Method for one-pot detection of microRNA based on RPA-Cas12a cis-cleavage fluorescent RNA aptamer biosensor

Abstract

The invention belongs to the technical field of biological analysis, and particularly relates to a one-pot method, Cas12a cis-cleavage, in-vitro transcription, a fluorescent RNA aptamer and application of the fluorescent RNA aptamer in microRNA detection in a material sample. The invention provides a method for sensitively and specifically detecting microRNA by connecting, identifying and triggering RPA (recombinase polymerase amplification) amplification and Cas12a cis-cleavage to realize large-scale amplification of a double-stranded DNA (deoxyribonucleic acid) amplicon and transcription of a functional fluorescent RNA aptamer, and combining a mechanism strategy of T7 transcription signal amplification and fluorescent aptamer signal amplification. The invention also provides a method for realizing rapid detection of miRNA-21 by optimizing the reaction process through a one-pot method. By utilizing the high sensitivity, high specificity and label-free characteristics of a detection platform for connecting and recognizing RPA amplification and Cas12a cis-cleavage biosensor through the one-pot method, the miRNA-21 concentration in cancer cells and patient serum samples can be quickly and effectively detected.

Description

Technical Field

[0001] The present invention relates to the technical field of bioanalysis, and in particular to a method for one-pot detection of microRNA based on an RPA-Cas12a cis-cutting fluorescent RNA aptamer biosensor. Background Art

[0002] A large number of studies have shown that miRNA participates in a series of complex biological processes by regulating the expression levels of related genes, and plays an important role in cell growth, differentiation and apoptosis. At the same time, as one of the emerging biomarkers abundant in human tissues and cells, miRNA has the function of inhibiting cell apoptosis and promoting cell proliferation. Its abnormal expression has also been found in various cancer tissues such as liver cancer, gastric cancer, lung cancer and breast cancer. Therefore, accurate and sensitive determination of miRNA expression levels can provide favorable evidence for cancer diagnosis, pathogenesis exploration and therapeutic intervention. In general, Northern blotting technology is a widely used standard method for analyzing miRNAs, but it has the disadvantages of insufficient sensitivity and large sample size. Therefore, due to the short sequence of miRNAs, high sequence homology between its family members, and the usually low concentration in the total sample, ultrasensitive detection of miRNAs is still a major technical challenge.

[0003] Specifically, the CRISPR system integrates molecular recognition elements and flexible signal transduction modules, making it a potential programmable platform for constructing DNA-based signal amplification schemes. To improve the sensitivity of the platform, current nucleic acid detection methods usually require a signal amplification step. Traditional amplification methods are based on polymerase chain reaction (PCR) and require a thermal cycler for delicate temperature regulation. However, these amplification methods usually require varying degrees of pretreatment to achieve target amplification and trigger subsequent target recognition reactions, which leads to the disadvantages of low specificity and high background signals. Recently, some ligation-dependent nucleic acid detection amplification methods based on thiophosphate terminal hairpin formation and self-excitation extension (PS-THSP), loop-mediated isothermal amplification (LAMP) and ligase chain reaction (LCR) have come into people's view. As the main mechanism for constructing biosensors in the CRISPR system, the exploration of Cas12a cis-cleavage activity remains to be developed. MangoRNA aptamers are relatively easy to design and encode, and they are used to combine with target dyes (TO1-Biotin) to produce bright fluorescent signals, which are used as the signal characterization system of this study.

[0004] Here, we demonstrate a one-pot cis-cleavage fluorescent biosensor for label-free detection of microRNA. By exploring a special isothermal amplification design of ligation-recognition-triggered RPA reaction, double-stranded DNA amplicons containing PAM sequences were generated. In the Cas12a cis-cleavage system, the transcription and cis-cleavage of the system were controlled by regulating the activities of Cas12a and T7 RNA polymerase in the double-stranded DNA amplicons. In addition, after the double-stranded DNA amplicons containing PAM sequences were cis-cleaved by Cas12a, functional Mango RNA that can bind to TO1-Biotin was continuously transcribed, thereby generating an amplified fluorescent output signal. By designing the reaction kinetics and optimizing the conditions, multiple reactions such as ligation recognition, RPA, and Cas12a cis-cleavage system were integrated into a collaborative coupling reaction system, creating a robust one-pot isothermal detection for miRNA analysis. Thanks to the cascade amplification effect of ligation-recognition-triggered RPA and the one-pot isothermal assay, this sensor can achieve sensitive detection within 40 minutes. In addition, it can effectively identify single-base mutations through specific ligases. This method has the advantages of high sensitivity, strong specificity, good versatility, and no background, and has great application potential in clinical miRNA analysis. More importantly, the one-pot detection technology of ligated recognition RPA amplification and Cas12a cis-cleavage fluorescent RNA aptamer biosensor for microRNA greatly simplifies the detection workflow, does not require professional instruments, and provides a highly adaptable method for clinical diagnosis. Summary of the invention

[0005] 1. The present invention provides a fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cleavage for one-pot detection of microRNA to solve the problems of low specificity, complex operation and high cost of existing methods. The method comprises:

[0006] (1) Providing a reaction mixture, wherein the reaction mixture includes a reaction mixture of a Mango probe, an Anti-Mango probe, T4 DNA ligase, a DNA polymerase, NEBuffer r2.1, and a T4 DNA ligation reaction buffer. The Mango probe includes a Mango sequence (yellow area) and a miRNA-21 specific recognition site (blue area). The Anti-Mango probe includes a recognition region (blue area) for specifically recognizing miRNA-21, an Anti-Mango region (orange area), and a crRNA sequence (cyan area);

[0007] (2) placing the reaction mixture of step (1) at a reaction temperature, wherein the Mango probe and the Anti-Mango probe bind to the miRNA-21 at the target recognition site under the action of base complementary pairing to form a three-stranded complex, and form an RPA amplicon with a PAM site under the action of T4 DNA ligase and DNA polymerase;

[0008] (3) Providing a reaction mixture for RPA amplification, wherein the reaction mixture includes a genomic forward primer, a reverse primer, SSB protein, a recombinase, and a reaction mixture of NEBuffer r2.1 reaction buffer. The forward primer contains a complete T7 promoter sequence (pink region);

[0009] (4) placing the reaction mixture of step (2) in the reaction mixture of reaction temperature (3), the RPA amplicon product of step (2) can be recognized by the primer sequence in the reaction mixture of step (3) and the SSB protein and further RPA amplified under the action of recombinase and DNA polymerase to obtain a large number of RPA amplicon products with T7 promoter;

[0010] (5) providing a reaction mixture of Cas12a and in vitro transcription, wherein the reaction mixture includes a reaction mixture of Cas12a protein, T7 RNA polymerase, rNTPs, RNase inhibitor, T7 RNA transcription buffer and NEBuffer2.1 reaction buffer;

[0011] (6) placing the reaction mixture described in step (4) in the reaction mixture described in reaction temperature (5), the large amount of RPA amplicon products with T7 promoter described in step (4) first bind to T7 RNA polymerase in the reaction mixture described in step (5) as a transcription template chain and transcribe Anti-Mango-crRNA, Anti-Mango-crRNA cannot bind to TO1-Biotin due to the closed secondary structure, further, Cas12a protein can process Anti-Mango-crRNA to form mature crRNA and self-provide to subsequent reactions, RPA amplicon with PAM site reacts with Cas12a-crRNA as an activation chain, activates the cis-cleavage activity of Cas12a protein and realizes cis-cleavage of RPA amplicon, finally, the cis-cleaved RPA amplicon is used as T7 transcription template again to transcribe Functional Mango RNA, bind to TO1-Biotin, and generate a strong fluorescent output signal;

[0012] (7) analyzing the amplified product to identify the target base type, including real-time polyacrylamide gel electrophoresis analysis and real-time fluorescence analysis, wherein the reaction mixture is provided before step (2), step (4) and step (6).

[0013] 2. The transcription process of producing fluorescent RNA aptamers (Functional Mango RNA) is regulated by the transcription mechanism and the Cas12a cis-cleavage mechanism. The miRNA-21-activated T4 DNA ligation reaction amplifies the signal output through RPA amplification of the RPA amplicon. The Anti-Mango-crRNA transcribed from the complete RPA amplicon reduces the background signal of the reaction by blocking the Functional Mango structure.

[0014] 3. according to the method described in claim 1-2, wherein the miRNA-21 is combined with the connection probe and completes T4DNA connection, DNA polymerase binds to the self-primer sequence of Mango probe to activate its polymerization reaction to form double-stranded DNA; when miRNA-21 is removed from the system, Mango probe and Anti-Mango probe cannot be connected to react. Meanwhile, SSB protein and recombinase recognize and bind to double-stranded DNA, and DNA polymerase binds to primers with T7 promoter to activate RPA amplification. The RPA amplicon produced by RPA amplification includes T7 promoter sequence (pink area), Mango RNA aptamer transcription template sequence (orange area), miRNA-21 recognition area (blue area), PAM site (purple area), Anti-Mango sequence (orange-red area) and crRNA transcription template sequence (cyan area). In the Cas12a cis-cutting system, by adjusting the activity of Cas12a and T7 RNA polymerase in double-stranded DNA amplicon, they are combined together. The CRISPR / Cas12a has pre-crRNA processing activity, which can be used to process the Anti-Mango-crRNA transcribed from the complete RPA amplicon into mature crRNA, thereby realizing the self-supply of crRNA to the Cas12a cis-cutting system. After the double-stranded DNA amplicon containing the PAM sequence is cis-cut by Cas12a, it will continuously transcribe the FunctionalMango RNA that can bind to TO1-Biotin, thereby generating a fluorescent output signal.

[0015] 4. The method according to any one of claims 1 to 3, wherein the fluorescence spectrum and PAGE results of the sensing system are used to verify the feasibility of miRNA-21 detection. By analyzing whether the Mango probe and the Anti-Mango probe can recognize miRNA-21 and whether the Mango probe and the Anti-Mango probe can be effectively connected after adding T4 DNA polymerase. Gel electrophoresis analysis shows that when the Mango probe and the Anti-Mango probe are not added to miRNA-21, no connection band is observed. When the Mango probe and the Anti-Mango probe only bind to miRNA-21, but no connection band is observed in the absence of T4 DNA ligase. Only when the Mango probe, the Anti-Mango probe, the T4 DNA ligase and the miRNA-21 are present, can the T4 DNA ligation reaction occur and a band with a predicted length be observed in the PAGE analysis. Subsequently, the DNA polymerase is used to bind to the self-primer position of the connection chain to polymerize the RPA amplicon without the T7 promoter, and a band with a predicted length is observed in the PAGE gel analysis. In addition, we used RPA amplification to introduce the forward primer with T7 promoter into the RPA amplicon, and observed the amplified band on gel electrophoresis. Next, we used non-denaturing gel electrophoresis to analyze the feasibility of the Cas12a cis-cutting system. When the RPA amplicon with T7 promoter activated the transcription reaction, a band at the corresponding position of Anti-Mango-crRNA was displayed in the gel electrophoresis, and then the double-stranded DNA after Cas12a cis-cutting transcribed functional Mango RNA. Finally, we combined the solution containing the reaction product with TO1-Biotin dye to detect the fluorescent signal. The fluorescence signal is very low when miRNA-21 is not present, and when miRNA-21 is added to the reaction, the fluorescence intensity increases. It shows that the system can work well and regulate the output of the fluorescent signal, achieving a seven-fold fluorescence ratio.

[0016] In some embodiments, the fluorescence intensity under different reaction conditions (NonCas12a, NonT7-RNAP andCas12a) was measured, and the results were consistent with expectations.

[0017] In some embodiments, the effect of RPA amplification at different times on fluorescence intensity is further analyzed. Gel electrophoresis shows that RPA forms a large number of RPA amplicons in a shorter time.

[0018] 5. according to the method described in any one of claims 1-4, wherein the one-pot detection of the detection object is realized, and a one-pot microRNA detection system is provided. In the three-step detection, it comprises a miRNA-21 sample to be tested, a T4DNA connection and DNA polymerization reaction system, an RPA index amplification reaction system and a Cas12a cis-cutting reaction system; wherein the T4 DNA connection and DNA polymerization reaction system include Mango probe and Anti-Mango probe, T4 DNA ligase, DNA polymerase and miRNA-21; the RPA index amplification reaction system includes recombinase, SSB protein and DNA polymerase; the Cas12a cis-cutting reaction system includes Cas12a protein and T7 RNA polymerase; afterwards, on the basis of three-step tandem detection, the detection step is included in the one-pot detection, the connection-recognition system, the RPA system and the CRISPR cis-cutting reaction system are combined in one reaction and the signal output is characterized using real-time fluorescence detection.

[0019] In some embodiments, although the workflow is simplified, the one-pot method slightly affects the detection sensitivity and speed, which is manifested in lower signal intensity detected when the reaction time is longer.

[0020] 6. The method according to any one of claims 1-5, wherein in order to obtain the best one-pot detection performance, we systematically studied and optimized several important aspects.

[0021] In some embodiments, different concentrations of monovalent cations in the buffer may inhibit the activity of the enzyme. We compared the effects of two different enzyme buffers on the detection effect. The results showed that NEBuffer r3.1 had a higher fluorescence value than NEBuffer r2.1, and the effect was better when both were present to achieve optimal performance.

[0022] In some embodiments, when Mg 2+ When the concentration of Mg reaches 20 mM, the optimal signal-to-noise ratio is obtained, and then it tends to be stable. 2+ The concentration is optimal.

[0023] In some embodiments, the reaction time of the one-pot method is optimized. When the reaction time of the one-pot method reaches 40 minutes, the best optimized signal-to-noise ratio is obtained, and then tends to be stable, so the reaction time of 40 minutes is optimal.

[0024] In some embodiments, the reaction temperature may affect the response signal of the biosensor. The results show that the fluorescence intensity at 37°C is better than that at 16, 42, and 65°C. Therefore, we set the temperature of the one-pot method to 37°C.

[0025] In some embodiments, when the concentration of Cas12a protein is 100 nM, the signal is strongest and the signal-to-noise ratio is best, so 100 nM is the optimal reaction concentration.

[0026] In some embodiments, when the concentration of Cas12a protein is 100 nM, the signal is strongest and the signal-to-noise ratio is best, so 100 nM is the optimal reaction concentration.

[0027] In some embodiments, the final reaction concentrations of T7 RNA polymerase, DNA polymerase, T4 DNA ligase, and recombinase are optimized to obtain optimal performance.

[0028] 7. The method according to any one of claims 1 to 6, wherein in order to analyze the performance of the sensor, it is subjected to fluorescence spectroscopy analysis.

[0029] In some embodiments, under the optimized conditions, the feasibility of the detection is confirmed by measuring the fluorescence signal. The final signal intensity is positively correlated with the concentration of miRNA-21 and increases significantly with the increase of miRNA-21 concentration. In addition, within the concentration range of 100aM to 100pM, the method has a good linear relationship, and the corresponding equation is F=244.20x-162.9(R 2 =0.9943), with a detection limit of 3.43aM, which is much lower than that of various miRNA analysis methods reported previously. This is mainly due to the signal amplification mechanism of the one-pot reaction scheme.

[0030] In some embodiments, under the optimized conditions, the specificity of the method was studied by fluorescence measurement and visual detection. One consequence of the short length of miRNA is that the sequences of mature miRNAs are very similar. Under the same experimental conditions, the fluorescence intensity of miRNA-155, miRNA-10b, miRNA-224, let-7a, let-7b and let-7c was very low compared with the high fluorescence intensity of miRNA-21. Similarly, the high homology of miRNA sequences also brings difficulties to accurate analysis. Effective identification of microRNAs with single-base mutations is of great significance for practical clinical applications. In order to test the sensitivity of this method, we synthesized three non-homologous microRNAs and three other homologous sequences as parallel controls. Among them, the mutation positions of mutant-11 and mutant-12 are located at both ends of the junction. However, no matter where the base mutation is located, no obvious signal is generated in the mutant group. In addition, the other signal intensities of microRNA are also much lower than those of the miRNA-21 group. These results fully demonstrate that this method has excellent specificity and can effectively identify single-base mutations. These results indicate that the integrated LRPA-CRISPR assay has excellent specificity in detecting miRNA-21.

[0031] 8. The method according to any one of claims 1-7, wherein in order to evaluate the practical application of this method, in some embodiments, the expression level of miRNA-21 in plasma samples and cell samples of breast cancer patients is analyzed using this detection method. First, miRNA-21 in four different cell line samples was measured using our method and RT-qPCR, including hepatocellular carcinoma cells (HepG2), breast cancer cell lines (MCF-7), cervical tumor cell lines (HeLa) and normal human breast epithelial cells (MCF-10A). The experimental results showed that the concentration of miRNA-21 in HepG2 cell line, MCF-7 cell line and HeLa cell line samples was significantly higher than that in MCF-10A cell line samples.

[0032] In some embodiments, the detection method is used to analyze miRNA-21 in serum samples, and total RNA is extracted from plasma samples of 8 breast cancer patients and 8 normal healthy people using a commercial RNA extraction kit, miRNA-21 is measured, and then the fluorescence signal is measured in real time. Subsequently, we estimate the concentration of miRNA-21 in each serum sample based on the standard curve drawn based on the relationship between miRNA concentration and fluorescence intensity. The results measured by the method are highly consistent with the clinical standard fluorescence RT-qPCR technology, and the signal intensity of breast cancer samples is higher than that of healthy samples.

[0033] Compared with the prior art, the one-pot detection of microRNA by the fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cleavage described in the present invention has the following advantages:

[0034] (1) The one-pot detection scheme described in the present invention greatly simplifies the operation and design process, the reaction time is short, and the operation is simple.

[0035] (2) The RPA signal amplification strategy based on ligation recognition RPA amplification and Cas12a cis-cleavage fluorescent RNA aptamer biosensor described in the present invention can achieve sensitive detection of miRNA-21 with a detection limit of 3.43aM.

[0036] (3) The fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cutting described in the present invention can effectively distinguish homologous sequences with single-base mutations through specific T4-DNA ligase, and can achieve specific detection of microRNA.

[0037] (4) The fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cleavage described in the present invention does not require fluorescent labeling, thereby reducing costs.

[0038] (5) The present invention discloses a fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cleavage, which not only has excellent stability in complex samples, but also can successfully analyze miRNA-21 in tumor cells and clinical patient serum samples. Using a one-pot approach, this method can become an effective strategy for simple and rapid miRNA sensors and is widely used in research and clinics. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a basic principle diagram of the present invention using a fluorescent RNA aptamer biosensor based on ligation recognition RPA amplification and Cas12a cis-cleavage for one-pot detection of miRNA-21.

[0040] Figure 2 Figure A in the middle is a schematic diagram of the design of the Cas12a cis-cutting system and the fluorescence signal output mechanism. Figure B is a 10% non-denaturing gel electrophoresis verification of the ligation-recognition trigger RPA system. Figure C is a 10% non-denaturing gel electrophoresis verification of the Cas12a cis-cutting activation Functional Mango system. Figure D is a comparative analysis of fluorescence spectra under different reaction component conditions.

[0041] Figure 3 Non-denaturing gel electrophoresis characterization of recombinase polymerase amplification (RPA) used for detection at different reaction times.

[0042] Figure 4 Figure A in the middle shows the secondary structure of Anti-Mango-crRNA predicted using NUPACK. Figure B shows the secondary structure of Functional Mango RNA predicted using NUPACK.

[0043] Figure 5 Figure A is a schematic diagram of the steps of the three-step method for detecting miRNA-21. Figure B is a schematic diagram of the detection procedure of the method for detecting miRNA-21 in a one-pot method. Figure C is the kinetic real-time fluorescence response of the three-step sensor to miRNA-21. Figure D is the kinetic real-time fluorescence response of the sensor to miRNA-21.

[0044] Figure 6 To optimize the experimental conditions of one-pot detection of different reaction buffers, the effects of three different reaction buffers on the detection performance were compared.

[0045] Figure 7 To optimize the experimental conditions for one-pot detection. Figure 6 Figure A shows the optimized Mg 2+ Figure B is the optimized reaction time. Figure C is the optimized reaction temperature. Figure D is the optimized Cas12a concentration. The error bars are the calculated results of three parallel experiments.

[0046] Figure 8 To optimize the experimental conditions for one-pot detection. Figure 7 Figure A shows the optimized transcription temperature. Figure B shows the optimized T7 RNA polymerase concentration. Figure C shows the optimized T4 DNA polymerase concentration. Figure D shows the optimized T4 DNA ligase concentration. Error bars are calculated from three parallel experiments.

[0047] Fig. 9 This is a feasibility analysis of one-pot detection. Figure 8 Figure A shows the fluorescence spectra of miRNA-21 at different concentrations. Figure B shows the fluorescence intensity of miRNA-21 at different concentrations. Figure C shows the linear relationship between Mango fluorescence intensity at 540nm and miRNA-21 concentration from 100aM to 100pM. Figure D shows the specificity study of miRNA-21 sequence detection by one-pot method.

[0048] Fig.10 This is a heat map showing the specificity of one-pot detection of miRNA-21 using probe point mutations. The base differences are marked in red.

[0049] Fig.11 To test the practical application of detecting miRNA-21 in different cell lysates. Fig.10Figure A in the middle shows the real-time fluorescence monitoring of different concentrations of miRNA-21 (10aM, 100aM, 1pM, 10pM and 100pM) using RT-qPCR. Figure B shows the linear relationship between the Ct value and the logarithm of the miRNA-21 concentration in the range of 10aM to 100pM.

[0050] Fig.12 To evaluate the expression level of miRNA-21 extracted from HepG2, MCF-7, HeLa, and MCF-10A cell lines by RT-qPCR.

[0051] Fig.13 This one-pot method is used for clinical application of amplification-free miRNA detection in clinical samples. Fig.12 Figure A shows a schematic diagram of the detection of miRNAs in human plasma samples and cell samples using this method. The illustrations were created using BioRender.com. Figure B shows the RT-qPCR detection of miRNA-21 extracted from HepG2, MCF-7, HeLa, and MCF-10A cell lines and the comparison of this method in biosensing applications. Figure C shows a heat map of the target miRNA-21 concentrations estimated by this method and the traditional RT-qPCR method. Figure D shows the miRNA-21 expression levels of patients and healthy donors analyzed by this method (blue) and RT-qPCR (black). The center line represents the median of the expression level, the boundaries of the box represent the interquartile range, and the whiskers represent the maximum and minimum values. DETAILED DESCRIPTION

[0052] Sequence Listing:

[0053]

[0054]

[0055] The technical solution of the present invention will be described clearly and completely below in combination with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, ordinary technicians in this field can follow the implementation without creative work, and all other embodiments obtained by them belong to the scope of protection of the present invention.

[0056] (1) Experimental reagents:

[0057] AmpliScribe T7 Flash Transcription Kit was purchased from Epicentre (WI, USA). T4 DNA ligase, DNA polymerase, Cas12a, and dNTPs were purchased from New England BioLabs (MA, USA). TwistAmpBasic Kit for RPA reaction was purchased from TwistDx Limited (Maidenhead, UK). 96-well plates were purchased from Greiner Bio-One (Shanghai, China). Diethyl carbonate (DEPC) water, non-denaturing PAGE kit, 4SGelRed dye, TBE buffer, TRE reagent, DNA labeling (25-500 bp), UNIQ-10 column Trizol total RNA purification kit (SK1321), and RNase inhibitor were purchased from Shanghai Sangon Biotechnology Co., Ltd. Ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai). miRNA Universal SYBR qPCR Master Mix was purchased from Vazyme Biotechnology (Nanjing). All oligonucleotide sequences used in this study were synthesized by Sangon and purified by HPLC. Loading buffer (10×) was purchased from TaKaRa Biotechnology (Dalian, China). TO1-Biotin was purchased from Applied Biological Materials Inc. Human breast cancer cell lines MCF-7, HeLa, and HepG2 were purchased from the Central Laboratory Cell Bank of Xiangya Hospital (Changsha, China), and MCF-10A cells were purchased from the Cell Bank of Type Culture Committee of the Chinese Academy of Sciences (Beijing, China);

[0058] (2) Ligation activation and recombinase amplification system

[0059] The proximity-induced ligation reaction was performed in a 20 μL mixture, which included 2 μL 10×T4 DNA ligase buffer, different concentrations of miRNA extracts, 1 μL 4 μM Mango probe, 1 μL 4 μM Anti-Mango probe, and 16 μL ddH2O. The mixed solution was annealed at 95°C for 5 minutes and then immediately placed in ice. Then, 1 μL T4 DNA ligase (200 U / μL) was mixed and incubated at 37°C for 15 minutes to complete the ligation reaction. The ligation product was polymerized using DNA polymerase. Specifically, 1 μL DNA polymerase (8 U / μL), 3 μL 10× DNA polymerase buffer, 1 μL MgSO4 (100 mM), and 5 μL ddH2O were added to the 20 μL ligation reaction solution to obtain 30 μL of polymerization solution, which was then incubated at 37°C for 15 minutes. Then mix 10 μL of the ligation product with 20 μL of RPA reaction system, which contains 5 μL ERA, 3 μL RPA reaction buffer, 1 μL RPA forward primer (4 μM), 1 μL RPA reverse primer (4 μM) and 10 μL H2O. Refer to the instructions of the kit for specific operations. Incubate the mixed system at 37°C for 20 minutes to obtain a large amount of specific amplification products;

[0060] (3) Self-supply crRNA for Cas12a cis-cleavage

[0061] CRISPR / Cas12a cis-cleavage experiments were performed using RPA amplicon products treated with a ligation recognition activation RPA system. In brief, the reaction was performed in 3 μl of 10×NEBuffer r2.1 and 3 μl of 10×T7 RNA transcription buffer, which contained: 1 μl of 2mM NTP (10mM each NTP), 1 μl of T7 RNA polymerase (1U / μl), 1 μl of RNase inhibitor (2U / μl) and 1 μl of Cas12a (100nM). The total volume of the complex was 30 μl and incubated at 37°C for 30 minutes. The complex was then mixed with the RNA synthase dye molecule TO1-Biotin (10 μM) in a buffer solution (10 μL, 40mM HEPES, pH=7.4, 100mM KCl, 10mM MgCl2). Technical replicates of each reaction were performed in 96-well plates, and the Mango fluorescence of the reaction was observed using the fluorescence monitoring function (495 / 515 nm) on a microplate reader (BioTek, Gen5 3.02. Fisher Scientific BTNEO2M);

[0062] (4) Steps of one-pot experiment

[0063] In the three-step protocol, 1 μL of 4 μM Mango primer and 1 μL of 4 μM Anti-Mango primer, 1 μL of 2 μM miRNA-21, 1 μL of 20 U / μL T4 DNA ligase and 1 μL of 0.5 U / μL DNA polymerase, 2 μL of 10×T4 DNA ligase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH = 7.5), 2 μL of 10×NEBuffer r2.1 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 1000 μg / ml recombinant albumin, pH = 7.9), and 11 μL of ddH2O were added to 20 μL of the solution and then kept at 16°C for 30 minutes. Then, the reaction solution was heated to 65°C for 10 minutes to inactivate T4 DNA ligase and DNA polymerase. Then mix 1 connection mixture with 20μl RPA reaction system, the RPA reaction system contains 5μL ERA, 3μL RPA reaction buffer, 1μL RPA forward primer (4μM), 1μL RPA reverse primer (4μM) and 10μL H2O. Refer to the instructions for the kit for specific operations. The mixed system is incubated at 37°C for 20 minutes to obtain a large amount of specific amplification products. The specific primer sequences used in this experiment are shown in the table. The last step is the same as the Cas12a cis-cutting system method, which is carried out at 37°C for 2 hours in a microplate reader and monitored every 1 minute.

[0064] In the one-pot protocol, a mixed reaction of 30 μL solution was set up, which contained the ligation recognition polymerization reaction, RPA and Cas12a cis-cleavage system. The entire reaction was mixed in 3 μL 10×T4 DNA ligase buffer, 3 μL 10×NEBufferr2.1, 3 μL 10×T7 RNA polymerization buffer, 3 μL RPA reaction buffer, 1 μL miRNA extract of different concentrations, 1 μL 4 μM Mango primer, 1 μL 4 μM Anti-Mango primer, 5 μL ERA, 1 μL RPA forward primer (4 μM), 1 μL RPA reverse primer (4 μM), 1 μL 2 mM NTPs (10 mM each NTP), 1 μL T4 DNA ligase (20 U / μL), 1 μL DNA polymerase (0.5 U / μL), 1 μL T7 RNA polymerase (1 U / μL), 1 μL Cas12a (100 nM), 1 μL RNase inhibitor (2 U / μL) and 2 μL ddH2O, and incubated at 37°C for 60 min to complete the one-pot reaction. Then, 30 μL of the complex was mixed with the fluorescent slow RNA aptamer dye TO1-Biotin (10 μM), and the reaction was carried out in a microplate reader at 37 °C for 2 h and monitored every 1 min;

[0065] (5) In vitro fluorescence assay

[0066] The transcription product was mixed with TO1-Biotin (10 μM) in a buffer solution (100 μL, 40 mM HEPES, pH = 7.4, 100 mM KCl, 10 mM MgCl2) and reacted at room temperature for 20 minutes. Fluorescence was measured at the corresponding excitation and emission wavelengths. The fluorescence signal was measured using an F-4600 fluorescence spectrophotometer. The experiment used a quartz cuvette with an optical path of 1.0 mm as a measuring container. The excitation wavelength of the fluorophore was 495 nm, the emission wavelength was 515-615 nm, and the slit width of the excitation and emission light was set to 5.0 nm. All experimental processes were carried out at room temperature;

[0067] (6) Native polyacrylamide gel electrophoresis (PAGE) analysis

[0068] Prepare 10% PAGE gel for electrophoresis analysis. 4 mL of ultrapure water, 100 μL of APS (10%), 2 mL of 5×TBE buffer, 4 mL of 30% Acr-bis (29:1) and 6 μL of TEMED were mixed and polymerized at room temperature for 1 hour, and then 1 μL of the resulting solution was mixed with 1 μL of 10× loading buffer and 8 μL of H2O to prepare the loading sample. Subsequently, electrophoresis was performed at 100 V in 1×TBE buffer for 90 minutes. The gel was then incubated in 4S GelRed dye (1 μg / mL) for 10 minutes and stained. The stained bands were imaged using 4S GelRed dye under ultraviolet excitation using the Tocan 240 gel imaging system (Shanghai Lingcheng Biotechnology Co., Ltd.);

[0069] (7) Detection steps of miRNA-21 in cells and serum samples

[0070] First, total RNA was extracted from different cell lines, healthy subjects, and sera of breast cancer patients according to the above method. Then, 2 μg of total miRNA was extracted from each sample and added to a total volume of 50 μL reaction system, which contained 1 μL 4 μM Mango probe, 1 μL 4 μM Anti-Mango probe, 1 μL 4 μM RPA forward primer and reverse primer, 0.1 mM dNTPs, 1 μL 20 U / μL DNA polymerase, 1 μL 20 U / μL T4 DNA ligase, 0.5 μL 5 U / μL recombinase, 5 μL 10×NEBuffer r2.1, and 5 μL 10×NEBuffer r3.1. Then, the solution was incubated at 16°C for ligation recognition activation RPA system reaction. After 40 minutes, 100nM Cas12a, 2mM rNTP (10mM each NTP), 1μL of 1U / μL T7 RNA polymerase, 1μL of 2U / μL Rnase inhibitor and RNA dye molecule TO1-Biotin (10μM) were added to the buffer to make the volume reach 100μL. Then, the self-supplied crRNA CRISPR / Cas12a cis-cutting system was reacted at 37°C for 30 minutes. The fluorescence intensity at 515nm (excitation 495nm) was measured using F-4600 or QuantStudio3;

[0071] (8) Steps for extracting total RNA from real samples

[0072] According to the product manual, total miRNA was isolated from cells and human serum samples using TRE reagent. First, add 1mL of TRE reagent to the real sample and mix thoroughly, then add 2 / 5 volume of RNase-free ddH2O to the above lysate and let stand at room temperature for 5 minutes. After centrifugation at 12000rpm for 15 minutes, aspirate the upper aqueous phase and transfer it to a clean centrifuge tube, then add an equal volume of isopropanol, mix and let stand at room temperature for 20 minutes. Then centrifuge at 12000rpm for 10 minutes and discard the supernatant. Add 1mL of 75% ethanol for washing and precipitation, then centrifuge at 12000rpm for 3 minutes and discard the supernatant. Finally, dry the RNA at room temperature for 5 minutes, then add 30μL of RNase-free ddH2O to completely dissolve the RNA. The resulting total RNA solution was stored at -80°C for subsequent experiments;

[0073] (9) Detection of the relative expression of miRNA-21 in different cell lines by reverse transcription real-time quantitative PCR (RT-qPCR)

[0074] cDNA was prepared using the miRNA 1st Strand cDNA Synthsis Kit (stem-loop). The reaction process was as follows: the extracted total RNA (2 μg) was added to the reaction system containing 10 μL of 2×miRNA P-RT mixed solution and 2 μL of miRNA P-RT enzyme mixture. DEPC-H2O was added to the solution to make the volume reach 20 μL. The solution was then incubated at 37°C for 60 minutes and then at 85°C for 5 minutes to inactivate the reverse transcriptase. The cDNA samples were stored at 4°C for future use. RT-qPCR was performed on QuantStudio 3 (Applied Biosystems), and the miRNA Universal SYBR qPCR Master Mix was used according to the protocol. U6 was used as an internal reference for miRNA-21. The 20 μL reaction solution contained 1 μL cDNA sample, 10 μL 2× miRNA qPCR master solution, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), and 8 μL nuclease-free water. The qPCR process was as follows: incubation at 95 °C for 30 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 30 seconds. Primers (miRNA-21 and U6) were synthesized by Sangon Biotechnology (Shanghai, China). The relative expression of miRNA-21 was calculated using 2 -ΔΔCt Method calculation;

[0075] (10) Cell culture

[0076] MCF-7, HeLa, and HepG2 cells were cultured in Dulbecco's modified Eagle's (DMEM, Invitrogen, US) containing 10% fetal bovine serum (FBS, Gibco, US) and 1% penicillin-streptomycin (Invitrogen, US). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The MCF-10A cell line represents non-tumorigenic human breast cells and is routinely cultured in low-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 mg / mL streptomycin, stabilized glutamine, and sodium pyruvate. To prevent mycoplasma contamination, 1× Mycokill was added to the growth medium. Cells were cultured at 37°C in a humidified atmosphere containing 5% (v / v) CO2. The growth medium of MCF-10A cells was refreshed every 2-3 days, and the cell line was used for subsequent experiments after culture.

[0077] Note: Serum samples of breast cancer patients and normal subjects were obtained from Changsha Maternal and Child Health Hospital and the Hunan Provincial Key Laboratory for Regional Hereditary Birth Defect Prevention and Control of Changsha Maternal and Child Health Hospital Affiliated to Hunan Normal University, which has been approved by the Ethics Committee of Changsha Maternal and Child Health Hospital Affiliated to Hunan Normal University (No.: EC-20240828-09).

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for one-pot detection of microRNA based on RPA-Cas12a cis-cleavage fluorescent RNA aptamer biosensor, characterized in that: The detection method comprises the following steps: (1) Designing Mango probes and Anti-Mango probes that can specifically recognize microRNA, wherein the Mango probe contains a complementary sequence and a Mango sequence that specifically recognize miRNA-21, and the Anti-Mango probe contains another complementary sequence that specifically recognizes miRNA-21, an Anti-Mango sequence, and a crRNA sequence; (2) Add the probe and the corresponding target miRNA-21 into the ligation recognition amplification reaction system, and the miRNA-21 hybridizes with the Mango probe and the Anti-Mango probe to form a three-strand complex. The connection characteristics of T4-DNA ligase are used to detect miRNA-21 by the connection reaction principle of the Mango probe and the Anti-Mango probe. The Mango probe with its own primer and the Anti-Mango probe complement each other under the action of DNA polymerase to form a complete DNA double strand; (3) The double-stranded DNA in step (2) is amplified by RPA to introduce the T7 promoter into the RPA amplicon. The 3' end of these forward primers is designed with a T7 sequence, which can not only promote the multiple amplification of double-stranded DNA amplicons by RPA, but also serve as the primer site of RPA. Therefore, DNA polymerase can promote the bidirectional extension of the forward primer and the reverse primer, thereby generating a large number of dsDNA amplicons with T7 promoter, PAM sequence and sequence that can be recognized by crRNA; (4) T7 RNA polymerase, rNTPs and Cas12a protein are added, where Anti-Mango-crRNA will be transcribed from the complete double-stranded DNA amplicon, processed into mature crRNA under the treatment of Cas12a protein and provided to Cas12a cis-cleavage reaction; (5) The exponentially amplified RPA amplicons can activate the target-dependent cis-cleavage mechanism of CRISPR / Cas12a, simultaneously linking highly specific target recognition and highly cis-cleavage of dsDNA amplicons; (6) At the same time, the cut double-stranded DNA amplicon is transcribed into the Functional Mango RNA aptamer under the action of T7 RNA polymerase. The Functional Mango RNA aptamer / TO1-Biotin dye complex serves as an output signal, which can further show the difference between the presence and absence of the miRNA-21 analyte.

2. The design of the Mango probe and Anti-Mango probe according to claim 1 is characterized in that The specificity of base complementary pairing is used to detect miRNA-21. In the absence of a target, the Mango probe and Anti-Mango probe cannot be ligated by T4 DNA ligase. Once miRNA-21 binds to the target recognition site of the probe, T4 DNA ligase can activate the ligation reaction.

3. The preparation method according to claim 1-2, characterized in that: The use of RPA double-stranded DNA amplification mechanism can effectively avoid the influence of Cas12a trans-cutting.

4. The preparation method according to claims 1-3, characterized in that: The Cas12a cis-cutting system is used to regulate the transcription of Functional Mango RNA aptamers for fluorescent signal output.

5. A method comprising using the miRNA-21 described in any one of claims 1 to 4 as a target for targeting Mango probe and Anti-Mango probe.

6. The method of claim 1 wherein the three-step detection process is optimized into an efficient one-pot detection process.

7. A rapid, universal, specific and sensitive RNA detection platform comprising any one of claims 1 to 6 for diagnosis of cancer cells and serum samples of patients.

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