the main chain fixed, and then performing a global optimization for 5000 steps in a simulated aqueous solution environment.

[0046] In some embodiments, the above-mentioned units or steps can achieve the effect through a computing device. In some embodiments, the units or steps can be concentrated on a single computing device. In some embodiments, the units or steps can be distributed on multiple computing devices, and multiple computing devices are linked by a network or other linking methods. In some embodiments, the computing device stores a program code for executing the above-mentioned units or steps, and the program code is stored in a storage device. In some embodiments, the units or steps can be prepared into integrated circuits separately, or they can be combined into a single integrated circuit.

[0047] The present invention provides a device, which includes a storage memory and a processor, wherein the storage memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the above-mentioned method for purifying and screening tumor glycoRNA, or the above-mentioned method for purifying polysaccharides in tumor glycoRNA is implemented.

[0048] Further, the memory includes read-only memory (ROM), phase-change random access memory (PRAM), static random access memory (SRAM), flash memory, random access memory (RAM), dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM), electrically erasable programmable read-only memory (EEPROM), static memory, and other types of random access memory, cache memory, registers, compact disk read-only memory (CD-ROM), digital versatile disk (DVD) or other optical storage devices, cassettes, other non-transitory media.

[0049] Furthermore, the processor includes a microprocessor, a microcontroller, a graphics processing unit (GPU), an application specific integrated circuit (ASIC), and a field programmable gate array (FPGA).

[0050] The present invention provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the computer program implements the above-mentioned method for purifying and screening tumor glycoRNA or the above-mentioned method for purifying polysaccharides in tumor glycoRNA.

[0051] The term "computer-readable storage medium" used in the present invention should be understood to include a single medium or multiple media (e.g., a centralized database or distributed database, and / or associated caches and servers) that store one or more instruction sets. The term "computer-readable storage medium" should also be understood to include any medium capable of storing or encoding an instruction set that is used to be executed by a machine and causes the machine to perform any one or more of the methods of the present invention. The term "computer-readable storage medium" should accordingly be understood to include, but is not limited to, solid-state memories and optical and magnetic media.

[0052] The present invention provides the use of the aforementioned probe set, the aforementioned glycoRNA purification and screening method, or the aforementioned glycoRNA polysaccharide purification method in the preparation of tumor glycoRNA purification, tumor glycoRNA screening, and tumor glycoRNA polysaccharide purification products.

[0053] Advantages and beneficial effects of the present invention:

[0054] The correspondence between the two components of glioma cell glycoRNA: small RNA and polysaccharides was found to clarify its specific composition; the three-dimensional structure of glioma cell glycoRNA was predicted using molecular docking simulation methods, providing a basis for further functional research on glycoRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure is the labeling and Nothern blot imaging result of glycoRNA, wherein A is the blot development result of anti-biotin streptavidin-HRP, B is the observation result of biotin signal in RNA samples of U87, LN229 and Hela cells treated with Ac4ManNAz, C is the effect of incubation / pre-incubation with DNase or RNase and RNase inhibitor (RNasin) on biotin signal, and D is the detection result of biotin signal in small RNA samples and large RNA samples in U87 and LN229 cells.

[0056] Figure 2 Figure 3 is a heat map, Venn diagram and pie chart of small RNA sequencing data of glycoRNA enriched in U87 and LN229 cells, where A is the small RNA expression analysis results in U87, LN229 and Hela cells with / without Ac4ManNAz treatment, B is the glycoRNA detection results in U87, LN229 and Hela cells, and C is the glycoRNA classification counting results in U87, LN229 and Hela cells.

[0057] Figure 3 It is a diagram of the analysis, screening and validation results of differentially expressed glycoRNA, where A is the RNA enriched in U87 and LN229 cells, and B is the qRT-PCR result.

[0058] Figure 4 Figure 1 is a graph showing the analysis of the glycans on glycoRNA U2 and U4 by liquid chromatography-mass spectrometry. A is the cluster analysis of U2 and U4 sugar chains in U87 and LN229 cells, B is the classification and percentage calculation of glycosides in LN229 and U87 cells, C is the ratio of tri-fucosylated glycosides detected in U87 and LN229 cells, D is the ratio of glycoside sialylation in U87 and LN229 cells, E is the ratio of mixed glycosides in U87 and LN229 cells, FG is the intersection of U2 and U4 glycoside sugar chain types in U87 and LN229 cell samples, HJ is the analysis of glycoside forms in U87 cells, KM is the analysis of glycoside forms in LN229 cells, and NP is U87-total, U87-U2, and U87-U4. QS is the analysis of the proportion of complex glycosides in the glycosides of the LN229-total, LN229-U2 and LN229-U4 groups.

[0059] Figure 5 This is the result diagram of analyzing and identifying the most abundant glycan components on glycoRNA U2 and U4. Among them, AB is the analysis of glycoside types in U87 and LN229 cells, and CH is the relative abundance ranking of glycosides in U2 or U4 samples.

[0060] Figure 6 The three-dimensional structure prediction results of glycoRNA U2 and U4 are shown in Figure 1, where A is the result of U2 / U4 covalent bond analysis and hydrogen bond analysis, and BE is the conformation of RNA-U2_LYL, RNA-U2_YLS, RNA-U4_MYL and RNA-U4_YLS.

[0061] 3D diagram, FI is the covalent binding result between RNA and glycoside.

[0062] Figure 7 Figure 1 is the qRT-PCR result after U2-ASO and U4-ASO treatment, where AD is the effect of U2-ASO on the expression level of U2 in U87 and LN229 cells, and EH is the effect of U4-ASO on the expression level of U4 in U87 and LN229 cells.

[0063] Figure 8 It is the qRT-PCR result diagram after U87 and LN229 cells were treated with Y5-ASO, where A is U87 cells and B is LN229 cells.

[0064] Fig. 9 The figures are the experimental results of CCK-8 and Ki67 after knocking down U2 and U4 in glioma cells, among which A is the cell activity detection result after knocking down U2 and U4 in U87, B is the cell activity detection result after knocking down U2 and U4 in LN229, and CD are the Ki67 cell fluorescence test results after using U2-ASO and U4-ASO in U87 and LN229 cells.

[0065] Fig.10 It is a systematic diagram of glycoRNA glycan analysis.

[0066] Fig.11 It is a system diagram for predicting the three-dimensional structure of glycoRNA.

[0067] Fig.12 It is a schematic diagram of a glycoRNA glycan analysis device or a glycoRNA three-dimensional structure prediction device. DETAILED DESCRIPTION

[0068] This specification describes specific embodiments. Other embodiments are within the scope of the invention. In some cases, the actions or steps described in the present invention can be performed in a different order than in the embodiments and still achieve the desired results.

[0069] Example

[0070] 1. Experimental methods

[0071] 1.1 Labeling of glycoRNA in cells

[0072] 1.1.1 Experimental Principle

[0073] Ac4ManNAz (N-acetyl-4-methylglucosamine-β-ethyl ester) is an azide-containing metabolic glycoprotein labeling reagent that can be taken up by cells and replace natural glucose molecules in cells, as a biomarker associated with glycosylation processes on the cell surface or in molecules. Ac4ManNAz, as a precursor sugar functionalized with a clickable azide group, is capable of metabolically labeling cells or animals, and once incorporated into cellular glycans, the azide sugar can react bioorthogonally with biotin probes for enrichment, identification, and visualization.

[0074] 1.1.2 Experimental procedures

[0075] Ac4ManNAz Catalog Number: HY-118297.

[0076] (1) Drug preparation: Ac4ManNAz (MedChemExpress, HY-W728531) was dissolved in DMSO to 500 mM, and then diluted to 100 mM with DMSO. GalNAc and Gal were dissolved in sterile water to 500 mM and 50 mM, respectively, and then diluted to 100 mM and 10 mM with sterile water.

[0077] (2) Cell treatment: U87, LN229 or Hela cells in 10 cm culture dishes were treated with Ac4ManNAz at a final concentration of 100 μM (1:1000 dilution, 10 ml culture medium plus 10 μl 100 mM Ac4ManNAz) for 0 and 24 h, and then the cells were harvested for RNA extraction. Gal ((Sigma, G0750) (10 μM, 1:1000 dilution) and GalNAc (Sigma, A2795) (100 μM, 1:1000 dilution) were used as culture medium supplements and added simultaneously with Ac4ManNAz.

[0078] 1.2 Extraction and purification of total cell RNA

[0079] (1) Extract total RNA using the Trizol method. Collect cells, lyse and mix in TRIzol, and incubate the sample at 37°C for 10 min to further denature non-covalent interactions. Then add 0.2 volumes of chloroform to extract RNA. Vortex to mix, and finally centrifuge at 4°C. Carefully aspirate the aqueous phase, transfer to a new tube, and mix with 2 volumes of anhydrous ethanol.

[0080] (2) Add the above solution containing total RNA to the Zymo RNA purification and concentration column (Zymo Research, 1017), centrifuge at 10,000 xg for 20 s, discard the waste liquid, wash once with 400 μL RNA preparation buffer and twice with 400 μL RNA washing buffer to purify the RNA, and then centrifuge at 10,000 xg for 20 s. Elute the RNA with two volumes of pure water. Add 1 μg of proteinase K to 25 μg of purified RNA, incubate at 37°C for 45 min, and purify again with the Zymo column. Zymo Spin IC and IIICG columns can bind 50-350ug of total RNA. Select the column in each experiment according to the amount of RNA to be purified.

[0081] 1.3 Purification of glycoRNA in cells

[0082] 1.3.1 Experimental Principle

[0083] The classic click reaction involves a copper-catalyzed azide-alkyne cycloaddition reaction to label and detect target molecules in cells or tissues. A disadvantage of this method is that the copper ions (Cu(II) and Cu(I)) generated in the presence of ascorbic acid or TCEP can harm cells, reduce the fluorescence of fluorophores, and impair their protein functions.

[0084] The key to copper-free click chemistry is DBCO (dibenzocyclooctyne). This eight-membered ring enables reactions with azide-modified molecules to occur without catalysts, temperature, or solvents, making it possible to study the surface of living cells and preventing copper-induced fluorescent proteins such as GFP in fixed and permeabilized cells. DBCO-Biotin contains DBCO and biotin groups, and can react with azide-labeled molecules or biomolecules through copper-free click chemistry to label the target molecule with biotin.

[0085] 1.3.2 Experimental procedures

[0086] (1) Proteinase K to remove protein contamination: Add 1 μg of proteinase K to every 25 μg of RNA and incubate at 37°C for 45 min.

[0087] (2) Column purification: Add 400 μl of binding buffer to the above RNA solution. Add Zymo RNA purification and concentration column, centrifuge at 10,000 x g for 20 seconds, discard the waste liquid, wash once with 400 μl RNA preparation buffer and twice with 400 μl RNA washing buffer, wash the RNA, discard the waste liquid, and centrifuge at 10,000 x g for 20 seconds. Place the purification column in a new RNA free EP tube, add 15-25 μl of RNase-free water to the center of the membrane, incubate for five minutes, centrifuge at 10,000 g for 20 seconds, and elute the RNA. Add the effluent back to the center of the membrane and centrifuge again. The effluent is the purified RNA.

[0088] (3) Biotinylated azide-labeled glycoRNA.

[0089] RNA in pure water was mixed with 1× volume of Gel Loading Buffer II (df GLBII, prepared as 95% formamide, 18mM EDTA, and 0.025% SDS) and 500 μM DBCO-biotin (typically these reactions were 10 μL dfGLBII, 9 μL RNA, 1 μL 10mM DBCO reagent stock). The reaction was incubated at 55°C for 10 min, after which 80 μL of water was added to stop the reaction, followed by 2× volume (200 μL) of RNA Binding Buffer and mixing, followed by 3× volume (300 μL) of 100% ethanol and mixing. Purification was performed using Zymo columns, as above, and analysis was performed by gel electrophoresis.

[0090] 1.4 Identification of glycoRNA by northern blot

[0091] 1.4.1 Experimental Principle

[0092] The RNA sample to be tested is subjected to agarose gel electrophoresis under denaturing conditions, and then the position in the gel is transferred to a nitrocellulose film or nylon membrane, fixed and then reacted with an RNA probe labeled with an isotope or other marker. The RNA sample must be denatured before and during electrophoresis, otherwise the molecular mass cannot be accurately determined. Denaturation is accomplished by formaldehyde and formamide, and glyoxal and dimethyl sulfoxide or methylbenzene (not recommended) can also be used.

[0093] 1.4.2 Experimental procedures

[0094] NorthernMax™ Kit Catalog Number: AM1940.

[0095] (1) Sample preparation: Add three times the volume of Formaldehyde Load Dye and one tenth the volume of 10× SYBR Gold nucleic acid gel dye (DMSO dilution, 1 ml DMSO plus 1 μl 10000× SYBR Gold nucleic acid gel dye) to the biotinylated glycoRNA. Incubate at 65°C for 15 min to denature the RNA and place on ice.

[0096] (2) Removal of RNase: Use RNaseZap to spray various instruments required for electrophoresis and membrane transfer, including electrophoresis tank, conical flask, gel plate, sample comb, measuring cylinder, etc., and use RNase-free water to wash away foam.

[0097] Prepare gel: Prepare 1% agarose formaldehyde denaturing gel. When cooled to about 55°C, add 18 ml of formaldehyde and 10 μl of 10000× SYBR Gold nucleic acid gel stain and mix well.

[0098] (3) Electrophoresis: Dilute 10× MOPS gel running buffer to 1× with RNase-free water and cover the gel with about 0.5-1 cm of running buffer. Add the above samples to a 1% agarose-formaldehyde denaturing gel and run electrophoresis at 110V for 45 min. Observe the total RNA with a UV gel imager.

[0099] (4) Transfer: Perform transfer according to the NorthernMax™ kit operating instructions.

[0100] (5) Development: After transfer, use ultraviolet light to crosslink RNA to the NC membrane. Irradiate the NC membrane directly with ultraviolet light, taking care not to dry the membrane. Soak the membrane in Odyssey Blocking Buffer, add streptavidin-HRP and incubate at 25°C for 30 min. Wash 3 times with TBST, 2 minutes each time.

[0101] (6) Photography: Use the Tanon 5200 series fully automatic chemiluminescence image analysis system for imaging analysis.

[0102] 1.5 Purification of small RNA from cells

[0103] 1.5.1 Experimental Principle

[0104] RNAzol (Sigma-Aldrich, R4533) is a highly efficient total RNA or small RNA extraction reagent that can be used for a variety of samples such as humans, animals, plants, bacteria and viruses. Based on the interaction of phenol and guanidine isothiocyanate with cellular components, RNAzol can separate RNA from other molecules in just one step, and purified RNA can be obtained without chloroform-induced phase separation.

[0105] 1.5.2 Experimental procedures

[0106] (1) Homogenization: Add 2 ml of RNAzol to each 15 cm culture dish of cells.

[0107] (2) DNA / protein precipitation: Vortex the mixture thoroughly. Add 0.4 ml of water for every 1 ml of RNAzol. Invert and mix. Let stand for 15 min. Centrifuge at 12,000 g for 15 min.

[0108] (3) mRNA precipitation: Add 0.4 times the volume of 75% ethanol to the supernatant, invert to mix, let stand for 10 minutes, and centrifuge at 12,000 g for 8 minutes.

[0109] (4) mRNA washing: Collect the supernatant into a new tube, add 0.4 ml of 75% ethanol to wash the precipitate, centrifuge at 8000g for 2 min, discard the supernatant, and wash twice.

[0110] (5) mRNA dissolution: Use DEPC water to dissolve mRNA.

[0111] (6) miRNA precipitation: Add 0.8 times the volume of isopropanol to the supernatant (step 3), invert to mix, let stand for 30 minutes, and centrifuge at 12,000 g for 15 minutes.

[0112] (7) miRNA washing: discard the supernatant, add 0.4 ml 70% isopropanol to the precipitate, and wash twice at 8000 g for 3 min.

[0113] (8) Dissolution of miRNA: Dissolve miRNA in DEPC water and purify using Zymo purification columns.

[0114] 1.6 Purification of glycoRNA using streptavidin magnetic beads

[0115] 1.6.1 Experimental Principle

[0116] The magnetic beads are streptavidin magnetic beads (Thermo Fisher Scientific, REF: 65001), which can specifically bind to biotin glycoRNA in small RNA. After sample preparation, Nothern blot electrophoresis, and membrane transfer, the membrane reacts with Hrp streptavidin, and color is developed due to the presence of biotin.

[0117] 1.6.2 Experimental procedures

[0118] (1) Purification of glycoRNA using streptavidin magnetic beads: 10 μL MyOne C1 streptavidin magnetic beads were blocked with 50 ng / mL glycogen in 500 μL Biotin Wash Buffer (10 mM Tris HCl pH7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Tween-20) and incubated at 25°C for 1 h. After blocking, the beads were centrifuged at 3000 rpm for 2 min at 4°C, placed on a magnetic rack, and the supernatant was discarded.

[0119] (2) Resuspend the magnetic beads in 500 μL Biotin Wash Buffer, add to the biotinylated miRNA, seal with sealing film, rotate and mix at 4°C for 2 h, centrifuge at 3000 rpm for 2 min, place on a magnetic rack, collect the supernatant, and subsequently pass through a purification column for loading.

[0120] (3) Wash the beads to remove unbound RNA: Wash twice with 1 mL Biotin Wash Buffer, 3 minutes each time.

[0121] (4) Elute glycoRNA: Incubate the biotinylated RNA with magnetic beads in 60 μl of 0.75 M NaCl, 10 mM EDTA, and 10 mM Tris-Cl (pH 7.4) (prepared in advance) at 22°C for 5 minutes. Elute the RNA by resuspending the magnetic beads in 1× volume of 5% b-mercaptoethanol (BME) solution (do not discard the solution from the previous step) and incubate at 22°C for 5 minutes. Centrifuge at 3000 rpm for 2 minutes, collect the supernatant, and purify the glycoRNA using a Zymo concentration purification column.

[0122] 1.7 Specific magnetic bead purification of glycoRNA

[0123] 1.7.1 Experimental Principle

[0124] The activated carboxyl group of EDC will react with the amino group, so the buffer system containing amino groups, such as Tris buffer, cannot be used. MES is usually used as a buffer system. EDC is very easy to get wet and lose its activity. Before each use, place the EDC reagent bottle in a desiccator to warm up to room temperature for at least half an hour, then open it to take out the solid and weigh it to avoid moisture entering the bottle.

[0125] The half-life of EDC in aqueous solution is measured in hours, so EDC solutions cannot be stored and must be prepared before use.

[0126] 1.7.2 Experimental procedures

[0127] 1.7.2.1 Magnetic Bead Preparation

[0128] (1) Prepare the following buffers: reaction buffer (RBuffer): 100 mM MES, pH 4.5; washing buffer (WBuffer): 1× PBS, pH 7.4; storage buffer (SBuffer): 1× PBS, pH 7.4 + 0.05% (w / v) Proclin 300.

[0129] (2) Mix the magnetic bead dispersion thoroughly, then transfer 200 μL of MagnaBind™ carboxyl-derivatized microbeads (containing 10 mg of magnetic beads) (Thermo Fisher Scientific, REF: 21353) into a microcentrifuge tube, place it on a magnetic rack, separate the magnetic beads by magnetic attraction, and remove the supernatant.

[0130] (3) Wash the magnetic beads 2-3 times with R Buffer, 200 μL each time, place them on a magnetic rack, separate the magnetic beads by magnetic attraction, remove the supernatant, and retain the magnetic beads.

[0131] (4) Pipette 300 μL R Buffer to dissolve 3 OD of amino oligo, add to the above magnetic beads, pipette and mix thoroughly, then place on a rotating mixer and rotate at room temperature for 30 min.

[0132] (5) Weigh 20 mg of EDC solid (company catalog number) and dissolve it in R Buffer cooled in an ice bath to make the EDC concentration 100 mg / mL.

[0133] (6) Pipette 150 μL of EDC solution and add it to the mixture in (4) and mix thoroughly.

[0134] (7) Pipette 50 μL R Buffer into the above reaction solution to a final volume of 500 μL. Then place the mixture on a rotating mixer and rotate overnight at room temperature.

[0135] (8) Separate the magnetic beads by magnetic attraction. Wash the magnetic beads 2-3 times with W Buffer, 500 μl each time. Disperse the washed magnetic beads in 1 mL of S Buffer (magnetic bead concentration is 10 mg / mL) and store at 4°C.

[0136] 1.7.2.2 Probe Binding

[0137] (1) The glycoRNA sample purified by streptavidin magnetic beads was denatured by incubation at 65°C on a vertical mixer for 10 min. The probe magnetic beads were added to the denatured sample, hybridized at 37°C for 30 min, denatured at 50°C for 5 min, and hybridized at 37°C for 90-180 min.

[0138] (2) Add the RAP sample-probe group sample to the two sets of magnetic beads respectively, and incubate them on a vertical mixer at room temperature for 30 min.

[0139] (3) Collect the magnetic beads on a magnetic rack and discard the supernatant.

[0140] (4) Add 500 μL Wash Buffer and wash at 37°C for 2–4 min. Collect the beads on a magnetic rack and discard the supernatant.

[0141] (5) Repeat the washing process four times.

[0142] (6) Collect the magnetic beads on a magnetic rack and discard the supernatant.

[0143] 1.7.2.3 RNA elution

[0144] (1) Add 50 μL RNA Elution Buffer to the magnetic beads, mix thoroughly, and heat at 95°C for 2 min.

[0145] (2) Collect the magnetic beads on a magnetic rack and transfer the supernatant to a new RNase-free centrifuge tube. Repeat step (1) once.

[0146] (3) Add the supernatant to the RNA purification column and add 400 μL Binding Buffer. Centrifuge at 10,000 g for 20 seconds and discard the waste liquid.

[0147] (4) Wash once with 400 μL prep buffer, wash twice with 400 μL wash buffer, empty once, and replace with Eppendorf tube for collection.

[0148] (5) Add 15 μL RNA-free water to the center of the filter membrane and incubate for 5 min. Centrifuge at 10,000 g for 20 s, repeat once, and collect the eluate.

[0149] 1.8 Glycan elution and purification

[0150] 1.8.1 Experimental Principle

[0151] Trifluoroacetic acid is the most widely used acid in polysaccharide hydrolysis, especially in structural analysis. Trifluoroacetic acid is not highly oxidizing and has little damage to polysaccharides. It also has a strongly electronegative -CF group, which attracts more hydrogen ions and catalyzes the hydrolysis of polysaccharides. Trifluoroacetic acid is better than inorganic acids because organic acids are sufficiently volatile and can be removed by freeze drying or vacuum distillation after hydrolysis, avoiding the traditional neutralization step and the problem of carbonization of sulfuric acid hydrolysis. However, it is highly toxic and has certain limitations. Trifluoroacetic acid is mainly used to hydrolyze plant cell wall polysaccharides, glycosaminoglycans and glycoproteins, and is widely used in polysaccharide component analysis.

[0152] 1.8.2 Experimental procedures

[0153] 1.8.2.1 Glycan release: Dilute the glycoRNA glycans with RNA free water, add 10 μl of PNGaseF (New England Biolabs, P0704S) and 10 μl of 10× GlycoBuffer 2 to a final volume of 100 μl, and incubate at 37°C for 18 hours. Cool to room temperature.

[0154] 1.8.2.2 Glycan Purification

[0155] (1) Slowly and at a consistent flow rate, pretreat each PGC-SPE column with the following reagents in the following order: 15 ml 1 M NaOH, 15 ml HPLC grade water, 15 ml 30% acetic acid, and 15 ml HPLC grade water.

[0156] (2) Wash the column three times with 15 ml of 50% acetonitrile / 0.1% trifluoroacetic acid (TFA), and then wash the column twice with 15 ml of 5% acetonitrile / 0.1% trifluoroacetic acid.

[0157] (3) Resuspend the entire sample in water to 500 μl-1 ml to allow it to penetrate the resin. Pass the column twice.

[0158] (4) Then wash the column once with 15 ml of 5% acetonitrile / 0.1% TFA.

[0159] (5) Elute the glycans with 4 × 1.5 mL, 50% acetonitrile / 0.1% TFA into a labeled 15 mL conical tube.

[0160] (6) Freeze the sample immediately on dry ice and then lyophilize to dryness (16-24 hours).

[0161] (7) After freeze-drying, dissolve in 1 ml of water, dry again in a 1.5 mL Eppendorf tube, and store at -20°C.

[0162] 1.9 Identification and analysis of glycans in glycoRNA

[0163] First, the glycans were cleaved and released from the glycoRNA. The glycoRNA was diluted with RNA-free water, and 10 μL PNGaseF (New England Biolabs, P0704S) and 10 μL 10×GlycoBuffer 2 were added to a final volume of 100 μL. Incubate at 37°C for 18 h. Second, the released glycans were purified. Each PGC-SPE purification column was pretreated with reagents in the following order: 15 mL 1M NaOH, 15 mL hplc-grade water, 15 mL 30% acetic acid, and 15 mL HPLC-grade water. The purification column was then washed with 15 mL 50% acetonitrile / 0.1% trifluoroacetic acid (TFA) and then with 15 mL 5% acetonitrile / 0.1% trifluoroacetic acid. The sample was suspended in water and purified twice using the pretreated PGC-SPE purification column. It was then immediately frozen on dry ice until lyophilized (16-24h). After freeze-drying, dissolve in 1 mL of water, dry again in a 1.5 mL EP tube, and store at -20°C. Third, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify and analyze the glycans in glycoRNA. The polysaccharide solution was dried in a vacuum concentrator and then dissolved in pure water. CH3I and DMSO were added to the solution, and NaOH micro-spiral columns were used for full methylation. The methylated glycans were extracted with chloroform and rinsed with water several times. The chloroform layer was then collected and concentrated to dryness in a vacuum. The LC separation parameters were set as follows: analytical column: 360 μm OD × 75 μm ID, 75 cm; packing: Phenomenex Jupiter C18, 5 μm, 300 Å; isolation column: 360 μm OD × 200 μm ID, 5 cm; packing: Phenomenex Jupiter C18, 5 μm, 300 Å; mobile phase: buffer A is a mixture of 99.9% H2O and 0.1% FA, and buffer B is a mixture of 99.9% ACN and 0.1% FA. The parameters of the tandem mass spectrometer were: mass resolution 22.5 k, data-dependent acquisition top 20, automatic gain control target value 1×10 5 , maximum ion injection time 200 ms, precursor ion isolation window 3 m / z, dynamic exclusion 50.0 s, step NCE 10%. Based on LC-MS / MS data, library identification was performed using Glyseeker software (FDR ≤ 1%).

[0164] 1.10 Prediction of glycoRNA three-dimensional structure

[0165] GlycoRNA was predicted in RNAStructure (https: / / rna.urmc.rochester.edu / RNAstructureWeb / ). Then, the corresponding secondary structure was obtained from the RNA sequence and RNAStructure, and the three-dimensional structure was constructed by 3dRNA (http: / / biophy.hust.edu.cn / 3dRNA). The constructed glycosylated RNA molecules were optimized in an aqueous solution environment using Gromacs 5.0 dynamics software. The force field used in the dynamics simulation process was Gomacs 54a7, and the water model was SPC. The temperature coupling of the simulation system was determined using the "Berendsen thermostat" method, and the pressure coupling was determined using the "manometer" method, with a reference pressure of 1.01×10 5 , periodic boundary conditions were used in all directions of the system. The optimization process was mainly divided into the following two steps: a. Fix the main chain and optimize the side chain for 5000 steps in a simulated aqueous solution environment; b. Global optimization for 5000 steps in a simulated aqueous solution environment.

[0166] 1.11 Knockdown of U2, U4, and Y5

[0167] Antisense oligonucleotides (ASO) were used to knock down U2, U4 and Y5 in glioma cells U87 and LN229. ASO sequences specific to U2, U4 and Y5 were designed and listed in Table 1. ASO was transfected using lipofectamineTM 3000. The detailed steps were referred to the instructions of the kit. The transfection concentration of ASO was 40nM. 48 hours after transfection, U87 and LN229 cells were collected and qRT-PCR experiments were performed to detect the content of U2, U4 and Y5.

[0168] Table 1 Sequences of U2-ASO, U4-ASO, and Y5-ASO

[0169]

[0170] The designed random primer sequences are listed in Table 2.

[0171] Table 2 Primer sequences

[0172]

[0173] 1.12 Glioma cell viability and proliferation assay

[0174] 1.12.1 CCK-8: Cells were seeded into 96-well plates at 1 × 10 cells per well. 3100 µl of culture medium was added to 10 cells, transfected with ASO, and cultured at 37°C for 24 hours. Then 10 µl of CCK-8 reagent was added and cultured at 37°C for 2 hours. Finally, the optical density at 450 nm (OD450) was measured using a microplate reader.

[0175] 1.12.2 Ki67: Cells were fixed with 4% paraformaldehyde for 30 min at 25°C, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA. Then, cells were incubated with anti-Ki-67 primary antibody (Abcam, Cambridge, UK) for 12 h at 4°C and AF488-conjugated goat anti-mouse IgG secondary antibody (Absin, Shanghai, China) for 1 h at 25°C. Cells were then observed and photographed using a fluorescence microscope (Olympus, Tokyo, Japan).

[0176] 2. Experimental results

[0177] 2.1 U87 and LN229 glioma cells are rich in glycoRNA, mainly small RNAs.

[0178] Ac4ManNAz is an azide-containing mannose derivative that can modify cell surface proteins through N-linked glycosylation. It is commonly used to study glycoproteins through in vivo metabolic labeling and chemical selective ligation. In this study, to determine whether glycoRNA exists in glioma cells, the researchers used Ac4ManNAz to label glycoRNA in glioma cells (U87 and LN229) for 24 hours. After RNA samples were extracted, purified, and treated with DBCO-biotin, they were separated by gel electrophoresis and blotted with anti-biotin streptavidin-HRP ( Figure 1 A) Before transferring the RNA to a membrane, the total RNA in the gel is visualized and contrasted with the streptavidin-bound RNA in the membrane. Figure 1 As shown in B, biotin signals were observed in RNA samples from U87, LN229, and Hela cells treated with Ac4ManNAz for 24 hours (24 hour group). However, no biotin signals were observed in RNA samples from U87, LN229, and Hela cells that were not treated with Ac4ManNAz (0 hour group). RNaseA (RNase) and DNase I (DNase) were used to determine whether the biotin signal came from RNA or DNA. When RNA samples were digested with RNase A before electrophoresis, the signal disappeared on the blot. However, digestion with DNase or RNase followed by preincubation with RNase and RNase inhibitor (RNasin) did not result in loss of signal ( Figure 1 C). In addition, the RNA samples treated with RNase showed no signal in the gel ( Figure 1C). These results indicate that the purified samples consisted primarily of RNA with little or no DNA contamination. To determine the approximate molecular weight range of glycoRNA, we isolated small (<200 bp) and large (>200 bp) RNA using the RNAzol Isolation Kit and then analyzed them by gel electrophoresis and western blot, respectively. The results showed that there was a clear biotin signal in the small RNA samples from U87 and LN229 cells, while there was almost no signal in the large RNA samples ( Figure 1 D). This suggests that the glycoRNA of glioma U87 and LN229 cells may be mainly composed of small RNAs. The slow migration of glycoRNA on the gel may be due to the sugar group affecting the mobility of RNA.

[0179] 2.2 Analysis of glycoRNA enriched in U87 and LN229 cells.

[0180] Subsequently, we sought to analyze the composition of glycoRNAs enriched in glioma cells. Through a series of processes, including Ac4ManNAz labeling, small RNA extraction, DBCO-biotin treatment, streptavidin magnetic bead purification, and Zymo column purification, glycoRNAs were purified from U87, LN229, and Hela cells and used for small RNA deep sequencing analysis. The RNA samples of U87, LN229, and Hela cells treated with Ac4ManNAz were named U87-24h, LN229-24h, and Hela-24h, respectively. The RNA samples of U87, LN229, and Hela cells that were not treated with Ac4ManNAz were used as input and were named U87-0h, LN229-0h, and Hela-0h, respectively. Analysis of small RNA expression showed that there were significant differences in the expression profiles of the three cell types ( Figure 2A). It is particularly noteworthy that the RNA expression levels were high in U87-24h and LN229-24h, but low in U87-0h, LN229-0h, Hela-24h, and Hela-0h. Several small nuclear RNAs (snRNAs), including U2, U4, and U1, attracted our attention: U2 and U4 were enriched in U87-24h and LN229-24h, but were barely detectable in Hela-24h, Hela-0h, U87-0h, and LN229-0h. U1 and Y5 (members of the Y5 RNA family) were enriched in Hela-24h, U87-24h, and LN229-24h, but low in Hela-0h, U87-0h, and LN229-0h. These RNAs may be candidate RNAs for subsequent studies. Then, the 0h group was used as the input group to screen out the RNAs that were upregulated in the 24h group in the three cell lines. The screening process followed the following principles: (1) P < 0.05, log2FC > 1, and UMI (0h) < 100; or (2) P < 0.05, UMI (24h) > 0, and UMI (0h) = 0. P < 0.05, UMI (24h) > 0, UMI (0h) = 0. The screened RNAs were considered to be glycoRNAs detected in the three cell types, excluding nonspecific RNAs. The numbers of glycoRNAs detected in U87, LN229, and Hela were 40, 24, and 39, respectively. Figure 2 B). The intersection of the RNA detected in the three cell types is plotted in a Venn diagram ( Figure 2 B). The number of RNA cross-talks between U87 and LN229, U87 and Hela, and LN229 and Hela were 11, 13, and 9, respectively ( Figure 2 B). Obviously, there are significant differences in the types of glycoRNAs among the three cell types (including two glioma cell lines, U87 and LN229). By classifying and counting the RNAs detected in the three cell types, we found a variety of small RNAs, including small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), Y RNA, and small caerulein-specific RNA (scaRNA). Figure 2 C).

[0181] Among these RNAs, some were enriched in both U87 and LN229, including U7, U1, U2, U3, U4, U6, Y5, 7SK, snoU13, and Metazoa_SRP ( Figure 3A). Among the ten types of RNA, several RNAs including Y5, 7SK, U1, U7, U3, U6, and Metazoa_SRP were also enriched in Hela. We selected U2 and U4 as candidate RNAs because they were enriched in U87 and LN229, but lacked enrichment in Hela. On the other hand, Y5 and U1 were selected as positive control RNAs because they were enriched in all three cell types. Next, the levels of U1, U2, U4, and Y5 were verified by quantitative RT-PCR in multiple cell lines including U87, LN229, Hela, MCF7, and HEK-293T. qRT-PCR results showed that U2 was enriched in U87 and LN229 cells, but not in Hela, MCF7, and HEK-293T cells (p value < 0.05 for U87, p value < 0.01 for LN229, and p values ​​> 0.05 for Hela, MCF7, and HEK-293T; Figure 3 B). U4 was enriched in U87, LN229, and Hela cells, but not in MCF7 and HEK-293T cells (p value < 0.01 for U87, p value < 0.05 for LN229, p value < 0.01 for Hela, and p values ​​> 0.05 for MCF7 and HEK-293T; Figure 3 B). Y5 was enriched in all five cell types (p value < 0.001 for U87, p value < 0.01 for LN229, p value < 0.05 for Hela, p value < 0.05 for MCF7, p value < 0.01 for HEK-293T; Figure 3 B). U1 was enriched in U87, LN229, and Hela cells, but not in MCF7 and HEK-293T cells (U87 p<0.05, LN229 p<0.001, Hela p<0.05, MCF7 and HEK-293T p>0.05; Figure 3 B). Through comprehensive analysis of RNA sequencing results, we identified U2 and U4 as potential glioma-associated glycoRNAs and further investigated them, while Y5 was designated as a positive control glycoRNA. U2 and U4 are both members of the snRNA family. snRNAs are divided into seven categories, including U1-U7, which are major components of the RNA spliceosome during post-transcriptional processing in eukaryotes and are involved in the processing of mRNA precursors.

[0182] 2.3 Identification of glycans on glycoRNA U2 and U4.

[0183] In order to gain a deeper understanding of the composition of U2 and U4 glycoRNA, it is necessary to further identify the sugars present on them. In this study, a sequence-specific RNA capture magnetic bead system was developed, which consists of carboxyl-derivatized magnetic beads and amino oligonucleotides. This system is designed to enrich specific RNA fragments, such as U2 and U4. Amino oligonucleotides are amino-linked oligonucleotide sequences that specifically target specific RNA fragments. The specific probe sequences for U2 and U4 are shown in Table 3.

[0184] Table 3 Specific probe sequences of sequence-specific RNA capture magnetic bead system

[0185]

[0186] The enriched glycoRNA U2 or U4 was subjected to a series of treatments, such as PNGaseF treatment and PGC-SPE column purification, to cleave, release, and purify the contained glycans. The glycan samples were detected and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). There were 6 groups of glycan samples, including glycans cleaved from total RNA of U2, U4, and U87 cells (U87-U2, U87-U4, U87-total), and glycans cleaved from total RNA of U2, U4, and LN229 cells (LN229-U2, LN229-U4, LN229-total). The total groups (U87-total and LN229-total) represent the glycans in the total glycan RNA that were not enriched by the sequence-specific RNA capture magnetic bead system. Unsupervised cluster analysis of sugar chains revealed significant differences in the clustering of U87-U2 and LN229-U2, and in the clustering of U87-U4 and LN229-U4 ( Figure 4 A). Next, the glycosides from LN229 and U87 cells were classified and the percentages were calculated. The detected glycosides included three-branched, four-branched, and two-branched glycosides, and there was no significant difference in the proportion of each glycoside detected in LN229 and U87 ( Figure 4 B) The percentage of tri-fucosylated glycosides detected in U87 was greater than that in LN229 ( Figure 4 C). Notably, the glycosides exhibited different degrees of sialylation, with significantly different ratios observed in the U87 and LN229 samples ( Figure 4 D). In addition, compared with the LN229 sample, the U87 sample had a higher proportion of mixed glycosides and a lower proportion of mixed glycosides ( Figure 4 E). The results showed that two glioma cell lines, U87 and LN229, exhibited different glycosidic compositions. The intersection of glycosidic groups in the U2 and U4 samples was then analyzed. Whether from U87 or LN229, there were significant differences in the types of glycans detected in the U2, U4, and total samples ( Figure 4FG). We further counted the types of sugar chains detected in each sample, and the results are shown in Figure 4 HM shows that among the glycosides detected in the U87 sample, the most common glycoside forms or modifications were complex glycoforms, sialylation, and fucosylation ( Figure 4 HJ). Among the glycosides detected in LN229 samples, the most common glycoside forms or modifications were fucosylation, sialylation, and complex glycoforms ( Figure 4 KM). Among the glycosides in the U87-total, U87-U2, and U87-U4 groups, complex glycoforms accounted for 93.1%, 88.7%, and 92.0%, respectively ( Figure 4 NP). Among the glycosides in the LN229-total, LN229-U2, and LN229-U4 groups, complex glycosides accounted for 57.7%, 77.8%, and 82.7%, respectively ( Figure 4 QS).

[0187] Complex glycoforms have more structural changes than mixed glycoforms, so the glycoside samples of U87 seem to have more structural changes than those of LN229. In addition, most of the glycosides detected in each sample were sialylated glycosides. According to the Sankey diagram analysis, the detected glycosides can be divided into six categories, including core fucose, branched and terminal fucose, non-fucose, core and branched fucose, branched fucose, and core, branched and terminal fucose ( Figure 5 AB). Most of these glycosides are non-dimeric and sialylated ( Figure 5 AB). Subsequently, the glycosides detected in each sample were ranked according to their relative abundance. The top-ranked glycosides in each group were N5H6F0S3(U87-total), N4H5F1S0(U87-U2), N5H6F1S2(U87-U4), N5H6F2S2(LN229-total), N4H5F1S2(LN229-U2), and N4H5F1S2(LN229-U4)( Figure 5 CH). Apparently, more than one glycan type is attached to U2 or U4.

[0188] 2.4 Prediction of the three-dimensional structure of glycoRNA.

[0189] The three-dimensional structure of U2 / U4, which has the highest degree of glycosylation, was predicted. Figure 6A shows the results of covalent bond analysis and hydrogen bond analysis. There are four RNA-glycoside systems, namely RNA-U2_LYL, RNA-U2_YLS, RNA-U4_MYL and RNA-U4_YLS. The binding energies of these systems are -2572.63, -2029.48, -2189.73 and -1918.81 KJ / mol-1, respectively. The covalent bond groups of RNA-U2_LYL, RNA-U2_YLS, RNA-U4_MYL and RNA-U4_YLS are G110, A4, A129 and G24, respectively. In addition, the number of hydrogen bonds of RNA-U2_LYL, RNA-U2_YLS, RNA-U4_MYL and RNA-U4_YLS are 20, 15, 22 and 22, respectively. Figure 6 A). The conformational 3D images of RNA-U2_LYL, RNA-U2_YLS, RNA-U4_MYL, and RNA-U4_YLS are shown in Figure 6 BE. It is noteworthy that the sugar chain structures are all bound in the helical groove between the RNA hairpin structures. The presence of the hairpin structure allows RNA U2 and U4 to form a helical groove domain. Glycosides are branched molecules that can form long-range claw structures and bind by embedding into the "hairpin" structure of RNA. Therefore, the spatial fit between glycosides and RNA is very high, and the steric hindrance effect during the binding process is very small ( Figure 6 BE). The covalent binding mode between RNA and glycoside is: the base G or A is amidated, and then the hydroxyl groups of the terminal polysaccharide are linked to obtain a covalently bound complex. Figure 6 As shown in FI, the sugar chain is covalently attached to the G or A base in the RNA helical groove through the terminal sugar unit in the amide bond. In addition, there are non-covalent hydrogen bonding interactions between the sugar chain and RNA. LYL interacts with the bases G110, G111, G112, G113, A114, A134, U135, C136, C137, A138, U140, C141, and C142 in RNA U2, forming 20 hydrogen bonds. In addition, G112, G114, C137, and A138 interact with the bases through multiple hydrogen bonds ( Figure 6 F). The bases G110, G111, G113, A114, A118, C136, A138, U140, C141, C142, and U143 in RNA U2 interact with the sugar YLS through 15 hydrogen bonds, while A114, C136, C141, and C142 also interact with the bases through multiple hydrogen bonds ( Figure 6G). MYL interacts with multiple bases in the RNA U4 helical groove, forming 22 hydrogen bonds with the following bases: A17, A18, U19, G21, U24, C48, A49, U53, U56, U57, U128, and A129. In addition, bases G21, C48, A49, and U128 form multiple hydrogen bonds with other bases ( Figure 6 H). YLS interacts with bases A17, A18, U19, G21, U23, C50, A51, A52, A55, U56, and G130 in RNA U4, forming 22 hydrogen bonds. In addition, A18, U19, G21, U23, and A129 interact with bases through multiple hydrogen bonds ( Figure 6 I).

[0190] 2.5 Knockdown of U2 and U4 inhibited the proliferation of U87 and LN229 cells.

[0191] We then investigated the role of glycoRNA U2 and U4 in glioma cell proliferation. The basis for studying their functions is that they can be knocked down or knocked out. However, since there are currently no specific inhibitors for glycoRNA, we decided to downregulate the expression of glycoRNA U2, U4 and Y5 by knocking down small RNA U2, U4 and Y5 using antisense oligonucleotides (ASOs). Antisense oligonucleotides are small fragments of single-stranded DNA or RNA analogs of 12 to 25 nucleotides. After entering the cell, they bind to complementary target mRNAs based on the principle of base pairing complementarity and inhibit the expression of target genes under the action of nuclear glycoRNA enzyme H1. ASOs targeting U2, U4 and Y5 were designed and named U2-ASO, U4-ASO and Y5-ASO. Negative control (NC) sequences were designed to have no affinity for known target RNAs, thereby preventing the induction of specific biological effects. In this study, the NC sequence was introduced to ensure that any biological changes observed were caused by the binding of specific ASO sequences to target RNAs, rather than due to nonspecific effects that occurred during the experiment. The sequences of U2-ASO, U4-ASO, and Y5-ASO are listed in Table 1. The results showed that U2-ASO could significantly reduce the expression level of U2 in U87 and LN229 cells, regardless of whether U6 or Gapdh was used as the internal reference gene (p<0.05 in U87 [U6 as the internal reference gene]; p<0.05 in U87 [Gapdh as the internal reference gene]; p<0.01 in LN229 [U6 as the internal reference gene]; p<0.001 in LN229 [Gapdh as the internal reference gene], Figure 7AD). Regardless of whether U6 or Gapdh was used as the internal reference gene, U4-ASO could significantly reduce the expression level of U4 in U87 and LN229 cells (p<0.01 in U87 [U6 as the internal reference gene]; p<0.01 in U87 [Gapdh as the internal reference gene]; p<0.0001 in LN229 [U6 as the internal reference gene]; p<0.0001 in LN229 [Gapdh as the internal reference gene], Figure 7 EH). Y5-ASO significantly reduced the expression of Y5 in both U87 and LN229 cells (p<0.0001 in U87, p<0.01 in LN229 [U6 as internal reference gene], Figure 8 ).

[0192] We then investigated the effects of U2, U4, and Y5 on the proliferation of U87 and LN229 glioma cells. Cell counting kit-8 (CCK-8) assay results showed that the cell viability of U2-knockdown U87 and U4-knockdown U87 cells was significantly reduced compared with that of U87 cells transfected with NC sequence (p<0.0001; p<0.001, Fig. 9 A). Similarly, the cell viability of U2-knockdown LN229 and U4-knockdown LN229 cells was significantly lower than that of LN229 cells transfected with NC sequence (p<0.0001; p<0.0001, Fig. 9 B). Ki67 is a nuclear antigen used to identify the state of cell proliferation. It is mainly expressed in the G1, S, and G2 phases of the cell cycle and in cells in interphase mitosis. However, it is not expressed in cells in the G0 phase, that is, quiescent cells. Given that its function is closely related to cell proliferation, Ki67 is often used to detect tumor growth index. The results of the Ki67 cell fluorescence test showed that compared with the NC group, both U2-ASO and U4-ASO significantly reduced the expression of Ki67, both in U87 cells and LN229 cells (U87 cells transfected with U2-ASO, p value <0.0001; U87 cells transfected with U4-ASO, p<0.0001; LN229 cells transfected with U2-ASO, p<0.0001; LN229 cells transfected with U4-ASO, p<0.0001, Fig. 9CD). However, the decrease in Y5 expression levels did not produce statistically significant changes in the proliferation of U87 and LN229 cells, as confirmed by CCK-8 assays and Ki67 cell fluorescence assays. The above results indicate that the reduction in U2 and U4 levels impedes the proliferation of U87 and LN229 glioma cells. However, the positive control RNA Y5 is ubiquitous in many types of cells, and its reduction does not affect the proliferation of U87 and LN229 cells. These findings suggest that U2 and U4 are involved in the proliferation of glioma cells, but it is unclear whether this function is performed by glycoRNA, free RNA, or both. Nevertheless, given the importance of U2 and U4 in glioma cell proliferation and the unique structure of glycoRNA, we speculate that glycoRNAU2 and U4 may have a key impact on the survival and growth of glioma cells.

Claims

1. A method for purifying and screening tumor glycoRNA, the method comprising: 1) Labeling glycoRNA in cells; 2) extracting and purifying the total cellular RNA labeled with glycoRNA to obtain purified total RNA; 3) Purifying small RNA from the purified total RNA to obtain purified small RNA; 4) Purify the purified small RNA using streptavidin magnetic beads to obtain crude purified glycoRNA; 5) Purify the crude purified glycoRNA using glycoRNA-specific magnetic beads, and elute to obtain purified tumor U2 and / or U4 glycoRNA; The glycoRNA-specific magnetic beads are prepared by the following method: The magnetic bead dispersion was thoroughly mixed, and then the carboxyl-derivatized microbeads were transferred, the magnetic beads were separated by magnetic attraction, and the supernatant was removed; the magnetic beads were washed 2-3 times with R Buffer, and the magnetic beads were separated by magnetic attraction, and the magnetic beads were retained; R Buffer was transferred to dissolve the amino oligo, and the amino oligo was added to the magnetic beads, mixed, and rotated at room temperature; EDC solution dissolved in R Buffer was added, mixed, R Buffer was added to make up the volume, and the reaction was rotated at room temperature, and the magnetic beads were separated by magnetic attraction, and the magnetic beads were washed 2-3 times with W Buffer to obtain glycoRNA-specific magnetic beads; In step 5), the crude purified glycoRNA is purified by glycoRNA-specific magnetic beads, which specifically includes the following steps: The glycoRNA sample purified by streptavidin magnetic beads was denatured by incubation at 65°C, and a probe set for specific purification of tumor glycoRNA was added to the denatured sample, wherein the probe set for specific purification of tumor glycoRNA was: glycoRNA U2 probe SEQ ID NO: 1 and / or glycoRNA U4 probe SEQ ID NO: 2, hybridized at 37°C for 30 min, denatured by incubation at 50°C for 5 min, and hybridized at 37°C for 90-180 min; the prepared glycoRNA-specific magnetic beads were added, mixed and incubated at room temperature for 30 min, the magnetic beads were collected and the supernatant was removed, Wash Buffer was repeatedly added, and the magnetic beads were collected and the supernatant was removed after shaking and washing at 37°C.

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