Biosensor for simultaneously detecting N6-methyladenosine at single molecule level as well as preparation method and application of biosensor

Abstract

The invention discloses a biosensor for simultaneously detecting N6-methyladenosine at a single molecule level and a preparation method and application of the biosensor, and the preparation method of the biosensor comprises the following steps: step 1, incubating a reaction solution containing mRNA, a MazF buffer solution and MazF to complete a MazF cleavage reaction; and 2, adding a reaction product in the step 1 into a reaction system containing the hairpin probe, a DSN buffer solution and DSN, and incubating to complete DSN digestion reaction. The biosensor provided by the invention can be used for simultaneously detecting two m6A sites in different mRNAs with single nucleotide resolution. Besides, the method can identify 0.01% RNA methylation level, can distinguish m6A level between cancer cells / tissues and normal cells / tissues, and has a great application prospect in cancer detection and RNA epigenetics research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis, and in particular relates to a biosensor for simultaneously detecting N6-methyladenosine at a single molecule level, and a preparation method and application thereof. Background Art

[0002] N6-methyladenosine (m 6 A) is the most common epigenetic RNA modification and is essential for RNA metabolism processes including translation, alternative splicing, translocation, and degradation. 6 METTL1 is dynamically modified by methyltransferases (e.g., METTL3, METTL14, and METTL16) and demethylases (e.g., ALKBH5 and FTO) and is involved in a variety of cellular processes including proliferation, differentiation, regulation, and immune responses. 6 A level may interfere with normal cell function and induce the appearance and progression of a variety of cancers, such as breast cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, prostate cancer, bladder cancer, ovarian cancer, and pancreatic cancer. Therefore, accurate and efficient detection of m 6 A is conducive to revealing its biological effects and promoting its clinical application.

[0003] Including m 6 High-throughput sequencing methods such as A-seq and MeRIP-Seq are best suited for analyzing transcriptome-wide mRNAs at a base resolution of 200 nt. 6 A. PA-m 6 Innovative sequencing methods such as A-seq and miCLIP can even improve the resolution to the single base level. However, these methods rely on m 6 A antibody for target recognition has poor reproducibility, low binding efficiency, and cross-reactivity to other types of RNA modifications. Alternatively, the newly developed DART-seq, MePMe-seq, GLORI, LEAD-m 6 A-seq and 4SedTTP-inlovled next-generation sequencing can be used to 6 A is an antibody-independent detection method. However, sequencing methods are limited by cumbersome procedures, large sample consumption, high false positive rates, and lack of quantification of specific sites. 6 In addition, a variety of biochemical assays have been designed to achieve m 6 The SCARLET method combines RNase H cleavage and ligation-assisted extraction to detect mRNAs in lncRNAs and mRNAs using thin-layer chromatography. 6 A state and fraction level, but the extremely time-consuming protocol and the involvement of radioactive materials greatly limit its widespread application. 6The SELECT method for A-mediated inhibition of DNA polymerase and ligase activity can quantify m 6 A.

[0004] A site is in rRNA, lncRNA and mRNA, but it requires precise control of thermal cycles to achieve polymerase chain reaction for signal amplification. Recently, a series of isothermal amplification techniques (e.g., exponential amplification reaction coupled with CRISPR cas12a, proximity ligation-triggered rolling circle amplification, reverse transcription-ligation-rolling circle amplification cascade reaction, rolling circle amplification with loop-mediated isothermal amplification, and proximity hybridization followed by primer exchange amplification) have been introduced for m 6 Sensitive quantification of A at a constant reaction temperature. Despite their good performance, these methods often suffer from complex analytical protocols, complicated probe designs, multiple discrete reaction steps, laborious washing / separation steps, and false positive signals caused by nonspecific ligation / amplification. Notably, m 6 A modifications may coordinately regulate cellular functions, and a disease may be associated with various mRNAs in different RNAs. 6 A site. For example, ASB2 and RARA mRNA 6 Reduced A levels inhibited the differentiation of leukemic cells and enhanced leukemogenesis, while the expression of miR-21-5p, miR-17-5p, let-7a-5p, and miR-200c-3p in gastrointestinal cancers 6 A modification is increased. Simultaneous detection of multiple site-specific m 6 A is highly desirable but remains a challenge. Here, a new fluorescent biosensor was developed for the simultaneous quantification of mRNAs in different mRNA strands with single-base resolution. 6 A. In this method, m 6 A protects the mRNA chain from digestion by MazF, and the remaining mRNA will trigger cyclic digestion of the detection probe, generating enhanced fluorescence signals to simultaneously detect multiple mRNAs at different RNA sites. 6 This method achieves subfemtomolar sensitivity and can accurately quantify endogenous m 6 A level. Summary of the invention

[0005] The present invention aims to provide a biosensor for simultaneously detecting N6-methyladenosine at the single molecule level, and a preparation method and use thereof, by constructing a biosensor for simultaneously detecting two mRNAs in different mRNAs at a single nucleotide resolution. 6 A site.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a biosensor for simultaneously detecting N6-methyladenosine at the single molecule level comprises the following steps:

[0008] Step 1, incubating a reaction solution containing mRNA, a MazF buffer and MazF to complete a MazF cleavage reaction;

[0009] Among them, mRNA is the ribonucleic acid that interacts with MazF;

[0010] MazF is a toxin protein in the toxin-antitoxin system of E. coli. It is an endonuclease that specifically cuts the 5' end of the ACA sequence in single-stranded RNA, but cannot cut double-stranded RNA, double-stranded DNA, or single-stranded DNA.

[0011] MazF buffer is the reaction buffer that comes with the MazF product;

[0012] Step 2, adding the reaction product of step 1 to a reaction system containing a hairpin probe, a DSN buffer and DSN, and incubating to complete a DSN digestion reaction;

[0013] Among them, DSN is a double-stranded specific nuclease that highly selectively degrades double-stranded DNA and DNA in DNA-RNA hybrids, has almost no activity against single-stranded nucleic acid molecules and double-stranded RNA, and can well distinguish single-base mismatches;

[0014] DSN buffer is the reaction buffer that comes with the DSN product.

[0015] In the step 1, 10 μL of reaction solution containing mRNA of different concentrations, 1×MazF buffer and 0.2 U MazF were incubated at 37° C. for 40 minutes.

[0016] In the step 2, the reaction product of step 1 was added to a 20 μL reaction system containing 200 nM hairpin probe, 1×DSN buffer and 0.2 U DSN, and incubated at 55° C. for 60 minutes.

[0017] The MazF buffer included 20 mM Na3PO4, 0.01% Tween 20.

[0018] The DSN buffer includes 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, pH 8.0.

[0019] The hairpin probe includes a probe for m 6 The hairpin DNA probe H1 for A-PALB2 assay and for m 6 The sequence of the hairpin DNA probe H2 for the A-CHEK2 assay is as follows:

[0020] The H1 sequence is: BHQ2-GCG CGT CCA TCT TTT TGT CCA GCC AGC AAC GCG C-Cy3; the H2 sequence is: BHQ2-GCG CGT CCA CCA CTT TGT CAA ACA GCT CAC GCG C-Cy5.

[0021] A biosensor for simultaneous detection of N6-methyladenosine at the single-molecule level.

[0022] The biosensor is used in preparing a biosensor for simultaneously detecting site-specific N6-methyladenosine at the single molecule level.

[0023] The biosensor simultaneously detects two mRNAs in different mRNAs with single-nucleotide resolution. 6 A site.

[0024] Two m 6 A sites are m 6 A-PALB2 and m 6 A-CHEK2, whose sequence is as follows:

[0025] m 6 The sequence of A-PALB2 is: UGC UGG CUG Gm 6 AC AAA AAG AUG G;

[0026] m 6 The sequence of A-CHEK2 is: GAG CUG UUU Gm 6 AC AAA GUG GUG G;

[0027] Lack of m 6 The sequences of A-PALB2 and A-CHEK2 modified are as follows:

[0028] The sequence of A-PALB2 is: UGC UGG CUG GAC AAA AAG AUG G;

[0029] The sequence of A-CHEK2 is: GAG CUG UUU GAC AAA GUG GUG G.

[0030] The biosensor is for m 6 The detection limit of A-PALB2 was 0.35 fM, m 6 The detection limit of A-CHEK2 was 0.25 fM.

[0031] Beneficial effect: In the present invention, m 6A protects RNA from being cleaved by MazF, and the remaining RNA subsequently activates the activity of duplex-specific nucleases, inducing cyclic digestion of the detection probe. A large number of fluorescent groups are released, which can be sensitively monitored by single-molecule counting. MazF is used to efficiently and specifically distinguish m 6 A. Avoid using commonly used mRNAs with poor reproducibility, limited binding efficiency, and cross-reactivity to other RNA modifications. 6 A antibody. The integration of double-strand-specific nuclease amplification and single-molecule detection achieves subfemtomolar sensitivity, eliminating the reported m 6 The proposed biosensor was successfully applied to simultaneously detect two mRNAs in different mRNAs with single nucleotide resolution. 6 In addition, the test can identify RNA methylation levels of 0.01% and can distinguish m between cancer cells / tissues and normal cells / tissues. 6 A level, which has great application prospects in cancer detection and RNA epigenetic research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 :A and B are existence (A) and non-existence (B) respectively 6 Schematic diagram of the principle of the biosensor in case A.

[0033] Figure 2 :A is the PAGE analysis of MazF cleavage products after SYBR Gold staining; Lane 1, m 6 A-PALB2+MazF; Lane 2, A-PALB2+MazF; Lane 3, A-PALB2; Lane 4, m 6 A-CHEK2+MazF; Lane 5, A-CHEK2+MazF; Lane 6, A-CHEK2; B is PAGE analysis of DSN digestion products by direct excitation of Cy5 and Cy3; Lane 1, m 6 A-PALB2; Lane 2, A-PALB2; Lane 3, m 6 A-CHEK2; Lane 4, A-CHEK2; C, A-PALB2 (black line) and m 6 Fluorescence spectrum of Cy3 in the presence of A-PALB2 (green line), the inset shows the response m 6 A-PALB2 and the visual image of the product of A-PALB2; D is A-CHEK2 (blue line) and m 6 Fluorescence spectrum of Cy5 in the presence of A-CHEK2 (red line), the inset shows the response m 6 Visual images of A-CHEK2 and the products of A-CHEK2.

[0034] Figure 3 :A to H are responses to A-PALB2+A-CHEK2 (A and E), m 6 A-PALB2+A-CHEK2(B and F), A-PALB2+m 6 A-CHEK2 (C and G) and m 6 A-PALB2+m 6 A- Single molecule imaging of CHEK2 (D and H); Cy3 and Cy5 signals are shown in green and red, respectively; scale bar is 5 μm; I is the effect of Cy3 on different concentrations of m 6 A is the response count of PALB2; J is the response of Cy5 to different concentrations of m 6 A-CHEK2 response counts.

[0035] Figure 4 :A is m 6 A-Cy3 signals generated by PALB2 and interfering RNAs; B-m 6 A-Cy5 signals generated by CHEK2 and interfering RNAs; C-Input and measured m 6 A-Correlation between PALB2 methylation levels; D is the input and measured m 6 A-Correlation between CHEK2 methylation levels.

[0036] Figure 5 :A is the site-specific m in the cells detected simultaneously 6 A is an overview; B is the Cy3 signal generated by different cell lines; C is the Cy3 signal generated by different numbers of HeLa cells; D is the Cy5 signal generated by different cell lines; E is the Cy5 signal generated by different numbers of MDA-MB-231 cells.

[0037] Figure 6 :A is the site-specific m detected simultaneously in clinical tissues 6 Overview of A; B and C are heat map (B) and box plot (C) of Cy3 signals generated by healthy tissues and cancerous tissues, respectively; D and E are heat map (D) and box plot (E) of Cy5 signals generated by healthy tissues and cancerous tissues, respectively. DETAILED DESCRIPTION

[0038] The present invention will be further explained below in conjunction with the embodiments.

[0039] Reagents and Materials:

[0040] All oligonucleotides were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and HPLC purified. MazF and 5×MazF buffer were purchased from Takara Bio (Dalian, China). Duplex-specific nuclease and 10×DSN buffer were purchased from New born (Shenzhen, China). Diethylpyrocarbonate (DEPC)-treated water (RNase-free) was purchased from Sangon Biotech Co. Ltd (Shanghai, China). MCF-7, BT474, HeLa, MDA-MB-231, MCF-10A, and HBE cells were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human tissue samples were obtained from Shandong Second Medical University (Weifang, China), and the experiments were approved by the Ethics Committee of Shandong Second Medical University.

[0041] Gel electrophoresis:

[0042] MazF-mediated cleavage and DSN-mediated digestion products were analyzed by 14% native polyacrylamide gel electrophoresis (PAGE) × 110 V buffer (9 mM Tris-HCl, 9 mM boric acid, 0.2 mM EDTA, pH 7.9) at a constant voltage of 110 V for 65 min. The gel was visualized by a Biorad ChemiDoc MP imaging system (Hercules, CA, USA).

[0043] Single molecule detection:

[0044] The reaction product was diluted 500 times in imaging buffer. After 10 μL of the sample was pipetted onto a coverslip, fluorescence images were obtained by total internal reflection fluorescence (TIRF) microscopy (Nikon, Ti-E, Japan). Cy3 fluorophore was excited by 561 nm laser, Cy5 fluorophore was excited by 640 nm laser, and photons were collected using an oil immersion objective (CFIApochromat TIRF 100×). Fluorescent spots were counted in a 400 × 400 pixel area using Image J software, and the average Cy3 and Cy5 counts were obtained by counting 9 frames.

[0045] N6-methyladenosine (m 6 A) is the most common epigenetic RNA modification and is widely involved in different physiological and pathological processes. However, it is difficult to simultaneously detect sequence-specific m 6 A remains a challenge. Here, a fluorescent biosensor was constructed for the simultaneous detection of site-specific m 6 A. In this biosensor, m 6A protects the RNA scaffold from being cleaved by MazF, and the remaining RNA subsequently activates the activity of the double-stranded specific nuclease, inducing cyclic digestion of the detection probe. As a result, abundant fluorescent groups are released, and they can be sensitively detected simultaneously by single-molecule counting. MazF is used to efficiently and specifically distinguish m 6 A. Avoid using commonly used mRNAs with poor reproducibility, limited binding efficiency, and cross-reactivity to other RNA modifications. 6 A antibody. The integration of double-strand-specific nuclease amplification and single-molecule detection achieves subfemtomolar sensitivity, eliminating the reported m 6 The present invention demonstrates the successful application of the proposed biosensor for the simultaneous detection of two mRNAs in different mRNAs with single nucleotide resolution. 6 In addition, the test can identify RNA methylation levels of 0.01% and can distinguish m between cancer cells / tissues and normal cells / tissues. 6 A level, which has great application prospects in cancer detection and RNA epigenetic research.

[0046] Embodiment 1:

[0047] A method for preparing a biosensor for simultaneously detecting N6-methyladenosine at the single molecule level comprises the following steps:

[0048] In step 1, 10 μL of reaction solution containing different concentrations of mRNA, 1× MazF buffer and 0.2 U MazF was incubated at 37° C. for 40 minutes to complete the MazF cleavage reaction.

[0049] Step 2: Add the reaction product to a 20 μL reaction system containing 200 nM hairpin probe, 1×DSN buffer and 0.2 U DSN, and incubate at 55° C. for 60 minutes to complete the DSN digestion reaction.

[0050] Among them, mRNA is the ribonucleic acid that interacts with MazF;

[0051] MazF is a toxin protein in the toxin-antitoxin system of E. coli. It is an endonuclease that specifically cuts the 5' end of the ACA sequence in single-stranded RNA, but cannot cut double-stranded RNA, double-stranded DNA, or single-stranded DNA.

[0052] DSN is a double-stranded specific nuclease that highly selectively degrades double-stranded DNA and DNA in DNA-RNA hybrids, has almost no activity against single-stranded nucleic acid molecules and double-stranded RNA, and can well distinguish single-base mismatches;

[0053] MazF buffer is the reaction buffer that comes with the MazF product.

[0054] DSN buffer is the reaction buffer that comes with the DSN product.

[0055] MazF buffer included 20 mM Na3PO4, 0.01% Tween 20.

[0056] DSN buffer consisted of 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, pH 8.0.

[0057] The biosensor is capable of simultaneously detecting site-specific N6-methyladenosine at the single-molecule level.

[0058] The biosensor simultaneously detects two mRNAs in different mRNAs with single-nucleotide resolution. 6 A site m 6 A-PALB2 and m 6 A-CHEK2, whose sequence is as follows:

[0059] m 6 The sequence of A-PALB2 is: UGC UGG CUG Gm 6 AC AAA AAG AUG G;

[0060] m 6 The sequence of A-CHEK2 is: GAG CUG UUU Gm 6 AC AAA GUG GUG G.

[0061] Lack of m 6 The sequences of A-PALB2 and A-CHEK2 modified are as follows:

[0062] The sequence of A-PALB2 is: UGC UGG CUG GAC AAA AAG AUG G;

[0063] The sequence of A-CHEK2 is: GAG CUG UUU GAC AAA GUG GUG G.

[0064] Hairpin probes include those for m 6 The hairpin DNA probe H1 for A-PALB2 assay and for m 6 The sequence of the hairpin DNA probe H2 for the A-CHEK2 assay is as follows:

[0065] The H1 sequence is: BHQ2-GCG CGT CCA TCT TTT TGT CCA GCC AGC AAC GCG C-Cy3; the H2 sequence is: BHQ2-GCG CGT CCA CCA CTT TGT CAA ACA GCT CAC GCG C-Cy5.

[0066] Example 2: The overall process is as follows Figure 1 As a proof of concept, PALB2 mRNA (m 6 A-PALB2) and CHEK2 mRNA (m 6 A-CHEK2) in two m 6 The A site is used as a model target. Figure 1 As shown in Scheme A, a hairpin DNA probe 1 (H1) labeled with BHQ2 and Cy3 at its two ends is used to 6 A-PALB2 assay, hairpin DNA probe 2 (H2) labeled with BHQ2 and Cy5 at both ends was used for m 6 A-CHEK2 assay, in which Cy3 and Cy5 signals are effectively quenched by nearby BHQ2 via FRET. The endoribonuclease MazF was used to identify m from A 6 A, it can efficiently cleave RNA containing the 5'-ACA-3' sequence, but not the 5'-(m 6 A) CA-3'. 6 The presence of the A modification can prevent MazF from cutting the RNA chain. The remaining intact RNA chain can bind to the detection probe, open the hairpin structure and form an RNA-DNA heteroduplex (22bp). DSN is used for signal amplification, and it has efficient cutting activity on DNA in the DNA-RNA duplex, but not ssDNA and RNA. H1 and H2 in the heteroduplex can be recognized and hydrolyzed by DSN, resulting in RNA release. The free RNA will hybridize with other hairpin probes to trigger the next round of DSN-catalyzed digestion of H1 and H2. Therefore, many detection probes are digested to release a large number of Cy3 and Cy5 fluorescent groups, which will be quantified by single molecule counting. It is worth noting that there is no obvious overlap in the Cy3 and Cy5 emission spectra, which allows the simultaneous detection of m at the single molecule level. 6 A-PALB2 and m 6 A-CHEK2. In contrast, the lack of m 6A-PALB2 and A-CHEK2 modified by A are sensitive to MazF, and they can be completely cleaved by MazF, releasing two 11-nt fragments. Since DSN-mediated cleavage requires a DNA-RNA duplex of at least 15 bp in length, the cleaved RNA is too short (11 nt) to activate DSN-catalyzed digestion of H1 and H2, and no fluorescence signal is observed ( Figure 1 Medium option B).

[0067] Example 3: Feasibility of verification experiment

[0068] 14% PAGE was used to verify the cleavage products catalyzed by MazF after SYBR Gold staining. Unmethylated A-PALB2 and A-CHEK2 lncRNA were used as controls. After adding MazF, the band of A-PALB2 ( Figure 2 A, Lane 3) disappears ( Figure 2 A, lane 2), accompanied by the appearance of a new band of smaller size, indicating that MazF effectively digested unmethylated A-PALB2. 6 The band of A-PALB2 remained unchanged after MazF treatment ( Figure 2 A, lane 1), indicating m 6 A-PALB2 cannot be cleaved by MazF. In addition, unmethylated A-CHEK2 ( Figure 2 A, lane 6) can be detected by MazF ( Figure 2 A, lane 5) cut, while m 6 A-CHEK2 is resistant to MazF ( Figure 2 A, lane 4). The results showed that MazF can recognize site-specific m 6 A. The present invention further uses 14% PAGE to analyze the DSN-catalyzed digestion products by direct stimulation of Cy3 and Cy5. 6 A-PALB2 ( Figure 2 In B, lane 1), a distinct Cy3 fluorescence band can be observed due to DSN-mediated H1 cleavage. In contrast, no band was detected in response to A-PALB2 ( Figure 2 B, lane 2), indicating that no H1 was cleaved. Similarly, the Cy5 fluorescent band can only be seen in response to m 6 A-CHEK2( Figure 2 B, lane 3) instead of A-CHEK2 ( Figure 2 B, lane 4), which indicates that m 6 A-CHEK2-induced H2 digestion. Fluorescence spectroscopy measurements showed that m 6 A-PALB2 but not A-PALB2 produced significantly enhanced Cy3 signals ( Figure 2Middle C), high Cy5 signal is caused by m 6 A-CHEK2 instead of A-CHEK2 ( Figure 2 D), demonstrating the proposed biosensor for multiple m 6 A. Feasibility of determination.

[0069] Example 4: m 6 A. Sensitivity of biosensor

[0070] Single-molecule detection is one of the most sensitive analytical techniques with advantages such as high signal-to-noise ratio and low sample consumption. 6 A triggered Cy3 and Cy5 signals. 6 In the case of A-PALB2, only Cy3 spots were detected ( Figure 3 B in 3 and F in 4). However, in the case of m 6 In the case of A-CHEK2, only Cy5 spots can be produced ( Figure 3 C in 3 and G in 3). When m 6 A-PALB2 and m 6 When A-CHEK2, Cy3 and Cy5 spots appear simultaneously ( Figure 3 In contrast, when m 6 A-PALB2 and m 6 In the absence of A-CHEK2, neither Cy3 nor Cy5 spots were observed ( Figure 3 The results show that this method is applicable to multiple m at the single-molecule level. 6 A determination. Under the optimal parameters, the sensitivity was tested by detecting the Cy3 and Cy5 signals generated by different concentrations of targets. The Cy3 signal increased with the increase of m 6 A-CHEK2 concentration increased, and it was 1×10 -15 and 1×10 -9 The number of pairs between M 6 A concentration showed a good linear correlation ( Figure 3 I). The linear equation is N = 527.9 + 31.9log10C (R 2 =0.997), LOD is 0.35fM. Figure 3 As shown in J, the Cy5 counting signal increases with the m 6 The A-PALB2 concentration increased with the increase of -15 to 1×10 -9 The logarithm of M 6 A-PALB2 concentration showed a good linear correlation. The linear equation was N = 730.1 + 44.8 log10C (R 2=0.980), and the LOD is 0.25fM. Therefore, this method achieves better performance than other methods. 6 The improved sensitivity is attributed to the following factors: (1) 6 A-mediated protection of mRNA from Maz-mediated cleavage 6 (1) specific conversion of the A signal into an amplifiable nucleic acid signal; (2) efficient signal release mediated by DSN-catalyzed cyclic cleavage of the detection probe; and (3) ultra-high sensitivity of single-molecule detection.

[0071] Example 5: Specificity detection

[0072] In this example, 5mC-modified RNA sequences (5mC-PALB2 and 5mC-CHEK2) and random RNA sequences (5hmC-RU, m 6 A-RU, 5mC-RU and RU) were used as interfering substances to further evaluate the specificity of the biosensor. Figure 4 As shown in A, only m 6 A-PALB2 produced high Cy3 signals, while other interferences produced low background signals that were no different from the control group. 6 The signal of A-PALB2 is higher than that of 5mC-PALB2, 5hmC-RU, and m 6 The signals of A-RU, 5mC-RU, RU, and A-PALB2 were 8.89-fold, 8.42-fold, 7.94-fold, 8.47-fold, 7.92-fold, and 9.19-fold higher, respectively. 6 A-CHEK2 induced a significantly enhanced Cy5 signal, but other interferences produced a low background signal that was no different from the control group. 6 The signal generated by A-CHEK2 is higher than that of 5mC-CHEK2, 5hmC-RU, and m 6 A-RU, 5mC-RU, RU, and A-CHEK2 generated signals that were 10.16-, 11.52-, 8.86-, 10.18-, 11.48-, and 12.06-fold higher ( Figure 4 (B) The results show that this biosensor is effective for m 6 A has good specificity. 6 A often coexists with unmethylated A in the transcriptome and is derived from m 6 A specifically distinguishes low-frequency mRNAs from mixtures of modified and unmodified RNA 6 A is challenging. Preparation of m 6 A-RNA and a mixture of A-RNA, and calculate the corresponding methylation levels. Where M is m 6 The measured m 6A-PALB2 level (X) increases from 0.01% to 100% ( Figure 4 C), and is linearly correlated with the input methylation level (Y), with the equation Y = 0.953X + 0.093 (R 2 =0.997). In addition, the measured m 6 A-CHEK2 levels (X) increased from 0.01% to 100% and were linearly correlated with the input methylation level (Y) according to the equation Y = 0.992X + 0.015 (R 2 =0.999). Notably, RNA methylation levels as low as 0.01% could be well distinguished, outperforming other m 6 A detection, such as fluorescence detection based on exponential amplification reaction (30%), m-based 6 Immunochemical detection of A antibodies (5%) and qPCR amplification detection (0.25%).

[0073] Example 6: m in human cells 6 Detection of A

[0074] To test the potential of this method for real sample analysis, this example detected endogenous mRNA extracted from cancer cell lines (i.e., HeLa, MCF-7, BT474, MDA-MB-231 cells) and normal cell lines (i.e., HBE and MCF-10A cells). 6 A-mRNA( Figure 5 Compared with the low Cy3 signals induced by MCF-10A and HBE cells, HeLa, MCF-7, BT474, and MDA-MB-231 cells produced significantly enhanced Cy3 signals, and the Cy3 signals decreased in the order of Hela>MCF-7>BT474>MDA-MB-231>HBE>MCF-10A cells ( Figure 5 Cy3 signal (N) is linearly correlated with the logarithmic HeLa cell number (X) from 10 to 10,000 cells ( Figure 5 C), the equation is N = 53.9 + 32.7log10X (R 2 =0.998). In addition, the Cy5 signals induced by MCF-7, HeLa, BT474, and MDA-MB-231 cells were higher than those induced by MCF-10A and HBE cells, and the Cy5 signals decreased in the order of MDA-MB-231>BT474>MCF-7>Hela>MCF-10A>HELA ( Figure 5 Cy5 signal (N) is linearly correlated with the logarithmic MDA-MB-231 cell number (X) according to the equation N = 49.9 + 24.9log10X (R 2 =0.997)( Figure 5Therefore, this method can quantify endogenous m 6 A modification, and can distinguish cancer cells from normal cells 6 A level.

[0075] Example 7: Detection of m in clinical breast tissue 6 A

[0076] The present invention further applies this method to detect site-specific m 6 A and analyzed RNA samples extracted from breast cancer (n = 7) and healthy (n = 5) breast tissues ( Figure 6 Heat map analysis showed that breast cancer tissue induced Cy3 ( Figure 6 B) and Cy5 ( Figure 6 The signal in D) is much higher than that induced by healthy breast tissue, indicating that m 6 A was more highly expressed in cancer. The average Cy3 signal generated by breast cancer tissue was 3.12 times higher than that generated by healthy tissue (p<0.001, Figure 6 In middle C), the average Cy5 signal generated by breast cancer tissue was 5.37 times higher than that generated by healthy tissue (p<0.001, Figure 6 (E) This method measures m in real clinical samples. 6 The outstanding capabilities of A make it useful for disease diagnosis and prognosis.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a biosensor for simultaneous detection of N6-methyladenosine at the single molecule level, characterized in that: The steps include: Step 1, incubating a reaction solution containing mRNA, a MazF buffer and MazF to complete a MazF cleavage reaction; Among them, mRNA is the ribonucleic acid that interacts with MazF; MazF is a toxin protein in the E. coli toxin-antitoxin system; MazF buffer is the reaction buffer that comes with the MazF product; Step 2, adding the reaction product of step 1 to a reaction system containing a hairpin probe, a DSN buffer and DSN, and incubating to complete a DSN digestion reaction; Among them, DSN is a double-strand specific nuclease; DSN buffer is the reaction buffer that comes with the DSN product.

2. The method for preparing a biosensor for simultaneous detection of N6-methyladenosine at the single molecule level according to claim 1, characterized in that: In the step 1, 10 μL of reaction solution containing mRNA of different concentrations, 1×MazF buffer and 0.2 U MazF were incubated at 37° C. for 40 minutes.

3. The method for preparing a biosensor for simultaneous detection of N6-methyladenosine at the single molecule level according to claim 1, characterized in that: In the step 2, the reaction product of step 1 was added to a 20 μL reaction system containing 200 nM hairpin probe, 1×DSN buffer and 0.2 U DSN, and incubated at 55° C. for 60 minutes.

4. The method for preparing a biosensor for simultaneous detection of N6-methyladenosine at the single molecule level according to claim 1, characterized in that: The MazF buffer includes 20 mM Na3PO4, 0.01% Tween 20; the DSN buffer includes 50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, pH 8.

0.

5. The method for preparing a biosensor for simultaneous detection of N6-methyladenosine at the single molecule level according to claim 1, characterized in that: The hairpin probe includes a probe for m 6 The hairpin DNA probe H1 for A-PALB2 assay and for m 6 The sequence of the hairpin DNA probe H2 for the A-CHEK2 assay is as follows: The sequence of H1 is: BHQ2-GCG CGT CCA TCT TTT TGT CCA GCC AGC AAC GCG C-Cy3; The H2 sequence is: BHQ2-GCG CGT CCA CCA CTT TGT CAA ACA GCT CAC GCG C-Cy5.

6. A biosensor for simultaneously detecting N6-methyladenosine at the single molecule level obtained by the preparation method of claim 1.

7. Use of the biosensor according to claim 6 in the preparation of a biosensor for simultaneous detection of site-specific N6-methyladenosine at the single molecule level.

8. The use according to claim 7, characterized in that: The biosensor simultaneously detects two mRNAs in different mRNAs with single-nucleotide resolution. 6 A site.

9. The use according to claim 8, characterized in that: Two m 6 A sites are m 6 A-PALB2 and m 6 A-CHEK2, whose sequence is as follows: m 6 The sequence of A-PALB2 is: UGC UGG CUG Gm 6 AC AAA AAG AUG G; m 6 The sequence of A-CHEK2 is: GAG CUG UUU Gm 6 AC AAA GUG GUG G; Lack of m 6 The sequences of A-PALB2 and A-CHEK2 modified are as follows: The sequence of A-PALB2 is: UGC UGG CUG GAC AAA AAG AUG G; The sequence of A-CHEK2 is: GAG CUG UUU GAC AAA GUG GUG G.

10. The use according to claim 9, characterized in that: The biosensor is for m 6 The detection limit of A-PALB2 was 0.35 fM, m 6 The detection limit of A-CHEK2 was 0.25 fM.

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