Method for analyzing intracellular glycosylated RNA (Ribonucleic Acid) by modifying retardation nucleic acid hybridization technology

Abstract

The invention discloses an intracellular glycosylated RNA (Ribonucleic Acid) analysis method for modifying a retardation nucleic acid hybridization technology. Analysis detection and in-situ imaging of intracellular glycosylated RNA (glycoRNAs) are realized on the basis of structural transformation of a target nucleic acid mediated fluorescent substrate nucleic acid chain. Specifically, a substrate chain with toehold is designed according to a sequence of microRNA-21 and a glycosylation modification site of the microRNA-21, and the sequence of the substrate chain contains a specific sequence of a Pepper aptamer capable of being combined with a fluorescent small molecule dye HBC. And the sequence design of the substrate meets the condition that when the substrate is hybridized with glyco-microRNA-21 to a glycosylation modification site, the structure of the generated hybridized compound can just generate an 'ON' fluorescence signal. The hybridization of the substrate sequence and the microRNA-21 is full hybridization, and the formed hybridization product cannot generate a fluorescence signal. According to the invention, the detection of low-abundance glycoRNAs under the interference of a large number of non-glycosylated RNAs is realized, and the intracellular in-situ imaging of the glycoRNAs is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of glycosylated RNA analysis and detection, and provides a method for analyzing glycosylated RNA in cells by using a modification blocking nucleic acid hybridization technology. BACKGROUND

[0002] Glycosylation is an important post-translational modification process of proteins and lipids, and is widely involved in biomedical processes such as immunity, tumors, and cancers. Recent studies have found that some non-coding RNAs can also be glycosylated to form glycosylated RNA (glycoRNAs), which plays an important role in immunity and cancer. Previously, by recognizing the bispecificity of RNA and glycan, a method for in situ imaging of glycoRNAs on the cell membrane surface has been developed, providing a basis for the study of the biological functions of glycosylated RNA. However, the expression level of glycoRNAs is relatively low, and the presence of a large number of non-glycosylated RNAs in the system causes high background interference, making it a great challenge to analyze and detect low-abundance glycoRNAs. Most importantly, there is currently a lack of in-cell imaging technology to study the origin and biosynthetic mechanism of glycoRNAs. Therefore, there is an urgent need for an effective analysis and detection method not only with high sensitivity but also for in situ imaging analysis of glycoRNAs in cells.

[0003] Aptamers are easy to synthesize and have reversible binding properties. By using the structural transformation of aptamers during nucleic acid binding, high-specificity recognition, signal conversion, and biological function activation of target molecules can be achieved. Fluorescent RNA (FLRNA) is a special aptamer that, by forming a specific three-dimensional structure, binds and inhibits the excited state of small molecule fluorescent dyes to induce fluorescence, and as the structure of FLRNA changes, its fluorescence signal also changes, thereby producing an ideal molecular switch. In addition, the introduction of chemical modifications of bases on RNA greatly changes the charge, steric hindrance, and hydrogen bond strength of nucleic acids, thereby significantly affecting the kinetics and thermodynamics of RNA binding. Therefore, it is expected that by using the blocking effect of nucleic acid modification, the hybridization process of nucleic acids can be affected, the hybridization structure of FLRNA can be changed to identify glycosylation modifications on nucleic acids, i.e., only when the modification occurs, the open state of the signal readout is produced, and the other states are inactive. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a method for analyzing glycosylated RNA in cells by using a modification blocking nucleic acid hybridization technology.

[0005] To achieve the above purpose, the present application provides the following technical solutions: The application provides a substrate chain for identifying glycosylation modified microRNA-21, a deoxynucleotide sequence of the substrate chain is shown as SEQ ID NO. 1, a deoxynucleotide sequence of microRNA-21 is shown as SEQ ID NO. 2, and a deoxynucleotide sequence of the glycosylation modified microRNA-21 is shown as SEQ ID NO. 3.

[0006] The application provides application of the aforementioned substrate chain in analysis and detection of intracellular glycoRNAs.

[0007] Preferably, the analysis and detection of intracellular glycoRNAs are achieved by using the blocking effect of glycosylation modification.

[0008] The application provides application of the aforementioned substrate chain in preparation of a cancer detection kit.

[0009] The application also provides an intracellular glycoRNA analysis method for modifying blocking nucleic acid hybridization technology, wherein cells, the aforementioned substrate chain and fluorescent small molecule dye HBC are mixed and cultured, the construction of a modification blocking nucleic acid hybridization technology module is completed, signal difference of "OFF"-"ON"-"OFF" is established, and analysis and detection of glycoRNA are completed.

[0010] Preferably, the signal difference of the fluorescent sensing technology of the modification blocking nucleic acid hybridization is achieved by using structural transformation of fluorescent RNA (FLRNA).

[0011] Preferably, the cells are mixed and cultured with the aforementioned substrate chain and fluorescent small molecule dye HBC, then are dyed by adding a dyeing solution, and finally, imaging of intracellular glycoRNA is observed under a fluorescence microscope.

[0012] Preferably, the cells include but are not limited to HeLa cells and MCF-7 cells.

[0013] Preferably, the intracellular glycoRNA shows bright green fluorescent signals.

[0014] The application also provides application of the aforementioned intracellular glycoRNA analysis method in preparation of a cancer detection kit.

[0015] The application has the following beneficial effects: The application provides a method for analyzing glycosylated RNA in cells by modifying nucleic acid hybridization technology, which realizes the analysis and in-situ imaging of glycosylated RNA (glycoRNAs) in cells based on the structure transformation of a fluorescent substrate nucleic acid chain mediated by target nucleic acids. Specifically, according to the sequence of microRNA-21 and the glycosylation modification site thereof, a substrate chain with a toehold is designed, and the sequence contains a specific sequence of a “Pepper” aptamer capable of being combined with a fluorescent small molecule dye HBC. The substrate sequence is designed to meet the structure of the hybrid complex generated when the substrate sequence is hybridized to the glycosylation modification site of glyco-microRNA-21, so that the “ON” fluorescent signal is generated. The hybridization of the substrate sequence to microRNA-21 is full hybridization, and the hybridization product cannot generate a fluorescent signal. The application realizes the detection of low-abundance glycoRNAs under the interference of a large amount of non-glycosylated RNA, and realizes the in-situ imaging of glycoRNAs in cells.

[0016] The application designs a substrate nucleic acid chain containing a specific sequence of a fluorescent molecule, when a target nucleotide is combined with an artificially designed RNA through sequence specificity, a toehold-mediated strand displacement induces structure transformation of a hybridization product, and a molecular switch is formed. Through special sugar modification of the RNA, the blocking effect of the sugar modification is exerted, the displacement of the chain is inhibited in the transition state of the chain hybridization, and a specific domain is generated to combine a fluorescent dye molecule to generate a fluorescent signal. In the absence of glycosylation modification, the chain displacement continues and destroys the specific domain of the fluorescent molecule, so as to avoid the interference of a large amount of normal non-glycosylated RNA. By using the chemical modification blocking nucleic acid hybridization technology, the whole system has the difference of “OFF”-“ON”-“OFF” fluorescent signals in different states, so that the low-abundance glycosylated modified RNA can be sensitively detected in the interference background of a large amount of non-glycosylated RNA. More importantly, unlike the imaging technology of glycoRNAs on the cell membrane which depends on metabolic labeling, the modification blocking nucleic acid hybridization technology based on glycosylation modification itself has high biocompatibility, so that the whole strategy can be carried out in living cells, thereby realizing the in-situ imaging of glycoRNAs in cells.

[0017] The application provides an in-vitro chemical synthesis method of glycoRNAs, and provides data support for in-vitro detection of glycoRNAs by the modification blocking nucleic acid hybridization technology, and also provides data support for in-situ imaging of glycoRNAs in cells.

[0018] The applicant uses the coupling reaction between an azido group and a cyclooctyne to connect the DBCO modified microRNA-21 and the azido modified glyco compound, and through the coupling reaction, the glyco-microRNA-21 is chemically synthesized as a glycoRNAs simulation target for in-vitro verification.

[0019] The in vitro verification method is as follows: the mixed solution after the substrate is hybridized with glyco-microRNA-21 and microRNA-21 is subjected to gel electrophoresis experiment to preliminarily observe whether the strategy has the possibility of hybridization; then, the mixed solution after the hybridization reaction of glyco-microRNA-21 and microRNA-21 with the substrate nucleic acid chain is added with fluorescent dye HBC, and the fluorescence response signal of the reactants is tested, so as to further verify the feasibility of the method for glycoRNAs analysis and detection under the optimized experimental conditions (reaction temperature is 35-40 ℃, magnesium ion condition is 10 mM), and to determine the effective concentration range.

[0020] The present application transfects the mixed solution of the substrate and the fluorescent dye HBC contained in the reaction system into cell culture, and the technology can be successfully applied to in situ analysis and detection and in situ imaging of glycoRNAs in cells.

[0021] The present application has the following advantages: (1) The technology of the present application for modifying and blocking nucleic acid hybridization does not depend on double recognition of the glycan part and the RNA part, but uses the blocking function of glycosylation to realize analysis and detection of glycosylated RNA under a large amount of non-modified RNA interference; (2) The present application can achieve single base level research, which plays an important role in studying the structure-property relationship of glycoRNAs molecules; (3) The sequence and structure-specific substrate nucleic acid design endows the method with excellent specificity, accuracy and encodability, providing a potential basis for glycosylation modification detection.

[0022] (4) The separation-free property and high biocompatibility enable the entire strategy to be successfully carried out in living cells without relying on biological metabolic labeling, thereby realizing in situ imaging of glycoRNAs in cells, and providing support for revealing the biosynthesis pathway of glycoRNAs in cells.

[0023] Other advantages, objects, and features of the present application will be in part apparent and in part pointed out hereinafter in the specification, and in part will be learned from the practice of the present application. The objects and other advantages of the present application can be achieved and obtained by the following description. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred detailed description of the present application will be combined with the drawings as follows, wherein: Figure 1 The schematic diagram of the principle of modifying and blocking nucleic acid hybridization for detecting target glycoRNAs.

[0025] Figure 2 To modify the gel electrophoresis results of nucleic acid hybridization techniques.

[0026] Figure 3 Fluorescence analysis for detecting glycoRNAs using modified and repressed nucleic acid hybridization technique is presented. In this paper, A represents the fluorescence response results achieved by this technique within a series of concentration ranges, and B represents the fluorescence verification results based on a series of glycosylated / non-glycosylated RNA ratio conditions of this technique.

[0027] Figure 4 The results of in situ imaging of glycoRNAs in different cells were obtained by modifying the nucleic acid hybridization technique. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments.

[0029] Example 1 Design principles of modified and repressed nucleic acid hybridization techniques for detecting glycoRNAs First, the applicant chemically synthesized glyco-microRNA-21 by coupling DBCO-miR21 and azide-modified mannose via a coupling reaction between an azide group and cyclooctyne. Furthermore, a substrate chain with a toehold was designed, containing a specific sequence of the "Pepper" aptamer that binds to the fluorescent small dye HBC. Binding occurs in the presence of the target substance, and as the chain shifts, a structural transformation of the hybridization complex is induced. As... Figure 1 As shown, in the absence of the target compound, the substrate nucleic acid strand cannot bind to HBC, exhibiting an "OFF" state (State 1). Subsequently, even after strand displacement initiates and toehold binding is complete, it remains a "no signal" state (State 2). However, when the strand displacement proceeds to the vicinity of the glycosylation site of glyco-microRNA-21, the hybridization product forms a specific domain that binds specifically to HBC, generating an "ON" signal in State 3. When microRNA-21 fully binds to the substrate in State 4, the target compound mediates complete hybridization of the substrate nucleic acid strand, also exhibiting an "OFF" signal. Therefore, a "OFF"-"ON"-"OFF" signal difference can be generated during the strand displacement hybridization process throughout the system. Utilizing this signal difference, the detection of low-abundance glyco-microRNA-21 can be achieved against a background of significant unglycosylated microRNA-21 interference.

[0030] Example 2 In vitro analysis and detection of glycoRNAs using modified and repressed nucleic acid hybridization techniques. 1) Gel electrophoresis analysis of glycoRNAs detection by modified blocking nucleic acid hybridization technology The lyophilized powder of nucleic acid chains was obtained by gene synthesis technology. After dilution with enzyme-free water, it was stored at -20 ℃. Before the experiment, each reactant was diluted to 1 µM, and the nucleic acid chain containing the hairpin structure was first annealed at high temperature to maintain a single configuration. The mixed system needs to be hybridized at 37 ℃ for 2 h in a PCR amplifier to form a hybrid complex. As shown in Figure 2 lanes 1, 2, and 3, only substrate, microRNA-21, and glyco-microRNA-21 were added, respectively. Lanes 4 and 5 were mixed systems, and the substrate was hybridized with the samples of glyco-microRNA-21 and microRNA-21 hybridization products, respectively. New bands with higher molecular weights appeared in lanes 4 and 5, and the brightness of the substrate band decreased, and the target band disappeared, confirming the successful hybridization of the substrate and the target. In addition, the intensity of each band in the gel electrophoresis experiment was quantitatively analyzed using image J software, and the results directly reflected the differences between glycosylation modification and non-glycosylation systems. For glycosylated nucleic acid chains, glyco-microRNA-21 only hybridizes to the glycosylation site with the substrate, and the energy change of the binding reaction is theoretically smaller than that of the stable full hybridization of the non-glycosylation system, making the reverse movement trend of the equilibrium reaction larger than that of the non-glycosylation system, resulting in a relatively small amount of glycosylation hybridization product. In summary, the difference in glycosylation of RNA in the hybridization reaction provides feasibility for the scheme.

[0031] 2) Fluorescence analysis of glycoRNAs detection by modified blocking nucleic acid hybridization technology Different concentrations of glyco-microRNA-21 and microRNA-21 were added to the substrate, and hybridization was incubated at 37 ℃ for 120 min in a PCR amplifier. After the reaction was completed, the fluorescence signal in the solution was detected by a fluorescence spectrophotometer. The excitation wavelength of the fluorescence instrument was 485 nm, the emission wavelength was selected as 530 nm, and the slit width was 10 nm. As shown in Figure 3As shown in Fig. 2A, under the optimized experimental conditions, the glycosylation system produced significantly higher signals than the non-glycosylation system, proving the successful detection of glyco-microRNA-21. From the graph of the relationship between the fluorescence signal and the concentration of glyco-microRNA-21 and microRNA-21, it can be seen that the fluorescence signal of the glycosylation-modified RNA shows a classic "S" type dose-effect relationship in the concentration range of 10 fM to 100 nM, and the binding of the unmodified RNA has a higher fluorescence signal, and the "OFF" fluorescence signal of microRNA-21 changes less with the concentration, proving the sensitive detection of glycoRNAs by the strategy in the series concentration range. In addition, in order to verify the effectiveness of the technology again, different proportions of microRNA-21 were added to the system with a fixed glyco-microRNA-21 concentration of 10 pM, and the fluorescence response results were determined. Figure 3 The results of Fig. 2B show that even if there is interference of non-glycosylation RNA in the glycosylation system, it will not cause significant changes in the fluorescence signal, and the system only responds to the binding of glycosylation RNA and the substrate. Example 3 Analysis and in situ imaging of glycoRNAs in cells by modified nucleic acid hybridization technology The HeLa, MCF-7 and MCF-10A cells used in the experiment were cultured in a culture box at 37 ℃ and 5% CO2, and the culture medium was DMEM complete medium (the components included DMEM basic medium, 10% fetal bovine serum and 1% penicillin-streptomycin double antibody). When the cells grew to 80% density, they were subcultured in 35 mm 2 dishes. After 24 h of culture to the appropriate density, the mixed solution (100 μL) composed of substrate oligonucleotide (2.5 μM, 10 μL), HBC (10 μM, 10 μL), liposome 3000 (10 μL) and complete medium was added to the culture dish for continuous culture. After 10 hours of incubation in the incubator, 20 μL of Hoechst 33342 was added for 25 minutes. Then the culture dish was washed with phosphate buffered saline (PBS, PH=8.0) buffer for 3 times, and then the culture medium was added to maintain cell activity. Then the imaging of glycoRNAs in cells was observed under a fluorescence microscope. Figure 4The results show that HeLa and MCF-7 cells with added substrate all show bright green fluorescent signals, while MCF-10A as normal cells show weak fluorescent signals. These results show that the modified blocking nucleic acid hybridization technology is used to first effectively respond to glycoRNAs in cells, prove the existence of glycoRNAs in cells, and different fluorescent intensities reveal that the expression of glycoRNAs in cancer cells is much higher than that in normal cells, indicating that glycoRNAs may be related to the occurrence of cancer.

[0032] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should be covered in the scope of the claims of the present application.

Claims

1. Substrate strand for the identification of glycosylation-modified microRNA-21, characterized in that, The deoxynucleotide sequence of the substrate chain is shown as SEQ ID NO. 1, the deoxynucleotide sequence of microRNA-21 is shown as SEQ ID NO. 2, and the deoxynucleotide sequence of the glycosylation modified microRNA-21 is shown as SEQ ID NO.

3.

2. The substrate chain of claim 1 is used in the analysis and detection of intracellular glycosylated RNA.

3. Use according to claim 2, characterized in that, The analysis and detection of intracellular glycosylated RNA is achieved by using the blocking effect of glycosylation modification.

4. The substrate chain of claim 1 is used in the preparation of a cancer detection kit.

5. A method for intracellular glycosylated RNA analysis that modifies the blocking nucleic acid hybridization technique, characterized by, The construction of the modification blocking nucleic acid hybridization technology module is completed by mixing the cells, the aforementioned substrate chain and the fluorescent small molecule dye HBC, the signal difference of "OFF"-"ON"-"OFF" is established, and the analysis and detection of glycosylated RNA is completed.

6. The method of intracellularly glycosylated RNA analysis according to claim 5, wherein, The signal difference of the fluorescence sensing technology of modification blocking nucleic acid hybridization is achieved by using the structural transformation of fluorescent RNA.

7. The method of intracellularly glycosylated RNA analysis according to claim 5, wherein, First, the cells are mixed with the aforementioned substrate chain and the fluorescent small molecule dye HBC, then staining solution is added for staining, and finally the imaging of intracellular glycosylated RNA is observed under a fluorescence microscope.

8. The method of intracellularly glycosylated RNA analysis according to claim 5, wherein, The cells include but are not limited to HeLa cells and MCF-7 cells.

9. The method of intracellularly glycosylated RNA analysis according to claim 5, wherein, The intracellular glycosylated RNA shows bright green fluorescence signals.

10. The intracellular glycosylated RNA analysis method of claim 5 is used in the preparation of a cancer detection kit.

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