Method for detecting and enriching glycosylated RNA (Ribonucleic Acid) based on in-vitro enzyme chemical labeling of alpha-2, 3, alpha-2, 6 type sialyltransferase

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for detecting and enriching glycosylated RNA (Ribonucleic Acid) based on in-vitro enzyme chemical labeling of alpha-2, 3, alpha-2, 6 type sialyltransferase. According to the application, sialidase is firstly used for digesting sialic acid at the tail end of a naturally generated glycoRNA sugar chain, and sialidase digestion is not needed for an RNA sample which does not have sialic acid at the tail end of the glycoRNA sugar chain but has potential sialic acid modification sites; sialic acid with a labeled group is transferred to a potential sialic acid modification site at the tail end of a glycoRNA carbohydrate chain by using alpha-2, 3, alpha-2, 6 type sialyltransferase, such as PmST1, PmST3, Ph2, 3siat and Pd26ST, so that glycoRNA with a detectable group can be directly obtained, or the detectable group is further introduced through subsequent reaction, so that subsequent signal detection or enrichment can be carried out; the RNA part can be further sequenced, and the glycosyl part can be further subjected to mass spectrum identification. The application provides a very efficient, simple and convenient detection means for detection and enrichment of sialylated glycoRNA, has the characteristics of high specificity and high sensitivity, and has a wide application prospect.

Description

Cross-reference to related applications

[0001] This application claims priority to Chinese Patent Application No. CN 202510124014.2, filed on January 26, 2025, entitled “A Method for In Vitro Enzyme Chemical Labeling Detection and Enrichment of Glycosylated RNA Based on α-2,3,α-2,6 Type Sialyltransferases”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application belongs to the field of biological technology, specifically, it relates to a method for in vitro enzyme chemical labeling detection and enrichment of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases. Background Technology

[0003] Glycosylation is one of the most important and widespread modifications in eukaryotes, regulating many vital physiological functions such as proper protein folding and transport, and signal transduction. Previously, it was generally accepted that glycosylation primarily occurs on proteins and lipids, while RNA is not glycosylated. However, in 2021, Carolyn R. Bertozzi and Ryan A. Flynn's team reported in *Cell* that mammalian-derived small RNA can undergo N-linked glycosylation, opening a new chapter in glycosylation research. The researchers used the metabolic labeling reagent Ac4ManNAz (CAS: 1213701-11-1) to incorporate sialic acid metabolism pathways in vivo, thereby enabling them to label all glycosylated biomolecules that may undergo sialylation, including glycoRNAs. Experiments have shown that the glycosyl groups of these glycoRNAs are N-linked to RNA. RNA sequencing results indicate that glycoRNAs are primarily small RNAs, including tRNA, yRNA, snRNA, and rRNA. Furthermore, researchers have confirmed through cellular sublocalization experiments that glycoRNAs are mainly located on the outer surface of the cell membrane, can be recognized by dsRNA antibodies, and can also serve as direct ligands for immunoglobulin-like lectin (Siglec) receptors, suggesting a potential important role in immune regulation. In 2023, a research team led by Zhang Ningning and Lu Jun from Yale University discovered that glycoRNAs on the cell surface can regulate neutrophil recruitment. In summary, glycoRNAs are potential molecules involved in immune regulation and signal transduction, and developing corresponding detection methods is of great significance for further research on their functions.

[0004] Currently, there are two main known methods for detecting glycoRNA. The team of Carolyn R. Bertozzi and Ryan A. Flynn used the metabolic labeling reagent Ac4ManNAz to label sialic acid on glycoRNA, thereby introducing an azide group onto the sialic acid. Then, through a copper-free click chemical reaction with DBCO-PEG4-Biotin, a biotin group was attached to the glycoRNA, allowing for subsequent detection or enrichment targeting the biotin group. The team of Lu Yi and Ma Yuan developed a glycoRNA in situ imaging method called ARPLA using proximity ligation assays. This method utilizes nucleic acid aptamers and RNA in situ hybridization to specifically recognize the glycosyl groups and RNA of glycoRNA, respectively. Then, DNA proximity ligation technology is used to generate a complete circular DNA, which serves as a template for rolling circle amplification, producing a long single-stranded DNA sequence. This sequence is then hybridized with a fluorescently labeled DNA probe to provide information on the location and abundance of glycoRNA.

[0005] The two current methods for detecting glycoRNA still have certain limitations: Regarding the metabolic labeling method used by Carolyn R. Bertozzi and Ryan A. Flynn's team, firstly, this method requires metabolic labeling with the labeling reagent Ac4ManNAz before glycoRNA generation, making it unsuitable for samples where metabolic labeling is not possible, such as clinicopathological tissues. Secondly, Ac4ManNAz is an azide, a highly toxic substance with flammable and explosive properties, posing certain risks during use, and certain concentrations of Ac4ManNAz can inhibit normal cell growth. Thirdly, the labeling efficiency of Ac4ManNAz as a metabolic labeling reagent is limited, and some glycoRNAs may remain unlabeled, causing some result bias. Finally, Ac4ManNAz itself, as a monosaccharide derivative, can affect normal glucose metabolism in vivo, thereby affecting the generation of glycosylated biomolecules.

[0006] The ARPLA in situ imaging method developed by Lu Yi and Ma Yuan's team also has certain limitations. The RNA in situ hybridization technique they use can only target specific RNA sequences, but the RNA composition of glycoRNAs is diverse. Therefore, this method can only detect glycoRNAs generated from specific small RNAs, which constitute only a small portion of the total glycoRNA signal and is not suitable for detecting the overall glycoRNA signal. Furthermore, this technique is limited to in situ imaging of glycoRNAs and cannot achieve glycoRNA enrichment. In addition, the nucleic acid aptamers and RNA in situ hybridization strands involved in this method, as well as the subsequent PLA technology, are costly and not suitable for widespread application.

[0007] Therefore, it is of great significance to develop a simple, specific, sensitive, and low-cost method for detecting naturally occurring glycoRNAs. Summary of the Invention

[0008] To address the shortcomings of existing detection methods, this application first digests the sialic acid at the ends of naturally occurring glycoRNA glycans using sialidase. For RNA samples with no sialic acid at the ends of the glycoRNA glycans but potential sialic acid modification sites, sialidase digestion is unnecessary, and the undigested glycoRNA can be used directly, thus obtaining glycoRNA with exposed sialic acid modification sites. Then, α-2,3,α-2,6 type sialyltransferases catalytically transfer sialic acid with a labeled group to the potential sialic acid modification sites at the ends of the glycoRNA glycans, thereby labeling the glycoRNA that can be modified by sialic acid. This directly yields glycoRNA with detectable groups (such as fluorescent groups or biotin), or further introduces detectable groups (such as fluorescent groups or biotin) through subsequent reactions (such as click chemistry), enabling subsequent signal detection or enrichment. Furthermore, it allows for sequencing of both the RNA and glycosyl moieties (referring to sequencing the RNA moieties and structural identification of the glycosyl moieties, such as mass spectrometry). This application provides a highly efficient and convenient detection method for the detection and enrichment of sialylated glycoRNAs. The glycoRNAs described in this application include all sialylated RNAs, regardless of their size, type, sequence, or the form and structure of glycosyl linkages; including glycoRNAs generated in cultured cells and glycoRNAs from other samples; other samples can be derived from various biological materials, including tissues and body fluids.

[0009] This application describes a method that involves digesting the sialic acid at the ends of naturally generated glycoRNA glycans with sialidase, followed by catalytic transfer of sialic acid with a labeled group to potential sialic acid modification sites at the ends of the glycoRNA glycans using α-2,3, and α-2,6 type sialyltransferases. This process directly yields glycoRNAs with detectable groups (such as fluorescent groups or biotin), or further introduces detectable groups (such as fluorescent groups or biotin) through subsequent reactions (such as click chemistry), enabling subsequent signal detection or enrichment. Furthermore, it allows for sequencing of both the RNA and glycosylation portions.

[0010] In a first aspect, this application provides a method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3, α-2,6 type sialyltransferases, comprising the following steps: 1) digesting all sialic acid at the glycan ends of glycoRNA in an RNA sample using sialyltransferase to obtain digested glycoRNA. For RNA samples with no sialic acid at the glycan ends but potential sialic acid modification sites, sialyltransferase digestion is not required, and the undigested glycoRNA can be used directly; 2) using α-2,3 or α-2,6 type sialyltransferase and CMP-Sialic acid with a labeling group to transfer the sialic acid with the labeling group to the potential sialic acid modification sites at the glycan ends of the digested or undigested glycoRNA to obtain glycoRNA with the labeling group.

[0011] This application provides a method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3, α-2,6 type sialyltransferases, comprising the following steps: 1) digesting all sialic acid at the terminal ends of the glycoRNA glycan chain in an RNA sample using sialyltransferase to obtain digested glycoRNA; 2) using α-2,3 or α-2,6 type sialyltransferases and CMP-Sialic acid with a labeled group to transfer the labeled sialic acid to potential sialic acid modification sites at the terminal ends of the digested glycoRNA glycan chain to obtain labeled glycoRNA.

[0012] The method described in this application first digests all sialic acid at the ends of the glycoRNA glycan chains in the RNA sample using sialidase, that is, removes the original α-2,3, α-2,6, α-2,8 and α-2,9 type sialic acid modifications. In short, it removes all sialic acid modifications and exposes potential sialic acid modification sites. Depending on the sialic acid modification at the terminal end of the glycoRNA glycan in the RNA sample, sialidases with different activities are selected. For example, if the glycoRNA glycan terminal is modified with α-2,3 or α-2,6 sialic acid, the sialidase used has α-2,3 or α-2,6 sialic acid activity; if the glycoRNA glycan terminal is modified with α-2,3 and α-2,6 sialic acid, the sialidase used has α-2,3 and α-2,6 sialic acid activity; if the glycoRNA glycan terminal is modified with α-2,8 and α-2,9 sialic acid, the sialidase used has α-2,3, α-2,6, α-2,8, and α-2,9 sialic acid activity. For RNA samples with no sialic acid at the terminal end but potential sialic acid modification sites, sialidase digestion is not required, and the undigested glycoRNA can be used directly. Subsequently, sialic acid with a labeled group is transferred to potential sialic acid modification sites at the ends of the glycoRNA glycans using α-2,3 or α-2,6 type sialyltransferases, resulting in labeled glycoRNA. This method can label all sialylated glycoRNAs, regardless of whether the sialyl-modifiable sites of the glycoRNA in the RNA sample have already been modified with sialic acid.

[0013] In some embodiments, the RNA sample may be derived from a variety of biological materials, including but not limited to human cells and tissues, mouse tissues, serum samples, fruit flies, Caenorhabditis elegans, zebrafish, plant samples, fungi, bacteria, etc.

[0014] In some embodiments, the sialidase has at least α-2,3 or α-2,6 sialidase activity; preferably, the sialidase has at least α-2,3 and α-2,6 sialidase activities; more preferably, the sialidase has α-2,3, α-2,6, α-2,8 and α-2,9 sialidase activities; and even more preferably, the sialidase is α-2-3,6,8,9 Neuraminidase A.

[0015] In some embodiments, the α-2,3 type sialyltransferase is a bacterial α-2,3 type sialyltransferase, such as PmST1, PmST3, Ph2,3siat, and its derivatives with corresponding mutants or truncated forms possessing α-2,3 type sialyltransferase activity, preferably the M144D mutant PmST1 and the truncated form Δ24Ph2,3sia. The α-2,6 type sialyltransferase is a bacterial α-2,6 type sialyltransferase, such as Pd26ST, and its derivatives with corresponding mutants or truncated forms possessing α-2,6 type sialyltransferase activity, preferably the truncated form Δ15Pd26ST.

[0016] In some embodiments, the labeling groups in the CMP-Sialic acid with the labeling groups include detectable groups (such as fluorescent groups, biotin) or groups (such as azide, alkynyl) that can be further introduced with detectable groups (such as fluorescent groups, biotin) through reactions (such as click chemistry, specifically copper-free click chemistry or copper-catalyzed click chemistry).

[0017] The fluorescent group is CY5, CY3, etc.; preferably CY5.

[0018] The CMP-Sialic acid with the labeled group can be azide-labeled CMP-azide-SialicAcid, alkynyl-labeled CMP-alkynyl-SialicAcid, biotin-labeled CMP-Biotin-SialicAcid, or fluorescently labeled CMP-CY5-SialicAcid.

[0019] In some embodiments, when the labeling group is a group that can be further introduced into a detectable group through a reaction, the method further includes introducing the detectable group onto sialic acid through a reaction involving the labeling group to obtain glycoRNA with the detectable group.

[0020] When the CMP-Sialic acid with the labeled group is azide-labeled CMP-azide-Sialic Acid, the method further includes introducing a detectable group (such as a fluorescent group or biotin) onto sialic acid via a copper-free click chemistry reaction of azide with a click chemistry reagent. The click chemistry reagent includes DBCO-PEG4-Biotin, DBCO-fluorescent groups (such as DBCO-CY5), and all other click chemistry reagents containing biotin or fluorescent groups. The concentration of the click chemistry reagent is 0.05-100 mM, 0.1-100 mM, preferably 0.5-10 mM, or 0.2-2 mM. df-GLBII buffer and the click chemistry reagent can be added, and the click chemistry reaction can be carried out at room temperature or above, for example, at room temperature - 80°C, preferably at 55°C, for a reaction time of 10 min or more (e.g., 10-60 min), after which water (such as DEPC water) is added to terminate the reaction.

[0021] When the CMP-Sialic acid with the labeled group is an alkyne-labeled CMP-alkynyl-Sialic Acid, one step of the method includes introducing a detectable group (such as a fluorescent group or biotin) onto sialic acid via a copper-catalyzed click chemistry reaction between the alkyne group and a click chemistry reagent. The click chemistry reagent includes N3-Biotin, N3-fluorescent groups (such as N3-CY5), and all other click chemistry reagents containing biotin or fluorescent groups. The concentration of the click chemistry reagent is 0.05-100 mM, 0.1-100 mM, preferably 0.5-10 mM, or 0.2-2 mM. CuSO4, TBTA, TCEP, and the click chemistry reagent can be added to carry out the click chemistry reaction.

[0022] In some embodiments, the method for in vitro enzymatic labeling of glycosylated RNA based on α-2,3, α-2,6 type sialyltransferases includes the following steps: 1) co-incubating the RNA sample and sialyltransferase in buffer solution A at a certain temperature for a period of time to obtain digested glycoRNA; for RNA samples with no sialic acid at the glycan ends but with potential sialic acid modification sites, sialyltransferase digestion is not required, and the undigested glycoRNA can be used directly; 2) co-incubating the digested or undigested glycoRNA, α-2,3 or α-2,6 type sialyltransferases, labeled CMP-Sialic Acid, metal ions, and optional RNase inhibitors in buffer solution B at a certain temperature for a period of time to obtain labeled glycoRNA. A method for in vitro enzymatic labeling of glycosylated RNA based on α-2,3, α-2,6 type sialyltransferases includes the following steps: 1) co-incubating an RNA sample and sialyltransferase in buffer solution A (e.g., 1×Glyco buffer 1) at a certain temperature for a period of time to obtain digested glycoRNA; 2) co-incubating the digested glycoRNA, α-2,3 or α-2,6 type sialyltransferase, labeled CMP-Sialic Acid, metal ions, and optionally an RNase inhibitor in buffer solution B at a certain temperature for a period of time to obtain glycoRNA with a labeled group. Preferably, the reaction system in step 2) contains an RNase inhibitor at a concentration of 0.01-10 U / μL, such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 U / μL, more preferably 1 U / μL.

[0023] In some embodiments, in the reaction system of step 1), the pH of the buffer solution A is 4-11, preferably 4.0-7.0, and more preferably 5.5. The buffer solution A is a Tris buffer (such as Tris·HCl), an acetate buffer (such as sodium acetate), a phosphate buffer (such as disodium hydrogen phosphate, sodium dihydrogen phosphate), a citrate buffer (sodium citrate), a HEPES buffer, a carbonate buffer (such as sodium bicarbonate, sodium carbonate), etc. Preferably, the buffer solution A is an acetate buffer system. The concentration of the buffer solution A is 5-200 mM, preferably 10-200 mM or 5-100 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, more preferably 40-60 mM, and most preferably 50 mM. For example, the buffer solution A is preferably 1×Glyco buffer 1.

[0024] In some embodiments, the RNA concentration in the reaction system of step 1) is 0.01-15 mg / mL, 0.01-10 mg / mL, or 0.1-10 mg / mL, preferably 0.5-1.25 mg / mL or 0.1-1 mg / mL, and more preferably 1.25 mg / mL.

[0025] In some embodiments, in the reaction system of step 1), the concentration of sialidase is 100-10000 U / mL, 500-5000 U / mL, preferably 1000-2000 U / mL, and more preferably 2000 U / mL.

[0026] In some embodiments, step 1) involves an incubation temperature of 4-40°C, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C, preferably 35-38°C, and more preferably 37°C. The incubation time is 10 min to 360 min, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 210, 240, 270, 300, 330, 360 min, preferably 60 to 240 min.

[0027] In some embodiments, in the reaction system of step 2), the pH of the buffer solution B is 6.5-11, preferably 7.0-8.0, and more preferably 7.0. The buffer solution B used for α-2,3 sialyl transferases (PmST1, PmST3, Ph2,3siat) preferably has a pH of 7.0, and the buffer solution B used for α-2,6 sialyl transferases (Pd26ST) preferably has a pH of 7.0. The buffer solution B is a Tris buffer (e.g., Tris·HCl), acetate buffer (e.g., sodium acetate), phosphate buffer (e.g., disodium hydrogen phosphate, sodium dihydrogen phosphate), citrate (sodium citrate) buffer, HEPES buffer, carbonate buffer (e.g., sodium bicarbonate, sodium carbonate), etc. Preferably, the buffer solution B is a Tris·HCl buffer or a sodium acetate buffer. The concentration of the buffer solution B is 5-200 mM, 20-200 mM, such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, preferably 50-150 mM, and more preferably 100 mM.

[0028] In some embodiments, in the reaction system of step 2), the metal ion is selected from Mg. 2+ Ca 2+ The metal ions are provided by their metal chlorides, metal bromides, metal nitrates, or metal sulfates, and the concentration of metal ions in the reaction system is 1-20 mM; preferably, the metal ions are provided by MgCl2, and the concentration of metal ions in the reaction system is 5-10 mM.

[0029] In some embodiments, the RNA concentration in the reaction system of step 2) is less than or equal to 100 mg / mL, such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / mL, preferably 0.01-10 mg / mL, more preferably 0.1-1 mg / mL.

[0030] In some embodiments, in the reaction system of step 2), the concentration of the α-2,3 or α-2,3 type sialyltransferase is 0.1-100 U / mL, such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 U / mL, preferably 0.1-10 U / mL or 1-10 U / mL.

[0031] In some embodiments, in the reaction system of step 2), the concentration of the labeled CMP-Sialic acid is 1µM-20mM, such as 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900µM or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20mM, preferably 1-15mM, more preferably 2-10mM.

[0032] In some embodiments, the incubation temperature in step 2) is 4-40°C, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, preferably 35-38°C, and more preferably 37°C. The incubation time for step 2) is 10 min to 360 min, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 210, 240, 270, 300, 330, 360 min, preferably 60 to 240 min.

[0033] In some embodiments, the method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases specifically includes the following steps: a) Extract total RNA from the sample to be tested; optionally, treat with proteinase K and purify the RNA sample; 1) Use sialidase to digest all the sialic acid at the ends of the glycoRNA glycans in the RNA sample to obtain digested glycoRNA, and then purify the RNA sample; for RNA samples with no sialic acid at the ends of the glycoRNA glycans but potential sialic acid modification sites, sialidase digestion is not required. 2) Using α-2,3 or α-2,6 type sialyltransferase and CMP-Sialic acid with a labeled group, the labeled sialyl acid is transferred to the potential sialyl modification site at the end of the sugar chain of the digested or undigested glycoRNA to obtain labeled glycoRNA; the RNA sample is then purified. b) Optionally, when the labeling group in the CMP-Sialic acid with the labeling group is a group that can be further introduced into a detectable group through a reaction, the detectable group is introduced into the sialic acid through a reaction involving the labeling group to obtain glycoRNA with a detectable group; the RNA sample is purified.

[0034] In some embodiments, the method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases specifically includes the following steps: a) Extract total RNA from the sample to be tested; optionally, treat with proteinase K and purify the RNA sample; 1) Use sialidase to digest all the sialic acid at the ends of the glycan chains of glycoRNA in the RNA sample to obtain digested glycoRNA; purify the RNA sample; 2) Using α-2,3 or α-2,6 type sialyltransferase and CMP-Sialic acid with a labeled group, the labeled sialyl acid is transferred to the potential sialyl modification site at the end of the sugar chain of the digested glycoRNA to obtain labeled glycoRNA; the RNA sample is then purified. b) Optionally, when the labeling group in the CMP-Sialic acid with the labeling group is a group that can be further introduced into a detectable group through a reaction, the detectable group is introduced into the sialic acid through a reaction involving the labeling group to obtain glycoRNA with a detectable group; the RNA sample is purified.

[0035] In some embodiments, the method for extracting total RNA in step a) and purifying the RNA sample, and the method for purifying the RNA sample in steps 1), 2), and b) can be a method commonly used in the art, such as the TRIzol method, magnetic bead method, or column method; preferably, the RNA is dissolved in DEPC water.

[0036] In some embodiments, in step a), after extracting total RNA, proteinase K should be added to digest potential glycosylated protein contamination, and the RNA sample should be purified again. Preferably, step a) is as follows: (1) Add an appropriate amount of TRIzol to the sample to be tested, shake to mix, and incubate at 37°C for 10 minutes; (2) Add chloroform at a ratio of 1:5, shake to mix, and let stand on ice; (3) Centrifuge at 4℃, remove the supernatant, add an equal volume of isopropanol, mix by inversion, and let stand at 4℃. (4) Centrifuge at 4℃, remove the supernatant, wash the precipitate with 70% ethanol, centrifuge again and repeat the washing once more; (5) Centrifuge at 4℃, remove supernatant, air dry at room temperature, dissolve in DEPC water to obtain total RNA solution; (6) Add proteinase K and digest at 37°C for 45 min; (7) Add an appropriate amount of TRIzol, repeat the RNA extraction steps again, and finally dissolve in DEPC water to obtain a total RNA solution.

[0037] In a second aspect, this application provides the use of the method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases as described in the first aspect, for detecting glycosylated RNA, enriching glycosylated RNA, sequencing the RNA portion of glycosylated RNA, or sequencing the glycosylated portion of glycosylated RNA (referring to structural identification, such as mass spectrometry identification).

[0038] This application provides the use of the method for in vitro enzymatic chemical labeling of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases as described in the first aspect, for detecting glycosylated RNA, enriching glycosylated RNA, sequencing the RNA portion of glycosylated RNA, or identifying the glycosylated RNA matrix profile.

[0039] In some embodiments, the detection of glycosylated RNA is performed by electrophoresis, fluorescence imaging, RNA blotting, ELISA, or a microplate reader to detect glycoRNAs with detectable groups.

[0040] When the detectable group is a fluorescent group, it can be detected by electrophoresis, fluorescence imaging, or by using a microplate reader; specifically, electrophoresis and fluorescence imaging can be performed on a formaldehyde-denaturing agarose gel, or the fluorescence signal can be detected directly in a microplate reader. Preferably, a 1% formaldehyde-denaturing agarose gel is used, and 2×RNA Loading is added to the glycoRNA sample containing the detectable group, followed by denaturation and electrophoresis at 115V for 30 minutes, and then fluorescence imaging is performed.

[0041] When the detectable group is biotin, detection can be performed by RNA blotting; for example, RNA blotting can be performed by electrophoresis, transfer, or chemiluminescence imaging. Specifically, in electrophoresis (e.g., in formaldehyde-denaturing agarose gel), RNA is transferred to an NC membrane, cross-linked with UV after transfer, blocked with blocking buffer, and incubated with Streptavidin-HRP (streptavidin-horseradish peroxidase). Chemiluminescence imaging is then performed using the bound HRP. Preferably, a 1% formaldehyde-denaturing agarose gel is used. 2×RNA Loading is added to the glycoRNA sample containing the detectable group, denatured, and then electrophoresed at 115V for 30 minutes. Similar to the Northern blotting experiment, RNA is transferred to a 0.45μm NC membrane; after transfer, cross-linked with UV, blocked with blocking buffer, incubated with Streptavidin-HRP, and washed with washing buffer; chemiluminescence imaging is then performed using the bound HRP to detect the glycoRNA signal.

[0042] In some embodiments, the enrichment of glycosylated RNA refers to obtaining biotinylated glycoRNA by the method described in the first aspect; and enriching the biotinylated glycoRNA.

[0043] glycoRNA can be enriched using streptavidin magnetic beads, followed by elution to obtain the enriched glycoRNA. Specifically, streptavidin magnetic beads are blocked with glycogen, and then biotin-containing glycoRNA is co-incubated with streptavidin magnetic beads (1-10℃, preferably 4-8℃). After washing away non-specific binding, elution is performed to dissociate the biotin on the glycoRNA from the streptavidin magnetic beads, resulting in purified enriched glycoRNA. Preferably, the specific steps are as follows: streptavidin magnetic beads are blocked with glycogen, and then biotin-containing glycoRNA is co-incubated with streptavidin magnetic beads at 4℃; non-specific binding is washed away with washing buffer, and then TRIzol is added, incubated at 70℃ for 10 minutes to dissociate the biotin on the glycoRNA from the streptavidin magnetic beads; the TRIzol is transferred, and RNA is extracted, during which glycogen is added to promote precipitation; finally, it is dissolved in DEPC water to obtain the enriched glycoRNA.

[0044] In some embodiments, the RNA portion sequencing or glycosylation portion sequencing of the glycosylated RNA refers to obtaining enriched glycoRNA according to the above method, and then performing RNA sequencing or glycosylation sequencing.

[0045] In some embodiments, the RNA portion sequencing of the glycosylated RNA or the glycoma profile identification of the glycosylated RNA refers to obtaining the enriched glycoRNA according to the above method, and then performing RNA sequencing or glycoma profile identification.

[0046] The specific steps of the RNA sequencing include: constructing a library of enriched glycoRNAs using an RNA library preparation kit, sequencing the RNA, and performing alignment analysis. Preferably, the RNA library is constructed using an RNA library preparation kit (such as the NEBNext® Small RNA Library Prep Set for Illumina), sequencing is performed using a sequencing platform (such as Illumina's NextSeq 500 instrument), and database alignment analysis is performed.

[0047] The specific steps of the glycosylation sequencing include: PNGase F digestion of the enriched glycoRNA, column chromatography separation, and mass spectrometry identification. Preferably, PNGase F (NEB) is used for overnight digestion at 37°C, glycosylation is performed using PGC SPE columns (Thermo Fisher Scientific), and mass spectrometry identification is performed using a Lumos Orbitrap mass spectrometer.

[0048] The specific steps for identifying the glycomolecular matrix include: PNGase F digestion of the enriched glycoRNA, column chromatography separation, and mass spectrometry identification. Preferably, PNGase F (NEB) is used for overnight digestion at 37°C, glycosyl groups are separated using PGC SPE columns (Thermo Fisher Scientific), and mass spectrometry identification is performed using a Lumos Orbitrap mass spectrometer.

[0049] In a third aspect, this application provides a method for detecting glycosylated RNA in vitro using α-2,3,α-2,6 type sialyltransferases, comprising the following steps: glycoRNAs with detectable groups are obtained by the method described in the first aspect; Detection of glycoRNAs with detectable groups.

[0050] In some implementations, glycoRNAs with detectable groups are detected by electrophoresis, fluorescence imaging, RNA blotting, ELISA, or a microplate reader.

[0051] In some embodiments, when the detectable group is a fluorescent group, detection can be performed by electrophoresis, fluorescence imaging, or using a microplate reader; specifically, electrophoresis and fluorescence imaging can be performed on a formaldehyde-denaturing agarose gel, or the fluorescence signal can be detected directly in a microplate reader. Preferably, a 1% formaldehyde-denaturing agarose gel is used, and 2×RNA Loading is added to the glycoRNA sample containing the detectable group, followed by denaturation and electrophoresis at 115V for 30 minutes, and then fluorescence imaging is performed.

[0052] In some embodiments, when the detectable group is biotin, detection can be performed by RNA blotting; for example, RNA blotting can be performed by electrophoresis, transfer, or chemiluminescence imaging. Specifically, electrophoresis (e.g., in formaldehyde-denaturing agarose gel) is performed to transfer RNA onto an NC membrane, followed by UV cross-linking, blocking with blocking buffer, and incubation with Streptavidin-HRP (streptavidin-horseradish peroxidase). Chemiluminescence imaging is then performed using the bound HRP. Preferably, a 1% formaldehyde-denaturing agarose gel is used. 2×RNA Loading is added to the glycoRNA sample containing the detectable group, followed by denaturation and electrophoresis at 115V for 30 minutes. Similar to Northern blotting, RNA is transferred onto a 0.45μm NC membrane; after transfer, UV cross-linking, blocking with blocking buffer, incubation with Streptavidin-HRP, and washing with washing buffer; chemiluminescence imaging using the bound HRP allows detection of the glycoRNA signal.

[0053] In a fourth aspect, this application provides a method for in vitro enzymatic chemical labeling and enrichment of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases, comprising the following steps: Biotin-containing glycoRNAs were obtained using the method described in the first aspect; Enrichment of glycoRNAs containing biotin.

[0054] In some embodiments, streptavidin magnetic beads can be used for enrichment, followed by elution to obtain enriched glycoRNA. Specifically, streptavidin magnetic beads are blocked with glycogen, and then biotin-containing glycoRNA is co-incubated with streptavidin magnetic beads (1-10°C, preferably 4-8°C). After washing away non-specific binding, elution is performed to dissociate the biotin on the glycoRNA from the streptavidin magnetic beads, purifying the enriched glycoRNA. Preferably, the specific steps are as follows: streptavidin magnetic beads are blocked with glycogen, and then biotin-containing glycoRNA is co-incubated with streptavidin magnetic beads at 4°C; non-specific binding is washed away with washing buffer, and then TRIzol is added, incubated at 70°C for 10 minutes to dissociate the biotin on the glycoRNA from the streptavidin magnetic beads; the TRIzol is transferred, and RNA is extracted, during which glycogen is added to promote precipitation; finally, it is dissolved in DEPC water to obtain the enriched glycoRNA.

[0055] In a fifth aspect, this application provides the use of the method for enriching glycosylated RNA as described in the fourth aspect in the RNA partial sequencing or glycosylation partial sequencing of glycoRNAs (i.e., structural identification, such as mass spectrometry identification).

[0056] This application provides the use of the method for enriching glycosylated RNA as described in the fourth aspect in RNA partial sequencing or glycomatrix identification of glycoRNAs.

[0057] In a sixth aspect, this application provides a method for RNA partial sequencing of glycoRNA, which includes obtaining enriched glycoRNA using the method for enriching glycosylated RNA described in the fourth aspect of this application, and then performing RNA sequencing.

[0058] In some embodiments, the specific steps of the RNA sequencing include: constructing a library of enriched glycoRNAs obtained by the method described in the fourth aspect using an RNA library preparation kit, sequencing the RNA using a sequencing platform, and performing alignment analysis. Preferably, the library is constructed using an RNA library preparation kit (such as the NEBNext® Small RNA Library Prep Set for Illumina), sequencing is performed using a sequencing platform (such as Illumina's NextSeq 500 instrument), and database alignment analysis is performed.

[0059] In a seventh aspect, this application provides a method for glycosylation sequencing (i.e., structural identification, such as mass spectrometry identification) of glycoRNA, which includes obtaining enriched glycoRNA using the method for enriching glycosylated RNA described in the fourth aspect of this application, and then performing glycosylation sequencing.

[0060] This application provides a method for identifying the glycoma spectrum of glycoRNA, which includes obtaining enriched glycoRNA using the method for enriching glycosylated RNA as described in the fourth aspect of this application, and then performing glycoma spectrum identification.

[0061] In some embodiments, the enriched glycoRNA obtained by the method described in the fourth aspect is subjected to PNGase F digestion, column chromatography separation, and mass spectrometry identification. Preferably, PNGase F (NEB) is used for overnight digestion at 37°C, glycosyl groups are separated using PGC SPEcolumns (Thermo Fisher Scientific), and mass spectrometry identification is performed using a Lumos Orbitrap mass spectrometer.

[0062] In an eighth aspect, this application provides a kit for the in vitro enzyme chemical labeling detection or enrichment of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases, comprising: Reagents for digesting sialic acid-modified RNA samples include sialidase as described in the first aspect, and optionally buffer solution A as described in the first aspect; Reagents for in vitro enzyme chemical labeling include α-2,3 or α-2,6 type sialyltransferases as described in the first aspect, CMP-Sialic acid with a labeled group as described in the first aspect; optional click chemistry reagents as described in the first aspect; optional buffer solution B as described in the first aspect; optional metal ions as described in the first aspect. Optional Streptavidin-HRP for detecting biotin-containing glycoRNAs; Optional streptavidin magnetic beads for enriching biotin-containing glycoRNAs.

[0063] In some embodiments, the kit also includes an RNase inhibitor.

[0064] In some embodiments, the kit also includes reagents and / or consumables for RNA extraction, such as those used in the TRIzol method, magnetic bead method, or column method.

[0065] Compared with the prior art, the beneficial effects of this application are as follows: (1) Compared with existing detection and enrichment technologies, this application is able to label, detect, enrich, and perform RNA sequencing analysis and glycosyl structure identification of glycoRNAs from all types of biological samples, including glycoRNAs from cultured cells, tissues, body fluids, and other samples. The sample sources include, but are not limited to, human cells and tissues, mouse tissues, serum samples, fruit flies, *C. elegans*, zebrafish, plant samples, fungi, and bacteria.

[0066] (2) The glycoRNA labeling method described in this application firstly digests all sialic acid at the end of the glycoRNA glycan chain in the RNA sample with sialidase to remove all sialic acid modifications and expose potential sialic acid modification sites. For RNA samples with no sialic acid at the end of the glycoRNA glycan chain but potential sialic acid modification sites, sialidase digestion is not required, and the undigested glycoRNA can be used directly. Then, sialic acid with the labeling group is transferred to the potential sialic acid modification sites at the end of the glycoRNA glycan chain by α-2,3 or α-2,6 type sialate transferase. This method can label all glycoRNAs that can be modified by sialylation, regardless of whether the sialic acid-modifiable sites of the glycoRNA in the RNA sample have been modified by sialic acid.

[0067] (3) The glycoRNA labeling method described in this application has the characteristics of high specificity and high sensitivity compared with the labeling methods of the prior art, and has no obvious by-products. Compared with metabolic labeling, this application does not require the addition of metabolic labeling reagents such as Ac4ManNAz during the cell culture stage, which can avoid their potential cytotoxicity and impact on cell metabolism, and reflect the true level of glycoRNA.

[0068] (4) The glycoRNA labeling detection and enrichment method described in this application uses inexpensive reagents, does not require expensive instruments, and is simple and convenient to operate, which is conducive to its widespread promotion and application.

[0069] (5) The glycoRNA labeling method described in this application can be directly detected or enriched by introducing sialic acid with a detectable group onto glycoRNA through an in vitro enzymatic chemical labeling reaction, such as a one-step reaction.

[0070] (6) Studies have shown that the glycoRNA labeling method described in this application has the best labeling effect when using Ph2,3siat and Pd26ST. Attached Figure Description

[0071] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 To understand the reaction mechanism of enzyme-catalyzed labeling using α-2,3,α-2,6 type sialyltransferases and azide-labeled sialic acid.

[0072] Figure 2-1 This diagram illustrates the workflow for in vitro enzyme-catalyzed labeling detection and enrichment of glycoRNA based on α-2,3,α-2,6 type sialyltransferases. Figure 1 .

[0073] Figure 2-2 Schematic diagram 2 shows the workflow for in vitro enzyme-catalyzed labeling detection and enrichment of glycoRNA based on α-2,3,α-2,6 type sialyltransferases.

[0074] For glycoRNA samples that lack sialic acid at the glycan ends but have potential sialic acid modification sites, sialidase digestion is unnecessary, and the undigested glycoRNA can be used directly.

[0075] Figure 3 To detect glycoRNAs in HCT116 cells using in vitro enzyme-catalyzed labeling based on α-2,3,α-2,6 type sialyltransferases.

[0076] Figure 4 To detect glycoRNAs in different cells using Pd26ST-based in vitro enzyme-catalyzed labeling.

[0077] Figures 5-1 to 5-7 To enable the detection of glycoRNAs in various biological samples using Pd26ST-based in vitro enzyme chemical labeling.

[0078] Figure 6-1 To detect glycoRNAs in rectal cancer patient tumor tissues using in vitro enzyme chemical labeling based on Ph2,3siat and Pd26ST.

[0079] Figure 6-2 Small RNA sequencing results of glycoRNAs enriched from rectal cancer patient tumor tissues using in vitro enzyme chemical labeling based on Ph2,3siat, and Pd26ST.

[0080] Figure 6-3 , Figure 6-4 The results of glycomatrix identification of glycoRNAs enriched from rectal cancer patient tumor tissues by in vitro enzyme chemical labeling based on Ph2,3siat and Pd26ST.

[0081] Figure 7-1 To compare the signal differences of glycoRNAs in tumor tissues and adjacent non-cancerous tissues of rectal cancer patients using in vitro enzyme chemical labeling based on Ph2,3siat.

[0082] Figure 7-2 To compare the signal differences of glycoRNA in tumor tissue and adjacent non-cancerous tissue of rectal cancer patients using in vitro enzyme chemical labeling based on Pd26ST.

[0083] Figure 8 To compare the signal differences of serum glycoRNAs from rectal cancer patients and healthy volunteers using in vitro enzyme chemical labeling based on Ph2,3siat. Detailed Implementation

[0084] The specific embodiments of this application are described in detail below. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0085] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0086] Features described or illustrated as part of one or more embodiments may be used in another or more embodiments to produce further embodiments.

[0087] Before describing this application in detail, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of this application, which is defined solely by the appended claims. For a more complete understanding of the application described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this application pertains.

[0088] definition Unless otherwise specified, the following terms used in this application shall have the following definitions.

[0089] Unless otherwise specified, the terms “comprising,” “including,” and “containing,” as well as similar expressions, shall be interpreted in an open and inclusive sense as “including but not limited to.”

[0090] The term “optional” means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

[0091] The term "glycosylated RNA" refers to RNA that has undergone glycosylation modification, i.e., ribonucleic acid with glycosylation modification.

[0092] The term "sialidase" refers to an enzyme that catalyzes the hydrolysis of all linear and branched non-reducing terminal sialic acid residues in glycan chains such as glycoproteins and oligosaccharides. It is an enzyme capable of hydrolyzing terminal sialic acid residues in glycan chains, thereby removing sialic acid from oligosaccharides or glycoconjugated molecules. The sialidase used in this application has at least α-2,3 or α-2,6 sialic acid activity, preferably at least α-2,3 and α-2,6 sialic acid activity, optionally α-2,8 and / or α-2,9 sialic acid activity, and preferably α2-3,6,8,9 Neuraminidase A. It catalyzes the hydrolysis of four types of sialic acid residues: α-2-3, α-2-6, α-2-8, and α-2-9. It can recognize and cleave sialic acid residues linked by α2-3, α2-6, α2-8, and α2-9 bonds on RNA-binding glycan chains or other glycosylated substrates, achieving efficient removal of terminal sialic acid from samples.

[0093] The term "α-2,3 type sialyltransferase" refers to a class of glycosyltransferases that catalyze the formation of α-2,3 type sialyl-modified sialyl, capable of catalyzing the transfer of cytidine-activated sialyl (CMP-Sia) to galactose residues on an acceptor substrate, and forming an α-2,3 bond between the 3-hydroxyl group of the galactose and the sialyl. The α-2,3 type sialyltransferases used in this application are bacterial α-2,3 type sialyltransferases, such as PmST1, PmST3, Ph2,3siat, and their derived mutants and truncated variants possessing α-2,3 type sialyltransferase activity.

[0094] The PmST1 used is the M144D mutant PmST1, with the following amino acid sequence, Seq ID No. 1: mktitlyldpaslpalnqlmdftqnnedkthprifglsrfkipdniitqyqnihfvelkdnrptealftildqypgnielnihlniahsvqlirpilayrfkhldrvsiqqlnlyddgsdeyvdlekeenkdisaeikqaekqlshylltgkikfdnptiaryvwqsafpvkyhflstdyfekaeflqplkeylaenyqkmdwtayqqltpeqqafyltlvgfndevkqslevqqakfiftgtttwegntdvreyyaqqqlnllnhftqaegdlfigdhykiyfkghprggeindyilnnaknitnipanisfevlmmtgllpdkvggvasslyfslpkekishiiftsnkqvkskedalnnpyvkvmrrlgiidesqvifwdslkqlggglehhhhhh。

[0095] The amino acid sequence of PmST3 is, Seq ID No.2: mdkfaeheipkavivagngeslsqidyrllpknydvfrcnqfyfeeryflgnkikavfftpgvfleqyytlyhlkrnneyfvdnvilssfnhptvdleksqkiqalfidvingyekylskltafdvylrykelyenqritsgvymcavaiamgytdiyltgidfyqaseenyafdnkkpniirllpdfrkektlfsyhskdidlealsflqqhyhvnfysispmsplskhfpiptveddcettfvaplkenyindillpphfvyeklgtivskksrfhsnlivrlirdllklpsalkhylkek。

[0096] The truncated form △24Ph2,3sia is used for Ph2,3siat, and its amino acid sequence is, Seq ID No.3: DSKHNNSDGNITKNKTIEVYVDRATLPTIQQMTQIINENSNNKKLISWSRYPINDETLLESINGSFFKNRPELIKSLDSMILTNEIKKVIINGNTLWAVDVVNIIKSIEALGKKTEIELNFYDDGSAEYVRLYDFSRLPESEQEYKISLSKDNIQSSINGTQPFDNSIENIYGFSQLYPTTYHMLRADIFETN LPLTSLKRVISNNIKQMKWDYFTTFNSQQKNKFYNFTGFNPEKIKEQYKASPHENFIFIGTNSGTATAEQQIDILTEAKKPDSPIITNSIQGLDLFFKGHPSATYNQQIIDAHNMIEIYNKIPFEALIMTDALPDAVGGMGSSVFFSLPNTVENKFIFYKSDTDIENNALIQVMIELNIVNRNDVKLISDLQ.

[0097] The term "α-2,6 type sialyltransferase" refers to a type of glycosyltransferase that catalyzes the formation of α-2,6 type sialyl-modified sialyl (CMP-Sia), enabling the transfer of cytidine-activated sialyl (CMP-Sia) to a galactose residue on an acceptor substrate, and forming an α-2,6 bond between the 6-hydroxyl group of the galactose residue and the sialyl residue. The α-2,6 type sialyltransferase used in this application is a bacterial α-2,6 type sialyltransferase, such as PPd26ST and its derived mutants and truncated forms possessing α-2,6 type sialyltransferase activity. Specifically, the truncated form △15Pd26ST is used, with the following amino acid sequence: SeqID No. 4: .

[0098] The term "CMP-Sialic acid" refers to cytidine 5'-monophosphate-N-acetylneuraminic acid, also known as cytidine 5'-monophosphate-sialic acid.

[0099] The term "CMP-Sialic acid with a labeled group" typically refers to sialic acid where the 9-hydroxyl group is replaced by a labeled group (such as -N3). For example, the structure of CMP-C9-Azide-Sialic acid is as follows: .

[0100] The term "fluorescent group" refers to a group that can emit light of another wavelength when excited by light of a certain wavelength, such as CY5 and CY3.

[0101] The term "RNA blotting" refers to RNA blotting, which involves denaturing and electrophoretically separating RNA, transferring it onto a solid support, and then hybridizing it with DNA probes to identify the size and abundance of specific RNA molecules.

[0102] The term "ELISA" refers to an enzyme-linked immune response.

[0103] The term "Glycogen" refers to a branched polysaccharide formed by the binding of glucose, with its glycosidic chain being of the α-type.

[0104] The term "proteinase K" refers to a powerful proteolytic enzyme isolated from Candida albicans, which has a very high specific activity.

[0105] The term "PNGase F" refers to an amidase that can cleave between the innermost GlcNAc and asparagine residues in high-mannose, heterozygous, and complex oligosaccharides.

[0106] All water used in this application is DEPC-treated water, and all instruments, test tubes, pipettes, etc., used have been treated with RNase removal reagent. M refers to the concentration in mol / L. The reagents used and their preparation are as follows: 1. 70% ethanol (volume ratio, aqueous solution).

[0107] 2. 100mM Tris·HCl (for example, 50mL): Dissolve 12.114g of Tris base in a certain amount of DEPC water, adjust the pH to the required level with concentrated hydrochloric acid, and then bring the volume to 50mL.

[0108] 3. 25mM CMP-C9-Azide-Sialic acid: 5mg CMP-C9-Azide-Sialic acid dissolved in 313μL DMSO, filtered through a 0.22μm filter membrane.

[0109] 4. "dye-free" Gel Loading Buffer II (df-GLBII, 1 mL as an example): Add 950 μL of formamide, 36 μL of 500 mM EDTA, 2.5 μL of 10% SDS, and 11.5 μL of DEPC water in sequence, and mix well.

[0110] 5. 10mM DBCO (dibenzocyclooctylene)-PEG4-Biotin: 5mg DBCO-PEG4-Biotin dissolved in 666μL DMSO and filtered through a 0.22μm filter membrane.

[0111] 6. 10mM DBCO-CY5: 5mg DBCO-CY5 dissolved in 538μL DMSO and filtered through a 0.22μm filter membrane.

[0112] 7. 20× SSC (taking 1L as an example): 175.3g NaCl, 88.2g trisodium citrate dihydrate, dissolved in 1L DEPC water, pH adjusted to 7.0 with concentrated hydrochloric acid, and autoclaved.

[0113] 8. 1×MOPS:10×MOPS buffer (0.2M 3-morpholinopropanesulfonic acid, 0.05M sodium acetate, 0.01M disodium ethylenediaminetetraacetate) can be diluted at a volume ratio of 1:10.

[0114] 9. Biotin Wash Buffer: Final concentration 10 mM Tris·HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.05% Tween-20.

[0115] 10. ChIRP Wash Buffer: Final concentration is 2 × SSC, 0.5% SDS.

[0116] 11. NT2 Buffer: 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.005% NP-40 (nonylphenyl polyethylene glycol).

[0117] 12. TBST: Dissolve 8g NaCl, 0.2g KCl and 3g Tris Base in 800mL distilled water, adjust the pH to 7.4 with concentrated hydrochloric acid, bring the volume to 1L with distilled water, and finally add 1mL Tween-20 and stir well.

[0118] 13. 500mM MgCl2 (taking 50mL as an example): 5.08g MgCl2·6H2O dissolved in 50mL DEPC water.

[0119] The PmST1 used in the examples has the amino acid sequence shown in Seq ID No. 1, the PmST3 has the amino acid sequence shown in Seq ID No. 2, the Ph2,3siat has the amino acid sequence shown in Seq ID No. 3, and the Pd26ST has the amino acid sequence shown in Seq ID No. 4.

[0120] Example 1 Detection of glycoRNAs derived from HCT116 cells using an in vitro enzyme chemical labeling method based on α-2,3 sialyl transferases (PmST1, PmST3, Ph2,3siat) and α-2,6 sialyl transferase (Pd26ST). 1. Extraction of total RNA from cells Total RNA was extracted using the classic TRIzol reagent. After lysis of the sample with TRIzol, it was extracted with chloroform, followed by precipitation of RNA from the aqueous phase with isopropanol. The precipitate was then washed twice with 70% ethanol, and finally dissolved in DEPC water to obtain a solution of total RNA. The specific steps are as follows: For HCT116 cells cultured adherently in 6cm culture dishes, aspirate the culture medium and wash the cells twice with pre-cooled 1×PBS. Discard the 1×PBS, add 1 mL of TRIzol (Invitrogen), and gently shake the culture dish to ensure the TRIzol is evenly distributed on the cell surface (cells can be detached using a cell scraper). Use a pipette to repeatedly pipette to detach the cells, then transfer the lysate containing the cells to a centrifuge tube, and pipette again until there is no obvious precipitate in the lysate (the solution is clear).

[0121] After standing at room temperature for 5-10 minutes, add 1 / 5 volume of chloroform to the above lysis solution, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0122] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0123] Add 1 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0124] Add 1 mL of TRIzol and extract RNA as described above. Dissolve the RNA in 10 μL of DEPC water to obtain the total RNA solution of HCT116.

[0125] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1 (NEB), 2 μL of α2-3,6,8,9Neuraminidase A (NEB), and DEPC water to the total RNA solution after digestion with proteinase K, and bring the volume to 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0126] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 volume of chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0127] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0128] 3. In vitro enzyme chemical labeling Take 25 μg of RNA from the HCT116 RNA solution and add α-2,3-sialyltransferase PmST1, PmST3, Ph2,3siat or α-2,6-sialyltransferase Pd26ST to a final concentration of 1 U / mL, RNase inhibitor (Novizan) to a final concentration of 1 U / μL, 3 μL of 25 mM CMP-C9-Azide-Sialic acid (Qiyue Biotechnology), and Mg2+ to a final concentration of 5 mM. 2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 8.0). The reaction was carried out at 37 °C for 3 h.

[0129] Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0130] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution of HCT116 containing azide-labeled glycoRNA.

[0131] 4. No copper-free click chemical reaction Add 10 μL of df-GLBII and 1 μL of 10 mM DBCO-PEG4-Biotin to the dissolved RNA solution, mix well; react at 55 °C for 10 min; stop the reaction by adding 80 μL of DEPC water.

[0132] Add 900 μL of TRIzol and vortex for 5-10 seconds; the subsequent RNA extraction steps are the same as above (among which 600 μL of supernatant is aspirated and isopropanol is added), and finally 10 μL of DEPC is added for dissolution.

[0133] 5. Formaldehyde denaturing gel electrophoresis, transfer To prepare a 1% formaldehyde denaturing gel: After washing and drying the beaker, add about 80 mL of DEPC water, weigh 0.6 g of agarose and add it to the beaker. Microwave on medium-high for 3 minutes, then remove and shake well. Continue heating until about 40 mL remains. Add 6 mL of 10×MOPS (Sangon Biotech) and shake well. Add 10.8 mL of 37% formaldehyde solution and shake well. Add 1.5 μL of SuperRed nucleic acid dye, pour into a gel casting plate with a comb inserted, and let stand for later use.

[0134] Add 10 μL of 2×RNA Loading (Beyotime) to the above RNA solution and mix well. Denature at 55°C for 10 min and then immediately place on ice for 3 min.

[0135] Place the gel in the electrophoresis tank, add 1×MOPS as the electrophoresis buffer, load the sample, and incubate at a constant voltage of 115V for 30 minutes; use the GelView 5000Plus intelligent gel imaging system (Boluteng) for gel imaging, such as... Figure 3 As shown on the left, the integrity of RNA is determined by the bands formed after RNA electrophoresis, indicating that the extracted RNA is intact.

[0136] Place a glass plate slightly larger than the gel in a petri dish as a platform, and add enough 20×SSC to cover the platform. Construct a transfer platform by cutting three pieces of transfer filter paper the same size as the platform and placing them on the platform surface, wetting them with 20×SSC. Place the gel on the filter paper surface and roll the surface with a glass rod to press out air bubbles. Cover the edges of the gel with four sheets of plastic wrap to prevent short circuits.

[0137] Cut a bio-Rad membrane (NC membrane) of similar size to the gel surface and wet it with DEPC water. Place the wetted NC membrane on the gel surface, remove air bubbles, and rinse the membrane surface with 20×SSC. Cut three transfer filter papers of similar size to the NC membrane and place them on the membrane surface, removing air bubbles. Then, neatly stack absorbent paper of similar size to the NC membrane on the surface of the filter paper, about 4 cm high. Place a glass plate on top of the absorbent paper and weigh it down with a weight (0.2–0.4 kg), and leave it overnight.

[0138] 6. Imaging Disassemble the transfer device, place the NC membrane on clean filter paper, and perform UV crosslinking at 0.18 J / cm. 2 Cross-linking; block with Odyssey Blocking Buffer on a horizontal shaker for 1 h; dilute Streptavidin-HRP (Streptavidin-Horderadish Peroxidase, Beyotime) 1:2000 in Odyssey Blocking Buffer (Licor) and incubate on a horizontal shaker for 30 min; wash 3 times with TBST for 5 min each time.

[0139] Prepare Immobilon ECL Ultra Western HRP Substrate developer (Merck), drop it onto the NC membrane, and image it in an Amersham ImageQuant 800 multifunction imager (GE).

[0140] like Figure 3 As shown on the right, several enzymes can effectively label sialylated glycoRNAs, with Ph2,3siat and Pd26ST showing better labeling effects.

[0141] Example 2 Detection of glycoRNAs from different cell sources using an in vitro enzymatic chemical labeling method based on α-2,6 sialyltransferase (Pd26ST). 1. Extraction of total RNA from cells Total RNA was extracted using the classic TRIzol reagent. After lysis of the sample with TRIzol, it was extracted with chloroform, followed by precipitation of RNA from the aqueous phase with isopropanol. The precipitate was then washed twice with 70% ethanol, and finally dissolved in DEPC water to obtain a solution of total RNA. The specific steps are as follows: For cells cultured in 6cm culture dishes, aspirate the culture medium and wash the cells twice with pre-cooled 1×PBS. Discard the 1×PBS, add 1 mL of TRIzol (Invitrogen), and gently shake the culture dish to ensure the TRIzol is evenly distributed on the cell surface (cells can be detached using a cell scraper). Use a pipette to repeatedly pipette to detach the cells, then transfer the lysate containing the cells to a centrifuge tube, and pipette again until there is no obvious precipitate in the lysate (the solution is clear).

[0142] After standing at room temperature for 5-10 minutes, add 1 / 5 volume of chloroform to the above lysis solution, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0143] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0144] Add 1 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0145] Add 1 mL of TRIzol and extract RNA as described above. Dissolve the RNA in 10 μL of DEPC water to obtain a total RNA solution.

[0146] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1 (NEB), 2 μL of α2-3,6,8,9Neuraminidase A (NEB), and DEPC water to the total RNA solution after digestion with proteinase K, and bring the volume to 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0147] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 volume of chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0148] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0149] 3. In vitro enzyme chemical labeling Take 25 μg of RNA from the total RNA solution and add 10 U / mL of α-2,6-sialyltransferase Pd26ST, 1 U / μL of RNase inhibitor (Novazia), 3 μL of 25 mM CMP-C9-Azide-Sialic acid (Qiyue Biotechnology), and 5 mM of Mg...2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 7.0). The reaction was carried out at 37 °C for 3 h.

[0150] Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0151] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution containing azide-labeled glycoRNA.

[0152] 4. No copper-free click chemical reaction Add 10 μL of df-GLBII and 1 μL of 10 mM DBCO-PEG4-Biotin to the dissolved RNA solution, mix well; react at 55 °C for 10 min; stop the reaction by adding 80 μL of DEPC water.

[0153] Add 900 μL of TRIzol and vortex for 5-10 seconds; the subsequent RNA extraction steps are the same as above (among which 600 μL of supernatant is aspirated and isopropanol is added), and finally 10 μL of DEPC is added for dissolution.

[0154] 5. Formaldehyde denaturing gel electrophoresis, transfer To prepare a 1% formaldehyde denaturing gel: After washing and drying the beaker, add about 80 mL of DEPC water, weigh 0.6 g of agarose and add it to the beaker. Microwave on medium-high for 3 minutes, then remove and shake well. Continue heating until about 40 mL remains. Add 6 mL of 10×MOPS (Sangon Biotech) and shake well. Add 10.8 mL of 37% formaldehyde solution and shake well. Add 1.5 μL of SuperRed nucleic acid dye, pour into a gel casting plate with a comb inserted, and let stand for later use.

[0155] Add 10 μL of 2×RNA Loading (Beyotime) to the above RNA solution and mix well. Denature at 55°C for 10 min and then immediately place on ice for 3 min.

[0156] Place the gel in the electrophoresis tank, add 1×MOPS as the electrophoresis buffer, load the sample, and incubate at a constant voltage of 115V for 30 minutes; use the GelView 5000Plus intelligent gel imaging system (Boluteng) for gel imaging, such as... Figure 4 As shown on the left, the integrity of RNA is determined by the bands formed after RNA electrophoresis, indicating that the extracted RNA is intact.

[0157] Place a glass plate slightly larger than the gel in a petri dish as a platform, and add enough 20×SSC to cover the platform. Construct a transfer platform by cutting three pieces of transfer filter paper the same size as the platform and placing them on the platform surface, wetting them with 20×SSC. Place the gel on the filter paper surface and roll the surface with a glass rod to press out air bubbles. Cover the edges of the gel with four sheets of plastic wrap to prevent short circuits.

[0158] Cut a bio-Rad membrane (NC membrane) of similar size to the gel surface and wet it with DEPC water. Place the wetted NC membrane on the gel surface, remove air bubbles, and rinse the membrane surface with 20×SSC. Cut three transfer filter papers of similar size to the NC membrane and place them on the membrane surface, removing air bubbles. Then, neatly stack absorbent paper of similar size to the NC membrane on the surface of the filter paper, about 4 cm high. Place a glass plate on top of the absorbent paper and weigh it down with a weight (0.2–0.4 kg), and leave it overnight.

[0159] 6. Imaging Disassemble the transfer device, place the NC membrane on clean filter paper, and perform UV crosslinking at 0.18 J / cm. 2 Cross-linking; block with Odyssey Blocking Buffer on a horizontal shaker for 1 h; dilute Streptavidin-HRP (Streptavidin-Horderadish Peroxidase, Beyotime) 1:2000 in Odyssey Blocking Buffer (Licor) and incubate on a horizontal shaker for 30 min; wash 3 times with TBST for 5 min each time.

[0160] Prepare Immobilon ECL Ultra Western HRP Substrate developer (Merck), drop it onto the NC membrane, and image it in an Amersham ImageQuant 800 multifunction imager (GE).

[0161] like Figure 4 As shown on the right, Pd26ST can effectively transfer sialic acid containing azide labeling onto glycoRNAs in various cells, enabling the effective detection of sialylated glycoRNAs.

[0162] Example 3 An in vitro enzymatic chemical labeling method based on α-2,6-sialyltransferase (Pd26ST) was used to detect glycoRNAs from human, bacterial, fungal, *C. elegans*, *Drosophila melanogaster*, *Arabidopsis thaliana*, and other organisms. 1. Extraction of total RNA from tissues Biological samples were collected: 50 mg of surgical tumor tissue samples from patients with esophageal, lung, and rectal cancer; 50 mg of mouse tissue samples; 10 fruit flies; 5000 *C. elegans* worms; 1 zebrafish; roots, stems, flowers, and leaves from 1 *Arabidopsis thaliana* plant; 25 mg of fungi; and 15 mg of bacteria. These samples were rapidly ground in liquid nitrogen and transferred to RNase-free EP tubes. 1 mL of TRIzol reagent was added. 200 μL of chloroform was added, and the mixture was shaken to mix for 30 seconds and incubated on ice for 2 minutes.

[0163] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube and add an equal volume (500 μL) of isopropanol. Mix thoroughly by inverting and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min. A white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible. Wash the precipitate with 1 mL of pre-chilled 70% ethanol. Centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant. Repeat the washing and centrifugation process, discarding the supernatant after each centrifugation. Air dry at room temperature for 5 min, then add 20 μL of DEPC water and dissolve on ice for 10 min to obtain the total RNA solution.

[0164] Add 0.5 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0165] Add 980 μL of TRIzol to the total RNA solution digested with proteinase K, vortex for 5-10 seconds, extract RNA according to the above steps, and finally add 10 μL of DEPC water to dissolve to obtain the proteinase K-treated RNA solution.

[0166] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1, 2 μL of α2-3,6,8,9Neuraminidase A, and DEPC water to the above-mentioned proteinase K-treated RNA solution, and bring the volume to 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0167] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 the volume of TRIzol in chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0168] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0169] Since there is no sialic acid modification at the sialic acid-modifiable sites of the RNA in the Arabidopsis thaliana sample, sialidase digestion is not required.

[0170] 3. In vitro enzyme chemical labeling Take 25 μg of RNA from each of the above total RNA solutions, add 10 U / mL of α-2,6-sialidyltransferase Pd26ST, 1 U / μL of RNase inhibitor, 3 μL of 25 mM CMP-C9-Azide-Sialic acid, and 5 mM MgSO4. 2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 7.0). The reaction was carried out at 37 °C for 3 h.

[0171] Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0172] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution containing azide-labeled glycoRNA.

[0173] 4. No copper-free click chemical reaction The copper-free click chemistry reaction was carried out using the same method as step 4 in Example 1.

[0174] 5. Formaldehyde denaturing gel electrophoresis, transfer A 1% formaldehyde denaturing gel was prepared using the same method as step 5 in Example 1, and gel electrophoresis imaging was performed, as shown below. Figure 5-1Left to Figure 5-7 As shown on the left, the transfer.

[0175] 6. Imaging Imaging was performed using the same method as step 6 in Example 1, such as... Figure 5-1 Right to Figure 5-7 As shown on the right, Pd26ST can effectively transfer sialic acid containing azide labeling to glycoRNAs in various biological samples, enabling the effective detection of sialyzable glycoRNAs.

[0176] Example 4 An in vitro enzymatic chemical labeling method based on α-2,3-sialate transferase Ph2,3siat or α-2,6-sialate transferase Pd26ST was used to detect and enrich glycoRNAs derived from tumor tissues of rectal cancer patients, followed by RNA sequencing analysis and mass spectrometry identification of glycosyl groups. 1. Extraction of total RNA from tissues Surgical tumor tissue samples were collected from rectal cancer patients. 50 mg of the tissue was added to liquid nitrogen and rapidly ground. The mixture was then transferred to an RNase-free EP tube and lysed with 1 mL of TRIzol reagent.

[0177] Add 200 μL of chloroform, shake to mix for 30 seconds, and let stand on ice for 2 minutes.

[0178] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube and add an equal volume (500 μL) of isopropanol. Mix thoroughly by inverting and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min. A white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible. Wash the precipitate with 1 mL of pre-chilled 70% ethanol. Centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant. Repeat the washing and centrifugation process, discarding the supernatant after each centrifugation. Air dry at room temperature for 5 min, then add 20 μL of DEPC water and dissolve on ice for 10 min to obtain the total RNA solution.

[0179] Add 1 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0180] Add 1 ml of TRIzol, and continue the RNA extraction steps as before. Finally, add 10 μL of DEPC water to dissolve and obtain the RNA solution treated with proteinase K.

[0181] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1 (NEB), 2 μL of α2-3,6,8,9Neuraminidase A, and DEPC water to the RNA solution digested with proteinase K to a final volume of 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0182] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 the volume of TRIzol in chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0183] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0184] 3. In vitro enzyme chemical labeling Take 25 μg of RNA from the above total RNA solution, add 1 U / mL of α-2,3-sialyltransferase Ph2,3siat or 1 U / mL of α-2,6-sialyltransferase Pd26ST, 1 U / μL of RNase inhibitor, 3 μL of 25 mM MMP-C9-Azide-Sialic acid, and 5 mM of MgSO4. 2+ One tube was filled with 100 mM Tris·HCl (pH 8.0) to a final volume of 30 μL; the other tube contained only 25 μg RNA and an RNase inhibitor at a final concentration of 1 U / μL, and was filled with 100 mM Tris·HCl (pH 8.0) to a final volume of 30 μL. The reaction was carried out at 37 °C for 3 h.

[0185] Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0186] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution containing azide-labeled glycoRNA.

[0187] 4. No copper-free click chemical reaction The copper-free click chemistry reaction was carried out using the same method as step 4 in Example 1.

[0188] 5. Formaldehyde denaturing gel electrophoresis, transfer Prepare a 1% formaldehyde denaturing gel using the same method as step 5 in Example 1, and perform gel electrophoresis imaging (e.g. Figure 6-1 (As shown on the left), transfer.

[0189] 6. Imaging Imaging was performed using the same method as step 6 in Example 1, such as... Figure 6-1 As shown on the right, Ph2,3siat or Pd26ST can effectively transfer sialic acid containing azide to glycoRNA in tumor tissue, enabling efficient detection of sialyzable glycoRNA.

[0190] 7. Enrichment of glycoRNAs Take 10 μL of MyOne C1 Streptavidin beads (Thermo Fisher Scientific) into an EP tube, add Biotin Wash Buffer to wash, place on a magnetic rack and let stand for 2 min, remove the buffer, add 1 mL of Biotin Wash Buffer, then add 2 μL of Glycogen, and block at room temperature for 1 h in a suspension apparatus.

[0191] Place on a magnetic rack and let stand for 2 minutes. Remove the buffer, add 800 μL of Biotin Wash Buffer, and then add 50 μg of Biotin-labeled glycoRNA obtained in step 4 (copper-free click chemistry). Incubate at 4°C for 2 hours.

[0192] Place on a magnetic rack and let stand for 2 minutes. Remove the buffer, add 1 mL of ChIRP Wash Buffer, and wash twice at room temperature for 3 minutes each time in a suspending apparatus.

[0193] Place on a magnetic rack and let stand for 2 minutes. Remove the buffer, add 1 mL of Biotin Wash Buffer, and wash twice at room temperature for 3 minutes each time in a suspending apparatus.

[0194] Place on a magnetic rack and let stand for 2 minutes. Remove the buffer, add 1 mL of NT2 Buffer, and wash twice at room temperature for 3 minutes on a suspending apparatus.

[0195] Place on a magnetic rack and let stand for 2 minutes. Remove the buffer, add 1 mL of TRIzol, and denature at 70°C for 10 minutes. Place on a magnetic rack and let stand for 2 minutes. Transfer the TRIzol to a new EP tube.

[0196] Repeat the RNA extraction steps, adding Glycogen to promote RNA precipitation during the RNA precipitation step; dissolve in 10 μL of DEPC in water.

[0197] 8. Small RNA library construction and sequencing of tissue-derived glycoRNAs The concentration and length distribution of Small RNA from the previous step were detected using an Agilent 2100 analyzer.

[0198] The RNA samples were used to construct libraries using the NEB Next® Small RNA (NEB) Diverse Library Preparation Kit, and then sequenced using a NextSeq 500 (Illumina) to obtain the glycoRNA sequence information. Some information can be found in [link to relevant documentation]. Figure 6-2 .

[0199] As can be seen from 6-2, glycoRNAs in tumor tissues are small RNAs, mainly composed of small RNAs such as rRNA, tRNA, and tsRNA.

[0200] 9. Mass spectrometry identification of glycosyl groups in tissue-derived glycoRNAs The enriched glycoRNA obtained in step 7 was digested overnight at 37°C with 8 μL of PNGase F (N-glycosidase F, NEB). The sample was then desalted using PGC SPE columns. The glycan structure was identified by LC-MS mass spectrometry; the results are shown below. Figure 6-3 , 6-4 .

[0201] Depend on Figure 6-3 , 6-4 It is evident that the N-glycans of glycoRNAs derived from tumor tissues are mainly complex, containing a large amount of sialic acid and fucose modifications, and are rich in N-glycan chains with multiple branches.

[0202] Example 5 In vitro enzymatic chemical labeling methods based on α-2,3 sialyl transferase (Ph2,3siat) and α-2,6 sialyl transferase (Pd26ST) were used to compare the signal intensity of glycoRNAs in tumor tissues and adjacent normal tissues. 1. Extraction of total RNA from tissues Three pairs of surgical tumor tissue and adjacent normal tissue samples were collected from clinical oncology patients. 50 mg of each sample was taken, rapidly ground in liquid nitrogen, and then transferred to RNase-free EP tubes. 1 mL of TRIzol reagent was added. 200 μL of chloroform was added, and the mixture was shaken for 30 seconds and incubated on ice for 2 minutes.

[0203] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube and add an equal volume (500 μL) of isopropanol. Mix thoroughly by inverting and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min. A white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible. Wash the precipitate with 1 mL of pre-chilled 70% ethanol. Centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant. Repeat the washing and centrifugation process, discarding the supernatant after each centrifugation. Air dry at room temperature for 5 min, then add 20 μL of DEPC water and dissolve on ice for 10 min to obtain the total RNA solution.

[0204] Add 1 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0205] Add 980 μL of TRIzol to the total RNA solution digested with proteinase K, vortex for 5-10 seconds, extract RNA according to the above steps, and finally add 10 μL of DEPC water to dissolve to obtain the proteinase K-treated RNA solution.

[0206] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1, 2 μL of α2-3,6,8,9Neuraminidase A, and DEPC water to the above-mentioned proteinase K-treated RNA solution, and bring the volume to 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0207] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 the volume of TRIzol in chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0208] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0209] 3. In vitro enzyme chemical labeling Take 25 μg of RNA from each of the three pairs of tumor and adjacent normal tissues extracted from the total RNA solutions mentioned above, and add α-2,3-siat type 2,3-sialate (Ph2,3siat) at a final concentration of 1 U / mL, an RNase inhibitor at a final concentration of 1 U / μL, 3 μL of 25 mM CMP-C9-Azide-Sialic acid, and Mg2+ at a final concentration of 5 mM. 2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 8.0). The reaction was carried out at 37 °C for 3 h.

[0210] Take 25 μg of RNA from each of the three pairs of tumor and adjacent normal tissues extracted from the total RNA solutions mentioned above, and add α-2,6-sialidyltransferase Pd26ST to a final concentration of 10 U / mL, RNase inhibitor to a final concentration of 1 U / μL, 3 μL of 25 mM CMP-C9-Azide-Sialic acid, and Mg2+ to a final concentration of 5 mM. 2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 7.0). The reaction was carried out at 37 °C for 3 h. Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0211] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution containing azide-labeled glycoRNA.

[0212] 4. Copper-free click chemistry reactions using click chemistry reagents with fluorescent groups. Add 10 μL of df-GLBII and 1 μL of 10 mM DBCO-CY5 to the dissolved RNA solution and mix well; (from this step onwards, all subsequent experiments must be conducted in the dark); react at 55°C for 10 min; add 80 μL of DEPC water to terminate the reaction.

[0213] Add 900 μL of TRIzol and vortex for 5-10 seconds; the subsequent RNA extraction steps are the same as above (among which 600 μL of supernatant is aspirated and isopropanol is added), and finally 10 μL of DEPC is added for dissolution.

[0214] 5. Detect fluorescence signals using an ELISA reader. Add the three pairs of RNA obtained in step 4 above to a black 96-well plate, and add 140 μL of water to bring the total to 150 μL. Use a microplate reader (revity) to detect the fluorescence intensity of CY5, with an excitation wavelength of 825 nm and an absorption wavelength of 880 nm.

[0215] See results Figure 7-1 Left, Figure 7-2 Left: Comparison of glycoRNA signal intensity in 3 tumors and 3 adjacent normal tissues.

[0216] 6. Formaldehyde denaturing gel electrophoresis and fluorescence imaging A 1% formaldehyde denaturing gel was prepared using the same method as step 5 in Example 1, and gel electrophoresis was performed. Gel imaging was conducted using a GelView 5000Plus intelligent gel imaging system. Figure 7-1 middle, Figure 7-2 As shown in the figure, the integrity of RNA is determined by the bands formed after RNA electrophoresis, indicating that the extracted RNA is intact.

[0217] Fluorescence imaging was performed using the Amersham ImageQuant 800 Multifunction Imager (GE), such as... Figure 7-1 right, Figure 7-2 As shown on the right, the location and intensity of the CY5 signal can be seen. The results indicate that the CY5 signal can be detected in both tumor and normal tissues, meaning that glycoRNA is present in both. Furthermore, the intensity of the CY5 signal is consistent with the results of gel electrophoresis.

[0218] Example 6 In vitro enzyme chemical labeling method based on α-2,3 sialyl transferase (Ph2,3siat) was used to compare the signal intensity of glycoRNA in the serum of tumor patients and normal volunteers. 1. Extraction of total RNA from serum Serum was collected from three clinical cancer patients and three normal volunteers, 500 μL from each. The 500 μL serum was divided into two portions, with 250 μL of serum added to each portion and 750 μL of TRIzol LS added. The mixture was shaken to mix.

[0219] Add 200 μL of chloroform, shake to mix for 30 seconds, and let stand on ice for 2 minutes.

[0220] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume (500 μL) of isopropanol and 1 μL of glycogen (Thermo Fisher Scientific), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, add another 0.5 mL of pre-chilled 70% ethanol to wash, and combine two serum samples from the same patient or healthy volunteer, centrifuge, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution.

[0221] Add 1 μL of proteinase K to the total RNA solution and digest at 37°C for 45 min.

[0222] Add 980 μL of TRIzol to the total RNA solution digested with proteinase K, vortex for 50-10 seconds, and then perform the same RNA extraction steps as before. Finally, add 10 μL of DEPC water to dissolve the RNA.

[0223] 2. Sialidase hydrolysis Add 2 μL of glyco buffer 1 (NEB), 2 μL of α2-3,6,8,9 Neuraminidase A, and DEPC water to the total RNA solution after digestion with proteinase K, and bring the volume to 20 μL. Incubate at 37°C for 1 hour to obtain total RNA containing glycoRNAs with sialic acid modification removed.

[0224] Add 980 μL of TRIzol, vortex for 5-10 seconds, let stand at room temperature for 5-10 minutes, then add 1 / 5 volume of chloroform to the above lysis buffer, shake to mix for 30 seconds, and let stand at room temperature for 2 minutes.

[0225] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new centrifuge tube, add an equal volume of isopropanol (500 μL), mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing process, centrifuge again, and discard the supernatant. Air dry at room temperature for 5 min, add 20 μL of DEPC water, and dissolve on ice for 10 min.

[0226] 3. In vitro enzyme chemical labeling The above 6 RNA solutions were respectively supplemented with α-2,3-sialyltransferase Ph2,3siat at a final concentration of 1 U / mL, RNase inhibitor (Novozymes) at a final concentration of 1 U / μL, 3 μL of 25 mM CMP-C9-Azide-Sialic acid (Qiyue Biotechnology), and Mg at a final concentration of 5 mM. 2+ The volume was brought to 30 μL with 100 mM Tris·HCl (pH 8.0). The reaction was carried out at 37 °C for 3 h.

[0227] Transfer the total reaction mixture to a new EP tube, add 970 μL of TRIzol, and vortex for 5-10 s; add 1 / 5 volume of chloroform to the above mixture, shake to mix for 30 s, and let stand at room temperature for 2 min.

[0228] Centrifuge at 12000g, 4℃ for 15 min. Transfer 500 μL of the supernatant to a new EP tube, add an equal volume of isopropanol (500 μL) and 1 μL of glycogen, mix by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g, 4℃ for 30 min; a white precipitate will appear at the bottom of the EP tube. Discard as much supernatant as possible, add 1 mL of pre-chilled 70% ethanol to wash the precipitate, and centrifuge at 12000g, 4℃ for 5 min. Discard the supernatant, repeat the washing and centrifugation, and discard the supernatant again. Air dry at room temperature for 5 min, add 9 μL of DEPC water, and dissolve on ice for 10 min to obtain the total RNA solution containing azide-labeled glycoRNA.

[0229] 4. Copper-free click chemistry reactions using click chemistry reagents with fluorescent groups. The RNA solution obtained in step 3 above was subjected to a copper-free click chemistry reaction in the same manner as step 4 in Example 3.

[0230] 5. Detect fluorescence signals using an ELISA reader. The six RNA solutions obtained in step 4 above were added to black 96-well plates, and the CY5 fluorescence intensity was detected using the same method as in step 5 of Example 3.

[0231] See results Figure 8 Left: Comparing the signal strength of glycoRNAs in the serum of cancer patients and healthy volunteers.

[0232] 5. Formaldehyde denaturing gel electrophoresis and fluorescence imaging Prepare a 1% formaldehyde denaturing gel using the same method as step 5 in Example 1, and perform gel electrophoresis.

[0233] Fluorescence imaging was performed using the Amersham ImageQuant 800 Multifunction Imager (GE), such as... Figure 8 As shown on the right, the location and intensity of CY5 signaling are visible. Compared with serum from normal volunteers, the expression of sialoglycoRNA in the serum of tumor patients is elevated.

[0234] The scope of protection of this application is not limited to the embodiments described above. Obviously, those skilled in the art can make various modifications and variations to this application without departing from the scope and spirit of the invention. If such modifications and variations fall within the scope of the claims of this application and their equivalents, then the intent of this application also includes such modifications and variations.

[0235] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

[0236] Furthermore, various different implementations of this application can be combined in any way, as long as they do not violate the spirit of this application, they should also be regarded as the content disclosed in this application.

Claims

1. A method for the in vitro enzymatic chemical labeling of glycosylated RNA based on an α-2,3, α-2,6 sialyltransferase, characterized in that, The process includes the following steps: 1) Digesting all sialic acid at the ends of the glycoRNA glycans in the RNA sample using sialidase to obtain digested glycoRNA; for RNA samples with no sialic acid at the ends of the glycoRNA glycans but potential sialic acid modification sites, sialidase digestion is not required, and the undigested glycoRNA can be used directly; 2) Using α-2,3 or α-2,6 type sialyltransferase and CMP-Sialic acid with a labeled group, the labeled sialic acid is transferred to the potential sialic acid modification sites at the ends of the digested or undigested glycoRNA glycans to obtain labeled glycoRNA.

2. The method of claim 1, wherein, The RNA samples may be derived from human cells and tissues, mouse tissues, serum samples, fruit flies, Caenorhabditis elegans, zebrafish, plant samples, fungi, and bacteria.

3. The method according to claim 1 or 2, characterized in that, The sialidase has at least α-2,3 or α-2,6 sialidase activity, preferably, the sialidase has at least α-2,3 and α-2,6 sialidase activities, more preferably, the sialidase is α-2-3,6,8,9 Neuraminidase A.

4. The method according to any of claims 1 to 3, characterized in that, The α-2,3 type sialyltransferases are PmST1, PmST3, Ph2,3siat, and their derivatives with corresponding mutants and truncated forms possessing α-2,3 type sialyltransferase activity, preferably the M144D mutant type PmST1 and the truncated form Δ24Ph2,3sia; the α-2,6 type sialyltransferase is Pd26ST, and its derivatives with corresponding mutants and truncated forms possessing α-2,6 type sialyltransferase activity, preferably the truncated form Δ15Pd26ST.

5. The method according to any one of claims 1 to 4, characterized in that, The labeling groups in the CMP-Sialicacid with the labeling groups include detectable groups, or groups that can be further introduced with detectable groups through reaction; Preferably, the detectable group is a fluorescent group or biotin; More preferably, the CMP-Sialic acid with the labeled group is azide-labeled CMP-azide-Sialic Acid, alkynyl-labeled CMP-alkynyl-Sialic Acid, biotin-labeled CMP-Biotin-Sialic Acid, or fluorescently labeled CMP-CY5-Sialic Acid.

6. The method of claim 5, wherein, The labeling group in the CMP-Sialic acid with the labeling group is a group that can be further introduced into a detectable group through a reaction. The method further includes introducing the detectable group onto sialic acid through a reaction involving the labeling group to obtain glycoRNA with a detectable group. Preferably, when the CMP-Sialic acid with the labeled group is azide-labeled CMP-azide-Sialic Acid, the method further includes introducing the detectable group onto sialic acid through a copper-free click chemistry reaction of azide with a click chemistry reagent; When the CMP-Sialic acid with the labeled group is an alkyne-labeled CMP-alkynyl-Sialic Acid, the method further includes introducing a detectable group onto sialic acid by a copper-catalyzed click chemistry reaction of the alkyne with a click chemistry reagent. More preferably, the click chemistry reagent without copper click chemistry reaction is DBCO-PEG4-Biotin or DBCO-fluorescent group; the click chemistry reagent with copper-catalyzed click chemistry reaction is N3-Biotin or N3-fluorescent group.

7. The method according to any of claims 1 to 6, characterized in that, Includes the following steps, 1) Incubate the RNA sample and sialidase in buffer solution A at a certain temperature for a period of time to obtain digested glycoRNA; for RNA samples with no sialic acid at the glycan ends but potential sialic acid modification sites, sialidase digestion is not required, and the undigested glycoRNA can be used directly. 2) The digested or undigested glycoRNA, α-2,3 or α-2,6 type sialyltransferase, labeled CMP-Sialic Acid, metal ions, and optional RNase inhibitors are co-incubated in buffer solution B at a certain temperature for a period of time to obtain labeled glycoRNA.

8. The method of claim 7, wherein, In the reaction system of step 1), the pH of the buffer solution A is 4-11, preferably 5.

5.

9. The method according to claim 7 or 8, characterized in that, In the reaction system of step 1), the buffer solution A is acetate buffer, Tris buffer, phosphate buffer, citrate buffer, HEPES buffer, or carbonate buffer, and the concentration of the buffer solution A is 5-200 mM; preferably, the buffer solution A is acetate buffer, and the concentration of the buffer solution A is 40-60 mM.

10. The method according to any of claims 7-9, characterized by, In the reaction system of step 1), the concentration of RNA is 0.01-15 mg / mL, and the concentration of sialidase is 100-10000 U / mL; preferably, the concentration of RNA is 0.5-1.25 mg / mL, and the concentration of sialidase is 1000-2000 U / mL.

11. The method according to any of claims 7-10, characterized by, In the reaction system of step 2), the pH of the buffer solution B is 6.5-11. Preferably, the pH of the buffer solution B used for α-2,3 type sialyltransferase is 7.0, and the pH of the buffer solution B used for α-2,6 type sialyltransferase is 7.

0.

12. The method according to any of claims 7-11, characterized by, In the reaction system of step 2), the buffer solution B is Tris buffer, acetate buffer, phosphate buffer, citrate buffer, HEPES buffer, or carbonate buffer, and the concentration of the buffer solution B is 5-200 mM; preferably, the buffer solution B is Tris·HCl or sodium acetate buffer, and the concentration of the buffer solution B is 50-150 mM.

13. The method according to any of claims 7-12, characterized by, Step 2) the metal ion is selected from Mg 2+ , Ca 2+ , provided as its metal chloride, metal bromide, metal nitrate, metal sulfate, at a concentration of 1-20 mM in the reaction system; preferably, the metal ion is provided as MgCl2, at a concentration of 5-10 mM in the reaction system.

14. The method according to any of claims 7-13, characterized by, In step 2), the concentration of RNA in the reaction system is 0.01-10 mg / mL, the concentration of α-2,3 or α-2,6 type sialyltransferase is 0.1-100 U / mL, and the concentration of labeled CMP-Sialic acid is 1 µM-20 mM; preferably, the concentration of RNA is 0.1-1 mg / mL, the concentration of α-2,3 or α-2,6 type sialyltransferase is 1-10 U / mL, and the concentration of labeled CMP-Sialic acid is 1-15 mM.

15. The method according to any of claims 7-14, characterized by, The incubation temperatures for steps 1)-2) are 4-40℃ and the incubation times are 10min-360min; preferably, the incubation temperatures are 35-38℃ and the incubation times are 60-240min.

16. The method according to any one of claims 1-15, characterized in that, Includes the following steps: a) Extract total RNA from the sample to be tested; optionally, treat with proteinase K and purify the RNA sample; 1) Use sialidase to digest all the sialic acid at the ends of the glycoRNA glycans in the RNA sample to obtain digested glycoRNA, and purify the RNA sample; for RNA samples with no sialic acid at the ends of the glycoRNA glycans but with potential sialic acid modification sites, sialidase digestion is not required. 2) Using α-2,3 or α-2,6 type sialyltransferase and CMP-Sialic acid with a labeled group, the labeled sialyl acid is transferred to the potential sialyl modification site at the end of the sugar chain of digested or undigested glycoRNA to obtain labeled glycoRNA; the RNA sample is then purified. b) Optionally, when the labeling group in the CMP-Sialic acid with the labeling group is a group that can be further introduced into a detectable group through a reaction, the detectable group is introduced into the sialic acid through a reaction involving the labeling group to obtain glycoRNA with a detectable group; the RNA sample is purified.

17. The use of the method according to any one of claims 1-16, characterized in that, It is used to detect glycosylated RNA, enrich glycosylated RNA, perform RNA partial sequencing of glycosylated RNA, or identify the glycoma spectrum of glycosylated RNA.

18. A kit for in vitro enzyme chemical labeling detection or enrichment of glycosylated RNA based on α-2,3,α-2,6 type sialyltransferases, characterized in that, It includes: The reagent for digesting sialic acid-modified RNA samples includes sialylase as described in claim 1 or 3, and optionally buffer solution A as described in any one of claims 7-9; Reagents for in vitro enzyme chemical labeling include α-2,3 or α-2,6 type sialyltransferases as described in claim 1 or 4; CMP-Sialic acid with a labeled group as described in claim 1 or 5; optionally, click chemistry reagents as described in claim 6; optionally, buffer solution B as described in any one of claims 7, 11-12; optionally, metal ions as described in claim 7 or 13. Optional Streptavidin-HRP for detecting biotin-containing glycoRNAs; Optional streptavidin magnetic beads for enriching biotin-containing glycoRNAs.

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