In-situ visual detection reagent for cell surface glycosylation RNA (Ribonucleic Acid) and application of in-situ visual detection reagent

Abstract

The invention discloses an in-situ visual detection reagent for cell surface glycosylation RNA and application thereof, the reagent comprises a glycosylation RNA recognition reagent and a multiple hybrid chain circulation reaction reagent, the glycosylation RNA recognition reagent comprises a nucleotide sequence Tsa of a 5'end modified DBCO functional group, a nucleotide sequence T2 for recognizing RNA and a connection sequence for connecting the Tsa and the T2; the multiple hybrid chain circular reaction reagent is composed of six nucleic acid sequences which are paired with one another. The whole reaction system does not use any enzyme, so that the system is less in interference factors; the method does not use biological enzyme, is isothermal in the whole process, is low in cost of used reagent equipment, is simple to operate, is easy to implement, and is easy to popularize.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, in particular to an in situ visualized detection reagent for cell surface glycosylated RNA, and also relates to the application of the reagent. BACKGROUND

[0002] Breast cancer is a common malignant tumor in women, which originates from breast duct or lobule cells. It is one of the few malignant tumors that can reduce mortality through early diagnosis. In the United States, the proportion of patients diagnosed with stage I breast cancer is 62%, while in China, the proportion is only 20%, and there are still a large number of patients with advanced breast cancer who cannot receive effective treatment because they are diagnosed too late. Therefore, through early screening, breast cancer patients are found in time, thereby improving the treatment effect and quality of life of patients, which is the key to preventing and controlling breast cancer. However, early breast cancer often does not have typical clinical symptoms and signs, and most of the lumps are painless, which can easily be ignored. The commonly used breast cancer screening methods include MRI, mammography and ultrasound examination, and other imaging auxiliary examinations, each of which has its own advantages and disadvantages, and often needs to be used in combination in clinical diagnosis, increasing the cost and time cost of patients for diagnosis. Compared with conventional imaging examination, genetic diagnosis has stronger specificity and does not need to be combined with examination.

[0003] Glycosylated RNA is a newly discovered biological molecule in recent years, and glycosylation is an important post-translational modification of proteins and lipids, and seriously affects the regulation of different cellular processes, including transcription, translation, protein transport, degradation and immunosuppression. Recent studies using blotting electrophoresis of RNA extracted from cells show that small RNAs on the surface of living cells are N-glycosylated and terminated with sialic acid (Sias). Mass spectrometry, high-performance anion exchange chromatography and RNA sequencing have also been applied to the in vitro detection of glycoRNA.

[0004] However, these in vitro analysis techniques discard the positional information on sialylated RNA, and the glycosylation sites and potential functions are still unclear. Recent studies have shown that location-specific glycan analysis has mainly focused on protein-specific glycans, and can be achieved through Raman imaging, photoacoustic imaging, mass spectrometry, and fluorescence imaging. These methods usually use metabolic strategies to label target glycans on the entire cell, and use protein recognition to activate the signal switch, which inevitably produces background interference from adjacent labeled glycans. In order to solve the problems of unknown location of glycosylated RNA and adjacent glycan interference and simplify the labeling procedure, a new strategy is urgently needed to visualize glycosylated RNA in situ, which further explores the function of glycosylated RNA and its relationship with breast cancer. SUMMARY

[0005] Therefore, the present application aims to provide a cell surface glycosylation RNA in situ visualization detection reagent, which can realize in situ visualization of low-abundance RNA specific glycan on the membrane of living cells, covalently bind DNA sequence to sialic acid through click chemistry, bind DNA sequence to target RNA through sequence specificity, then base pair the two-part DNA sequence to realize multiple hybridization chain reaction (HCR), assemble branched DNA copolymer with high molecular weight and high signal gain, and then amplify the signal to enable sensitive in situ visualization of low-abundance glycosylation RNA on the membrane of living cells, laying a foundation for future exploration of glycosylation RNA function and anabolism.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] The cell surface glycosylation RNA in situ visualization detection reagent comprises a glycosylation RNA recognition reagent and a multiple hybridization chain reaction reagent, the glycosylation RNA recognition reagent comprises a 5'-end modified DBCO functional group nucleic acid sequence Tsa, an RNA recognition nucleic acid sequence T2 and a connecting sequence connecting Tsa and T2, and the multiple hybridization chain reaction reagent is composed of 6 pairs of nucleic acid sequences.

[0008] Preferably, the 5'-end modified DBCO functional group nucleic acid sequence Tsa is shown as SEQ ID NO. 1, the RNA recognition nucleic acid sequence T2 is shown as SEQ ID NO. 2 or SEQ ID NO. 3, and the connecting sequence is shown as SEQ ID NO. 11; and the nucleic acid sequences of the multiple hybridization chain reaction reagent are shown as SEQ ID NO. 5-SEQ ID NO. 10.

[0009] Preferably, the reagent further comprises 50 mM Mg 2+ HEPES buffer.

[0010] Preferably, in the multiple hybridization chain reaction reagent, H1 and H2 form H1-H2, H3 and H4 form H3-H4, and H5 and H6 form H5-H6, and the concentration ratio of H1-H2, H3-H4 and H5-H6 is 3:2:1.

[0011] Preferably, in the multiple hybridization chain reaction reagent, H1, H4 and H6 are labeled with a fluorescent group.

[0012] The application has the beneficial effects that a new method for visualizing glycosylated RNA in situ is disclosed, and the specific binding to the glycosylated RNA glycosyl part and the RNA part is used to realize the accurate identification of the glycosylated RNA. After the accurate identification, the multiple hybrid chain cycle reaction (HCR) forms the branched DNA copolymer with high molecular weight and high signal gain, and realizes the in-situ visualization of the glycosylated RNA. In addition, the whole reaction system realizes the use of no enzyme, so that the system is less disturbed by factors; and the application does not use biological enzymes, and the whole process is isothermal, the reagent equipment cost is low, the operation is simple and easy to implement, and is easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to make the purpose, technical scheme and beneficial effects of the application clearer, the application provides the following drawings for illustration:

[0014] Figure 1 It is a patent principle diagram;

[0015] Figure 2 It is a non-denaturing polyacrylamide gel electrophoresis diagram;

[0016] Figure 3 It is a hybrid chain reaction condition optimization diagram;

[0017] Figure 4 It is the sensitivity of the method of the patent;

[0018] Figure 5 It is a cell enzyme-free isothermal multiple hybrid chain cycle reaction feasibility diagram. DETAILED DESCRIPTION

[0019] The application will be further described below in combination with the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not as a limitation on the application.

[0020] The detection method of the application is to covalently combine the DNA sequence to the sialic acid through click chemistry, to combine the DNA sequence to the target RNA through sequence specificity, then the DNA sequences of the two parts are base-paired to realize the multiple hybrid chain reaction cycle (HCR), to assemble the branched DNA copolymer with high molecular weight and high signal gain, and then to amplify the signal, so that the low-abundance glycosylated RNA on the living cell membrane can be sensitively visualized in situ.

[0021] Example 1

[0022] A kind of enzyme-free isothermal multiple hybrid chain cycle system is designed, which is used to image bioactive molecules in living cells in situ by assembling branched DNA copolymer with high molecular weight and high signal gain based on multiple hybrid chain cycle reaction (HCR), and its principle is as follows Figure 1During the detection process, the DBCO (dibenzocyclooctyne) functional group at the 5' end of Tsa specifically binds to the sugar moiety of the glycosylated RNA, and the 5' end of T2 specifically binds to the target RNA. Tsa and T2 are then connected via a connector to form a complex. The 5' ends of Tsa and T2 then bind to the 5' end of H1. The bound 3' end of H1 base pairs with H2, and the excess portion of H2 base pairs with the bound H3. The remainder of H3 then binds to H4, and the remainder of H4 binds to H5. H5 then partially binds to H4 and H6, and H6 partially binds to H5 and H3. Finally, H2, H3, H4, H5, and H6 cyclically bind to achieve multiple hybridization chain reaction (HCR) cycles.

[0023] The specific sequences used are shown in Table 1.

[0024] Table 1. Sequences involved in the invention

[0025]

[0026]

[0027] Using the above sequences, we developed a click chemistry strategy to label the sugar moieties of glycosylated RNAs and a hierarchical encoding (HieCo) strategy to label target RNAs. The latter strategy overcomes the distance limitation of fluorescence resonance energy transfer (FRET) to achieve in situ release of fluorescent quenchers for signal readout from target proteins.

[0028] First, determine whether the hybridization chain reaction can occur. The specific steps are as follows:

[0029] (1) T mimic (As a replacement for the complex of TSA, T2 and H1 linked to glycosylated RAN during the detection process), H1, H2, H3, H4, H5, and H6 were diluted to a solution with a concentration of 10 μM, and H1-H6 were heated to open the DNA hairpins for use.

[0030] (2) The experimental group added the above DNA chain into small EP tubes at volumes of 1μl, 3μl, 3μl, 2μl, 2μl, 1μl, and 1μl, while the control group did not add T mimic , and then 50mM Mg 2+ The total volume was made up to 100 μL with HEPES buffer.

[0031] (3) Prepare polyacrylamide gel.

[0032] (4) After assembling the electrophoresis device, add the samples in the following order: add DNA marker to well 1, add T to well 2 mimic, hole 3 plus H1, hole 4 plus H2, sequentially plus H6, hole 9 plus control group, hole 10 plus experimental group, 1 μl sample mixed with 5 μl dye was added to the gel hole, electrophoresis was carried out at 110 V / 60 min.

[0033] (5) After electrophoresis, the gel was placed in gelred solution for 5 min.

[0034] (6) Imaging was performed using Bio-Rad ChemiDoc XRS system (Bio-Rad, USA).

[0035] The results are shown in Figs. Figure 2 A and B. The results show that hole 10 forms a clear signal at the starting position, indicating that the HCR reaction occurs successfully, forming a high molecular weight polymer.

[0036] Further verification of the recognition and binding of the glycosyl moiety to the target sequence through click chemistry.

[0037] (1) Prepare 10 μM T SA , T2, connector solution, 10 μM H1, H2, H3, H4, H5, H6 solution.

[0038] (2) Sample 4 added the above reagents to the small ep tube according to the volume of 1 μl, 1 μl, 1.5 μl, 3 μl, 3 μl, 2 μl, 2 μl, 1 μl, 1 μl, sample 1-3 respectively less T SA , T 2-2 , connector one, and then use 50 mM Mg 2+ HEPES buffer to make up the total volume to 100 μl.

[0039] (3) Prepare polyacrylamide gel.

[0040] (4) After assembling the electrophoresis device, according to the sample addition order: hole 1 plus DNA marker, hole 2-5 sequentially plus sample 1-4. Electrophoresis was carried out at 110 V / 60 min.

[0041] (5) After electrophoresis, the gel was placed in gelred solution for 5 min.

[0042] (6) Imaging was performed using Bio-Rad ChemiDoc XRS system (Bio-Rad, USA), and the results are shown in Figs. Figure 2 C and D. The results show that only the positive group forms a band at the sample addition site, indicating that the hybridization chain reaction forms a high molecular polymer, and the reaction occurs successfully.

[0043] Example 2

[0044] Next, the experimental conditions are optimized using fluorescence. The method for optimizing the fluorescence-labeled strand is as follows:

[0045] (1) Prepare 10 μM of the labeled fluorescent and unlabeled fluorescent DNA strands H1-H6.

[0046] (2) Select 2 labeled fluorescent DNA strands and 4 unlabeled ones to form a combination. The positive group will add T mimic , H1, H2, H3, H4, H5, H6 in the volume of 1 μl, 3 μl, 3 μl, 2 μl, 2 μl, 1 μl, 1 μl into a small ep tube, and the negative group does not add T mimic , and then add 50 mM Mg 2+ HEPES buffer to make the total volume 100 μl. Each group is repeated for 3 samples.

[0047] (3) Select 3 labeled fluorescent DNA strands and 3 unlabeled ones to form a combination. The reaction volume is the same as above. Each group is repeated for 3 samples.

[0048] (4) Put the samples into a metal bath and react at 25°C for 4 hours.

[0049] (5) Measure the fluorescence of the samples using a fluorescence spectrophotometer, calculate the average fluorescence and standard deviation of each group, and compare the fluorescence difference between the positive group and the negative group of each combination.

[0050] By comparing the difference, the combination with the largest fluorescence difference between the positive group and the negative group is H1, H4, and H6 labeled with fluorescence( Figure 3 , A-C).

[0051] Optimize the reaction concentration of the DNA strands. The specific steps are as follows:

[0052] (1) Prepare 10 μM of the labeled fluorescent H1, H4, H6 and unlabeled fluorescent DNA strands H2, H3, H5.

[0053] (2) H1-H2, H3-H4, H5-H6 are respectively 1:1:1, 1:2:2, 2:1:1, 2:2:1, 3:2:1, 4:2:1 with 1 μl T mimic reaction (positive group), and the negative group does not add T mimic , and then add 50 mM Mg 2+ HEPES buffer to make the total volume 100 μl. Each group is repeated for 3 samples.

[0054] (3) Put the samples into a metal bath and react at 25°C for 4 hours.

[0055] (4) Measure the fluorescence of the samples using a fluorescence spectrophotometer, calculate the average fluorescence and standard deviation of each group, and compare the fluorescence difference between the positive group and the negative group of each combination.

[0056] The difference value H1-H2, H3-H4, H5-H6 reaction ratio of 3:2:1, the positive group and the negative group fluorescence difference value is the largest Figure 3 , D).

[0057] Optimization of hybridization chain reaction temperature, the specific steps are as follows:

[0058] (1) preparation of 10 μM labeled fluorescent H1, H4, H6 and non-labeled fluorescent DNA chain H2, H3, H5.

[0059] (2) H1-H2, H3-H4, H5-H6 is divided into positive group and negative group according to 3:2:1, and then 50 mM Mg 2+ HEPES buffer solution to make up the total volume to 100 μl. Each group of 3 samples.

[0060] (3) set 6 temperature gradient: 20℃, 25℃, 30℃, 35℃, 37℃, 40℃. 6 groups of samples are placed in the metal bath, and the corresponding temperature is set to react for 4 hours.

[0061] (5) using fluorescence spectrophotometer to measure the fluorescence of the sample, calculate the average value and standard deviation of each group, and compare the fluorescence difference value of each group.

[0062] The difference value H1-H2, H3-H4, H5-H6 reaction ratio of 3:2:1, the positive group and the negative group fluorescence difference value is the largest Figure 3 , E).

[0063] After the above experiment, the optimal reaction conditions are confirmed: the fluorescence intensity difference value is the largest when the DNA chain H1, H4, H6 is labeled with fluorescent group, the concentration ratio of H1-H6 is 3:2:1, and the reaction is carried out at 25℃.

[0064] Example 3

[0065] Verify the sensitivity, the specific method is as follows:

[0066] (1) configuration of a series of T mimic concentration: 10 3 fmol, 10 4 fmol, 10 5 fmol, 10 6 fmol, 10 7 fmol, 10 8 fmol, 10 9 fmol

[0067] (2) preparation of 10 μM labeled fluorescent H1, H4, H6 and non-labeled fluorescent DNA chain H2, H3, H5.

[0068] (3) Mix H1-H2, H3-H4, H5-H6 at 3:2:1, total 7 groups, add 1 μl T mimic to make the final concentration 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 fmol, and then add 50 mM Mg 2+ HEPES buffer to make the total volume 100 μl, and repeat 3 samples for each group.

[0069] Collect the fluorescence value of each group, and calculate the linear regression equation and R value according to the average value, such as Figure 4 .

[0070] Example 4

[0071] Verify the feasibility of HCR reaction in cells, the specific method is as follows:

[0072] (1) Prepare 10 μM T SA , T 2-2 , connector solution, 25 μg / μl BSA solution, 10 mg / ml salmon sperm DNA (SSD) solution, 10 μM H1, H2, H3, H4, H5, H6 solution, wherein H1, H4, H6 are labeled with fluorescence.

[0073] (2) Inoculate 10 5 MCF-7 cells in an imaging dish, a total of 7 dishes, and incubate at 37°C overnight.

[0074] (3) Replace the culture medium of 6 imaging dishes with culture medium containing 100 μM Ac4ManNaz, except for the blank group (sample 1), and incubate at 37°C for 48 hours.

[0075] (4) Configure the solution: sample 7: 1 μL T SA , 1 μL T 2-2 , 1.5 μL connector, 1 μL SSD, 1 μL BSA. Sample 2-5 respectively add less T SA , T 2-2 , connector one by one, and then add Tris-HCL buffer to make 100 μL.

[0076] (5) After 48h, wash the cells with PBS buffer for 3 times, add the solution prepared in step (4), and incubate at 37°C for 1h.

[0077] (6) Configuration solution: H1-H6 were reacted according to 3:2:1, and 5 portions were prepared by adding Tris-HCL buffer solution to 100 μL.

[0078] (7) After incubation for 1 h, the cells were washed with PBS for 3 times, sample 2-5 and sample 7 were added with the solution prepared in step (5), sample 1 and 6 were added with 100 μl PBS buffer, and incubated at 37℃ for 2 h.

[0079] (8) Washed with PBS for 3 times, added with 100 μl PBS, and observed by confocal microscope, the results were shown in Figure 5

[0080] According to the confocal imaging results, only sample 7 was positive, which indicated that HCR reaction could occur in living cells, and only when Ac4ManNaz, T SA , T 2-2 , connector, H1-H6 all existed.

[0081] The above-mentioned examples are only preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by the person skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.​

Claims

1. An in situ visualization detection reagent for cell surface glycosylated RNA, characterized by: The reagents include a glycosylated RNA recognition reagent and a multiple hybridization chain cycling reaction reagent. The glycosylated RNA recognition reagent includes a nucleic acid sequence Tsa with a DBCO functional group modified at the 5' end, a nucleic acid sequence T2 for recognizing RNA, and a connecting sequence connecting Tsa and T2. The multiple hybridization chain cycling reaction reagent is composed of 6 mutually paired nucleic acid sequences.

2. The in situ visualization detection reagent for cell surface glycosylated RNA according to claim 1, characterized in that: The nucleic acid sequence Tsa of the 5'-end modified DBCO functional group is shown as SEQ ID NO.1, the nucleic acid sequence T2 for recognizing RNA is shown as SEQ ID NO.2 or SEQ ID NO.3, and the connecting sequence is shown as SEQ ID NO.11; the nucleic acid sequence of the multiple hybridization chain cycling reaction reagent is shown as SEQ ID NO.5 to SEQ ID NO.

10.

3. The in situ visualization detection reagent for cell surface glycosylated RNA according to claim 1, characterized in that: The reagent also includes 50 mM Mg 2+ HEPES buffer.

4. The in situ visualization detection reagent for cell surface glycosylated RNA according to claim 1, characterized in that: In the multiple hybridization chain cycle reaction reagent, H1 and H2 constitute H1-H2, H3 and H4 constitute H3-H4, and H5 and H6 constitute H5-H6. The concentration ratio of H1-H2, H3-H4 and H5-H6 is 3:2:

1.

5. The in situ visualization detection reagent for cell surface glycosylated RNA according to claim 4, characterized in that: H1, H4 and H6 in the multiple hybridization chain cycling reaction reagents are labeled with fluorescent groups.

6. Use of the detection reagent according to any one of claims 1 to 5 in detecting glycosylated RNA on the surface of cells.

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