Glycosylated RNA imaging method based on RNA and sugar dimetabolism co-labeling

Abstract

The invention discloses a glycosylation RNA (Ribonucleic Acid) imaging method based on RNA and sugar dimetabolism co-labeling. Metabolic markers Ac4ManNAz and 5-EU are taken as examples, azide and alkynyl are marked on cell surface sugar and RNA respectively, then DBCO-AF647 and N3-AF488 are connected with the azide and the alkynyl respectively through click chemistry, and two fluorophores mark the same glycosylated RNA to generate FRET signals. A laser confocal microscope is used for detection, and a green cell image of a fluorescence donor channel, a red cell image of a fluorescence receptor channel and a yellow cell image of an FRET channel can be observed respectively, so that accurate positioning of RNA, sugar and glycosylated RNA on the cell surface is realized. The invention constructs a convenient, sensitive and accurate cell surface glycosylation RNA fluorescence imaging technology, and is expected to become a powerful tool for analyzing biological functions of glycosylation RNA.

Description

Technical Field

[0001] The present invention relates to glycosylated RNA (GRNA) detection and bioanalysis technology, and in particular to a glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling. Background Art

[0002] Glycosylation is an essential post-translational modification of biological macromolecules in all forms of life, mainly occurring in proteins and lipids to form glycoproteins and glycolipids. This modification affects structural folding, intracellular transport, and immune recognition by mediating molecular interactions of glycosylated substrates. Flynn et al. (Cell, 2021) recently discovered that RNA is a new glycosylation substrate, thus forming glycosylated RNA (glycoRNA). Glycosylated RNAs are displayed on the cell surface, and structural analysis shows that they are small RNAs modified by N-glycosyl groups, mainly with sialic acid as the end. The discovery of glycosylated RNA challenges people's traditional understanding of glycosylation and reveals a new RNA modification pathway, both of which have biological and clinical significance. However, the function of glycosylated RNA is still poorly understood. There are only reports that glycosylated RNA is involved in cell-to-cell interactions, mediating interactions and immune responses between neutrophils and endothelial cells, and the specific biological functions have not been described, which forces us to further clarify the function of glycosylated RNA and urgently need to develop technologies to analyze its distribution and abundance.

[0003] Currently, in vitro detection technologies for glycosylated RNA include Northern blotting, mass spectrometry, and RNA sequencing. However, these in vitro technologies have their limitations, including the need for a large number of cells and the risk of glycosylated RNA degradation during long-term treatment.

[0004] In addition, glycosylated RNA can also be detected by in situ fluorescent cell imaging. The Lu group (Nat Biotechnol, 2023) developed an ARPLA that uses proximity ligation-triggered rolling circle amplification of sialic acid aptamers and RNA-binding probes, which can be used to image U1, U35a, and Y5 glycosylated RNAs in cells. The Ju group (Journal of the American Chemical Society, 2024) proposed a hierarchical encoding strategy that was combined with hybridization chain reaction to visualize Y5 glycosylated RNA. Because these methods rely on specific nucleic acid probes, they can only observe glycosylated RNAs of partially known RNA sequences and require complex nucleic acid probe design and procedures related to DNA nanotechnology, which limits their compatibility with tissue imaging and reduces their universality in standard biomedical laboratories. Therefore, we urgently need to develop powerful imaging methods to visualize total glycosylated RNA on the cell surface and elucidate their distribution in cells and tissues, thereby helping us reveal their biological functions. Summary of the invention

[0005] Purpose of the invention: The present invention proposes a glycosylated RNA imaging method based on RNA and sugar dual metabolic co-labeling. The present invention proposes to combine metabolic labeling, click chemistry and fluorescence resonance energy transfer (FRET) signal output to construct a cell imaging technology for total glycosylated RNA on the cell surface, and simultaneously achieve accurate positioning of cell surface RNA, sugar and glycosylated RNA.

[0006] Technical solution: The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling of the present invention comprises the following steps:

[0007] (1) Using metabolic marker Ac 4 ManNAz and 5-EU were incubated with cells;

[0008] (2) taking out the incubated cells, washing them with a buffer solution, and incubating the cells with DBCO-AF647;

[0009] (3) Wash away free DBCO-AF647 with buffer solution and add N 3 -AF488、Cu 2+ Incubation of reagents with cells;

[0010] (4) After rinsing with buffer solution, cells were imaged using a laser confocal microscope.

[0011] The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, step (3) the Cu 2+ The reagent is selected from CuSO 4 .

[0012] In the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, step (2) is to allow the azide labeled on the sugar to be linked to AF647 through a copper-free click chemistry reaction; step (3) is to allow N 3 -AF488 is linked to the alkyne on the U base in RNA via a copper-catalyzed azide-alkyne cycloaddition reaction.

[0013] The principle of the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling is that AF488 and AF647 are FRET donors and acceptors. Since AF488 and AF647 are adjacent, the emission of AF488 is absorbed by AF647, thereby generating a FRET signal for in situ glycosylated RNA imaging.

[0014] In the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, the buffer solution is a PBS buffer solution.

[0015] The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, the cells in step (1) are selected from tumor cells, including human breast cancer cells MCF-7, human normal breast cells MCF-10A, human liver cancer cells HepG2, human cervical cancer cells Hela, human neuroblastoma cells IMR-32, and rat pheochromocytoma cells PC-12.

[0016] In the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, DBCO-AF647 in step (2) is incubated on ice, the incubation concentration is 30-70 μM, and the incubation time is 2-15 min; the incubation temperature in step (3) is 35-38° C., and the incubation time is 25-35 min.

[0017] The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, the laser confocal conditions in step (4) are: 478-498nm excitation, 500-550nm receiving RNA fluorescence signal; 600-650nm laser excitation, 662-737nm receiving sugar fluorescence signal; 478-498nm laser excitation, 662-737nm receiving glycosylated RNA fluorescence signal.

[0018] A diagnostic reagent comprising Ac 4 ManNAz,5-EU,DBCO-AF647,N 3 -AF488 and Cu 2+ .

[0019] The application of the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling in in vitro diagnosis of tumor cells.

[0020] Further preferably, a glycosylated RNA imaging method based on RNA and sugar dual metabolic co-labeling is provided, wherein the imaging method utilizes metabolic labeling and click chemistry reaction to connect FRET donors and acceptors to cell surface RNA and sugars, and a FRET signal will appear when the donor and acceptor are labeled on the same glycosylated RNA.

[0021] The metabolic markers include Ac 4 ManNAz and 5-EU, FRET donors and acceptors include AF488 and AF647.

[0022] The cell imaging method of dual metabolic labeling of cell surface nucleic acids and sugars has the following operating steps:

[0023] (1) Using metabolic marker Ac 4 ManNAz and 5-EU were incubated with cells for 48 h.

[0024] (2) The cells were taken out after incubation and washed three times with 1× PBS buffer solution, and the cells were incubated with DBCO-AF647. The azide labeled on the sugar was linked to AF647 through a copper-free click chemistry reaction.

[0025] (3) Wash away free DBCO-AF647 with 1× PBS buffer solution and wash with N 3 The AF488 kit is incubated with cells to attach to the alkyne on the U base in RNA via a copper-catalyzed azide-alkyne cycloaddition reaction.

[0026] (4) After washing with 1× PBS buffer solution, the cells were imaged using a laser confocal microscope. Since AF488 and AF647 are adjacent, the emission of AF488 is absorbed by AF647, thereby generating a FRET signal for in situ glycosylated RNA imaging.

[0027] In the step (1), the metabolic marker Ac 4 The concentrations of ManNAz and 5-EU were 50 μM and 250 μM, respectively.

[0028] The 1×PBS buffer solution in step (1) is a phosphate buffer solution, the main component of which is NaH 2 PO 4 、Na 2 HPO 4 and NaCl.

[0029] The tumor cells in step (1) include human breast cancer cells MCF-7, human normal breast cells MCF-10A, human liver cancer cells HepG2, human cervical cancer cells Hela, human neuroblastoma cells IMR-32, and rat pheochromocytoma cells PC-12. The cells were resuscitated and inoculated in RPMI-1640 medium containing 10% fetal bovine serum and double antibodies, and incubated at 37°C, 5% CO 2 The cells were cultured in an incubator and passaged every 2 to 3 days. The cells with good growth status (about 1.5×10 5 The cells were inoculated in 35 mm confocal culture dishes and washed three times with 1× PBS buffer solution (pH=7.4) the next day before incubation with metabolic markers.

[0030] The DBCO-AF647 in step (2) is incubated on ice at a concentration of 50 μM for 10 min.

[0031] The incubation temperature in step (3) is 37° C. and the incubation time is 30 min.

[0032] The laser confocal conditions in step (4) are as follows: using a ×40 objective lens, excitation at 488nm, receiving RNA fluorescence signals at 500-550nm (AF488 channel), excitation at 640nm laser, receiving sugar fluorescence signals at 662-737nm (AF647 channel), and excitation at 488nm, receiving glycosylated RNA fluorescence signals at 662-737nm (FRET channel).

[0033] The principle diagram of the present invention is shown in the attached Figure 1 The present invention comprises two functional molecules: (1) a metabolic marker, represented by Ac 4 ManNAz and 5-EU are examples. 4 ManNAz enters the sugar biosynthesis pathway and uses N 3 Labeling sialic acid-rich glycosylated RNA. 5-EU enters RNA de novo synthesis, and the U base in the newly generated RNA labels the alkyne group; (2) FRET pair, AF488 and AF647 are used as examples in the figure. 3 -AF488 and FRET acceptor DBCO-AF647. After cells were co-cultured with metabolic markers, glycans and RNA were respectively 3 Then, DBCO-AF647 was introduced into the 3 The reaction is localized to sialic acid on the cell surface (step 2). After removing free DBCO-AF647, the reaction is performed with N 3-AF488 is linked to the alkyne on the U base in the RNA sequence (step 3). Since AF488 and AF647 are on the same glycosylated RNA and within 10 nm of each other, the emission of AF488 is absorbed by AF647, thereby generating a FRET signal for in situ glycosylated RNA imaging. In this invention, AF488 locates cell surface RNA, AF647 recognizes cell surface sialic acid, and the FRET signal visualizes the glycosylated RNA on the cell surface, thereby achieving simultaneous imaging of RNA, sialic acid, and glycosylated RNA.

[0034] Beneficial effects: (1) Combined with the metabolic labeling strategy, sequence-independent total glycosylated RNA imaging is achieved; (2) The mechanism of the present invention for identifying glycosylated RNA is that the metabolic marker can specifically label RNA and sugar, and then use two click chemistry reactions to connect the fluorescent group; (3) The present invention can image specific sugars, RNA and glycosylated RNA by changing the signal channel, thereby improving the flexibility and accuracy of detection; (4) Glycosylated RNA imaging from the cell to the tissue level can be achieved, providing intuitive evidence for its existence and distribution in major tissues, reflecting the potential for exploring biological functions, and facilitating the future detection of dynamic changes in glycosylated RNA in living cells, organoids and tissues; (5) The present invention is simple to operate and can be used by laboratories and researchers without professional knowledge. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The schematic diagram is a glycosylated RNA imaging method based on RNA and sugar dual metabolic co-labeling;

[0036] Figure 2 The feasibility of the present invention; (a: laser confocal images of cells treated with each element; b: using Ac 4 Cells were treated with ManNAz, 5-EU, or both, followed by DBCO-AF647 and N 3 -Flow cytometric analysis of AF488 treatment);

[0037] Figure 3 The specificity and universality of the present invention for analyzing glycosylated RNA on the cell surface (a: fluorescence intensity of cells treated with RNase, glycosidase and glycosylation inhibitor and then treated with the present method; b: laser confocal images of the present method on different cell lines);

[0038] Figure 4 This is the application of the present invention at the organoid and tissue levels (a: the present invention locates glycosylated RNA in organoids; b: the present invention locates glycosylated RNA in normal lung tissue and lung nodules). DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The experimental methods described in the examples are conventional methods unless otherwise specified. 4 ManNAz was obtained from Jinan Shanmu Biopharmaceutical Technology Co., Ltd. 5-EU, BeyoClick TM EU-488 RNA synthesis detection kit, RNase T1, and PNGase F were obtained from Bio-Tech (Shanghai, China). DBCO-AF647 was obtained from Kangfuno Biotechnology (Xi’an, China). a2-3,6,8,9 neuraminidase A (NA) was purchased from NEB (UK). RNase A was provided by Sangon (Shanghai, China). Tunicamycin (TM) and benzyl-2-acetamido-2-deoxy-AD-pyranogalactoside (BG) were purchased from MacLean (Shanghai, China). Laser confocal imaging images were obtained from a Nikon laser confocal microscope (Japan), and fluorescence intensity data were obtained from the Synergy H1M microplate reader (USA) of Berton Instruments, Inc., USA. All cell culture supplies were purchased from Jiangsu KeyGen Biotech Co., Ltd. (Nanjing, China).

[0041] Example 1

[0042] 1.5×10 MCF-7 cells were seeded on a 35 mm glass-bottom confocal dish. 5 Before each step, the cell samples were washed three times with 1× PBS buffer solution. 4 ManNAz (50 μM) and 5-EU (250 μM) were incubated for 48 h. Then, the cells were treated with DBCO-AF647 (500 μL, 50 μM) on ice for 10 min. 3 -AF488, CuSO 4 (This experiment uses BeyoClick TM EU-488 RNA Synthesis Detection Kit can also be used with other 3 -AF488, CuSO 4The cells were treated with a kit at 37°C for 30 minutes. Finally, the cells were placed under a laser confocal microscope for bright field and dark field photography. The fluorescence signals of RNA were received at 500-550 nm (AF488 channel) using 488 nm laser excitation and 662-737 nm laser excitation (AF647 channel). The fluorescence signals of glycosylated RNA were received at 662-737 nm (FRET channel) using 488 nm laser excitation. The results are shown in Table 1. Figure 2 The fifth row of a.

[0043] The control group lacked 5-EU and Ac 4 ManNAz, DBCO-AF647 or N 3 -AF488, respectively Figure 2 The first, second, third, and fourth rows of a.

[0044] MCF-7 cells (about 2×10 5 ) were inoculated in 6-well plates and allowed to adhere overnight. The old culture medium was discarded and the cells were washed three times with 1× PBS buffer solution. 4 ManNAz (50 μM) and 5-EU (250 μM) were incubated together for 48 h. After metabolic labeling, cells were incubated with 50 μM DBCO-AF647 on ice for 10 min. 3 -AF488 kit was used to treat the cells for 30 min. Finally, the cells were dispersed in 1× PBS buffer solution for flow cytometric analysis. Flow cytometric data were analyzed using FlowJo software. Flow cytometric analysis showed that the number of FRET-positive cells increased significantly, 11.5 times compared with cells labeled with RNA alone and 35.9 times compared with cells labeled with sugar alone. Figure 2 b.

[0045] Example 2

[0046] Validation experiment for specificity and universality of glycosylated RNA analysis

[0047] MCF-7 cells (about 2×10 5 ) were inoculated in confocal culture dishes and allowed to adhere overnight. The old culture medium was discarded and the cells were washed three times with 1× PBS buffer solution. The cells were treated with RNaseA (0.02μg / μl) or RNase T1 (1U / μl) at 37°C for 30min to digest the RNA molecules of glycosylated RNA. In the glycosidase treatment experiment, the cells were treated with PNGase F (0.1U / μl) or NA (0.1U / μl) at 37°C to cleave the sugars of glycosylated RNA. In the glycosylation inhibitor treatment experiment, 50μM Ac was added to the culture medium. 4When metabolically labeled with ManNAz and 250 μM 5-EU, cells were cultured with 2 μM TM or 500 μM BG for 48 h. We then used the present invention to perform staining and imaging and used ImageJ (Fiji) to calculate the average fluorescence intensity of the cells. The results are shown in Figure 3 a. Among them, in the RNaseA and RNase T1 treatment groups, the FRET signal decreased by 67.74% and 69.37%, respectively, indicating that this method relies on RNA to generate FRET signals. At the same time, when MCF-7 cells were treated with PNGase F and NA, the FRET signal decreased by 66.45% and 67.94%, respectively, while the sugar signal disappeared and the RNA signal was still detectable. These results show that the presence of glycans is a basic condition for the effective imaging of glycosylated RNA by this method. In addition, after the cells were treated with the N-glycosylation inhibitor TM, the FRET signal decreased by 60.54%, while after the cells were treated with the O-glycosylation inhibitor BG, the FRET signal did not decrease significantly. Because glycosylated RNA belongs to N-sugar, it is sensitive to N-glycosylation inhibitors and is least affected by O-glycosylation inhibitors. These research results show that this method can specifically target RNA and sugar molecules of glycosylated RNA, thereby verifying its specificity in in situ imaging of glycosylated RNA.

[0048] Ac was used in different cell lines (Hela, HepG2, LM3, IMR-32, PC-12) 4 ManNAz (50 μM) and 5-EU (250 μM) were incubated for 48 h. Then, the cells were treated with DBCO-AF647 (500 μL, 50 μM) on ice for 10 min. 3 -AF488 kit was used to treat cells at 37°C for 30 minutes. Finally, the cells were placed under a laser confocal microscope for bright field and dark field photography. The fluorescence signals of RNA were received at 500-550 nm (AF488 channel) using 488 nm laser excitation. The fluorescence signals of sugar were received at 662-737 nm (AF647 channel). The fluorescence signals of glycosylated RNA were received at 662-737 nm (FRET channel) using 488 nm laser excitation. The results are shown in Figure 2. Figure 3 As shown in b, we can see that the signals corresponding to RNA, glycans, and glycosylated RNA are enhanced in all cell lines, indicating the universality of this method.

[0049] Example 3

[0050] Localization of glycosylated RNA at the organoid and tissue level

[0051] With Ac 4ManNAz (50 μM) and 5-EU (250 μM) were cultured in neural induction medium for 48 h, fixed with 4% paraformaldehyde after metabolic labeling, and then rinsed three times with PBS for 10 min each. Subsequently, the organoids were immersed in 20% sucrose solution, which was replaced with 30% sucrose solution after overnight at 4 °C. After overnight, the organoids were embedded and frozen and sectioned. Tissue sections were incubated with DBCO-AF647 (100 μM) at room temperature for 10 min. After rinsing three times with PBS, the sections were incubated with N-containing 3 -AF488 kit for 30 min. Finally, the samples were placed under a laser confocal microscope for bright field and dark field photography, with 488 nm excitation and 500-550 nm receiving RNA fluorescence signals (AF488 channel). 640 nm laser excitation and 662-737 nm receiving sugar fluorescence signals (AF647 channel). 488 nm excitation and 662-737 nm receiving glycosylated RNA fluorescence signals (FRET channel). Figure 4 The distribution of glycosylated RNA in organoids can be observed in a.

[0052] The experimental mice were divided into two groups (3 mice in each group): control group and model group. The model group was injected with 4T1 cells (1×10 7 The tumor metastasis model was established in 100 μL of 5-EU (20 mg / ml) and the control group was injected with PBS. After tumor metastasis, 5-EU (100 μL, 20 mg / ml) was injected intraperitoneally, followed by intraperitoneal injection of Ac once a day for three consecutive days. 4 ManNAz (5 mg / kg). DBCO-AF647 (0.16 mmole / kg) was injected into the tail vein 3 hours before the mice were killed on the 4th day for in vivo glycan labeling. The main organ lungs were collected and washed with saline. They were fixed in 4% paraformaldehyde and sucrose dehydration solution. The frozen sections were permeabilized with 0.1% Triton for 30 min and then rinsed three times with PBS. Then, the samples were incubated at 37°C with N 3 -AF488 kit for 30 minutes. Finally, the samples were placed under a laser confocal microscope for bright field and dark field photography, with 488nm excitation and 500-550nm receiving RNA fluorescence signals (AF488 channel). 640nm laser excitation, 662-737nm receiving sugar fluorescence signals (AF647 channel). 488nm excitation, 662-737nm receiving glycosylated RNA fluorescence signals (FRET channel), it can be observed that the abundance of glycosylated RNA on the nodules in the model group is low, indicating that the technology of the present invention can locate glycosylated RNA at the organ or tissue level, thereby achieving the localization and imaging of tumor cells. The results are shown in Figure 4 b.

Claims

1. A glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling, characterized in that: The steps include: (1) Incubating cells with metabolic markers Ac4ManNAz and 5-EU; (2) taking out the incubated cells, washing them with a buffer solution, and incubating the cells with DBCO-AF647; (3) Wash away the free DBCO-AF647 with buffer solution, add N3-AF488, Cu 2+ Incubation of reagents with cells; (4) After rinsing with buffer solution, cells were imaged using a laser confocal microscope.

2. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: Step (3) Cu 2+ The reagent is selected from CuSO4.

3. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: Step (2) is to connect the azide labeled on the sugar to AF647 through a copper-free click chemistry reaction; step (3) is to connect N3-AF488 to the alkyne on the U base in RNA through a copper-catalyzed azide-alkyne cycloaddition reaction.

4. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: The principle of step (4) is that AF488 and AF647 are FRET donors and acceptors. Since AF488 and AF647 are adjacent, the emission of AF488 is absorbed by AF647, thereby generating a FRET signal for in situ glycosylated RNA imaging.

5. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: The buffer solution is a PBS buffer solution.

6. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: The cells in step (1) are selected from tumor cells, including human breast cancer cells MCF-7, human normal breast cells MCF-10A, human liver cancer cells HepG2, human cervical cancer cells Hela, human neuroblastoma cells IMR-32, and rat pheochromocytoma cells PC-12.

7. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: The DBCO-AF647 in step (2) is incubated on ice at a concentration of 30-70 μM for 2-15 min. The incubation temperature in step (3) is 35-38° C. for 25-35 min.

8. The glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1, characterized in that: The laser confocal conditions in step (4) are: 478-498nm excitation, 500-550nm receiving RNA fluorescence signal; 600-650nm laser excitation, 662-737nm receiving sugar fluorescence signal; 478-498nm laser excitation, 662-737nm receiving glycosylated RNA fluorescence signal.

9. A diagnostic reagent, characterized in that Contains Ac4ManNAz, 5-EU, DBCO-AF647, N3-AF488 and Cu 2+ .

10. Use of the glycosylated RNA imaging method based on RNA and sugar dual metabolism co-labeling according to claim 1 in in vitro diagnosis of tumor cells.

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