Method for detecting breast cancer miR-155 marker by allosteric ribozyme driven crRNA switch sensor

Abstract

The invention provides a method for detecting a breast cancer miR-155 marker by using an allosteric ribozyme driven crRNA switch sensor. The sensor is mainly composed of two modules, namely a self-locking inactive Locked crRNA module and an allosteric hammerhead ribozyme module. The Locked crRNA is designed into a hairpin structure with a destroyed repeated region by extending the 3'end of the crRNA. The extended sequence effectively inhibits the assembly of the crRNA and the Cas12a, so that the activity of the CRISPR / Cas12a is inactivated. The design of the allosteric hammerhead ribozyme comprises an allosteric module for target detection and a hammerhead ribozyme sequence. In the absence of miR-155, a prolonged target recognition fragment at the 5'end of hammerhead ribozyme is complementarily paired with a basic group of a flanking arm part, so that the trans-lysis activity of the ribozyme is inhibited; under the condition that miR-155 exists, allosteric hammerhead ribozyme is converted into active conformation, then Locked crRNA is subjected to catalytic cracking in a sequence specificity mode to release natural crRNA, and a CRISPR / Cas12a system is activated to output a detection signal, so that sensitive detection of the breast cancer miR-155 marker is realized. The method is low in background interference, high in specificity, easy to operate and programmable, can be applied to quantitative detection of the miR-155 marker in serum of cancer cells and breast cancer, and has a wide application prospect in field detection and biomedical research.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis, and in particular relates to a method for detecting a breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor. Background Art

[0002] MiR-155 is a multifunctional miRNA that plays an important role in a variety of biological processes. It plays a key role in the body's hematopoiesis, immune response, muscle development, and fat differentiation. In the field of oncology, miR-155 has attracted particular attention because its expression level is abnormal in a variety of malignant tumors, including breast cancer, and is closely related to the occurrence, invasion, and metastasis of tumors. In 2024, the Nobel Prize in Physiology or Medicine was awarded to scientists who have made outstanding contributions to the field of miRNA research. They revealed a new principle of gene regulation and provided new strategies for disease diagnosis and treatment. At present, the detection methods of miRNA mainly include real-time fluorescence quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), microarray, and Northern blot. Although these methods are highly sensitive, the operation process is cumbersome and costly. Therefore, the development of a simpler and more cost-effective miRNA detection technology has become an urgent problem that needs to be solved.

[0003] The development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) has set off a research boom in the field of medical diagnosis with its advantages such as high specificity and programmability. In particular, type 2 V CRISPR / Cas12a can bind to the guide CRISPR RNA (crRNA) and indiscriminately cleave non-target ssDNA after specifically recognizing double-stranded DNA (dsDNA) containing the adjacent motif (protospacer and acent motif, PAM), showing significant reverse cleavage ability. However, the limitation of the CRISPR / Cas12a system is that it cannot directly recognize RNA sequences, which leads to many biosensors usually using reverse transcription and template amplification technologies to improve the sensitivity and accuracy of detection, such as recombinase polymerase amplification (RPA), rolling cycle amplification (RCA), exponential amplification reaction (EXPAR), etc. These isothermal amplification technologies improve the detection sensitivity and can convert RNA targets into DNA products. However, the complex amplification process is not only time-consuming, but also prone to non-specific amplification due to non-target or aerosol contamination, thus affecting the reproducibility and reliability of the analysis results. In addition, the CRISPR / Cas12a system also relies on signal transduction strategies to detect non-nucleic acid targets, such as small molecules, proteins, and pathogens. However, the existing information conversion strategies are limited to target detection related to DNA enzymes and aptamers, and there is still the problem of insufficient specificity. Therefore, screening more molecular recognition tools with better specificity is an important part of achieving rapid on-site detection in future biomedicine and treatment.

[0004] Hammerhead ribozymes are small RNA molecules that can catalyze specific RNA cleavage reactions. It has a unique secondary structure consisting of a central catalytic core and two arms that bind to the substrate RNA through base pairing and cut the adjacent sequence NUH↓ (N is any base, H is A, C or U). Hammerhead ribozymes have shown broad application potential in the field of mRNA therapy due to their high sequence specificity and catalytic efficiency, and are being used to develop innovative therapies for specific genetic diseases, viral infections and cancers. In addition, hammerhead ribozymes are also being combined with CRISPR technology to improve the accuracy and efficiency of gene editing by cutting specific RNA molecules. Utilizing the RNA cleavage activity of hammerhead ribozymes, CRISPR-based detection strategies can eliminate the nucleic acid amplification step, providing the possibility of developing rapid on-site detection methods. Summary of the invention

[0005] The present invention aims to overcome the problems of cumbersome operation steps, high cost, insufficient specificity and sensitivity in the existing miR-155 detection, and provides a method for detecting breast cancer miR-155 markers by driving a crRNA switch sensor with an allosteric ribozyme. The allosteric ribozyme is carefully designed to couple the target recognition sequence with the 5' flanking arm of the hammerhead ribozyme to form a hairpin structure through hybridization. The presence of the target molecule induces conformational changes in the allosteric module and destroys the hairpin structure, restoring the ribozyme catalytic RNA cleavage activity. Then, the activated ribozyme specifically cuts the restriction site of the locked crRNA hairpin structure, releases the natural crRNA and activates the CRISPR / Cas12a function for signal reporting. Thereby achieving sensitive detection of breast cancer miR-155 markers.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect of the present invention, an allosteric ribozyme driven crRNA switch sensor is provided, wherein the sensor comprises BR miR-155 , substrate Locked crRNA, Lba Cas12a, double-stranded DNA activator and FQ Reporter; wherein the BR miR-155 The invention relates to an allosteric hammerhead ribozyme that specifically recognizes miR-155 (sequence: 5'-ACC CCU AUC ACG AUU AGC AUUAAU CGU GAU CUG AUG AGU CCG GUA GCG GAC GAA AUG UUU G-3', SEQ ID NO: 1); the substrate Locked crRNA contains a ribozyme cleavage site (sequence: 5'-UAA UUU CUA CUA AGU GUA GAU CAA GGAACU GAU UAC AAA CAU AAU CAC GAA UCU ACA CGG CGU AGA-3', SEQ ID NO: 2); and the double-stranded DNA activator (sequence: 5'-GAT CTA TTG TCT TGT TTG TAA TCA GTT CCT TGT AAA TAT TGAATT TGA TAT GAC AGA GT-3', SEQ ID NO: 3).

[0008] A preferred technical solution is that the substrate Locked crRNA contains a ribozyme cleavage site NUH (N is any base, H is A, C or U).

[0009] In a preferred technical solution, the sequence of the FQ Reporter is as follows: 5'-FAM-TTATT-BHQ-3'.

[0010] The second aspect of the present invention provides a method for detecting the breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor as described in the first aspect, comprising the following steps:

[0011] S1, the BR miR-155 After mixing with miR-155 solution and incubating, miR-155-BR is formed through hybridization reaction. miR-155 complex, restoring the cleavage activity of the hammerhead ribozyme;

[0012] S2, miR-155-BR in step S1 miR-155 The complex and the substrate Locked crRNA are mixed evenly, and the hammerhead ribozyme recognizes and cleaves the substrate to obtain the crRNA cleavage product;

[0013] S3, LbaCas12a is mixed with the hammerhead ribozyme cleavage product crRNA of step S2, and then the reaction mixture is added to continue incubation to perform CRISPR / Cas12a cleavage reaction;

[0014] S4. The reaction product of step S3 is used to detect the fluorescence signal by F7000 fluorescence spectrophotometer.

[0015] According to a preferred technical solution, in step S1, the hybridization reaction buffer contains 10 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 100 mM KCl; the reaction temperature is 37°C; and the reaction time is 30 min.

[0016] According to a preferred technical solution, in step S2, the ribozyme cleavage reaction temperature is 25° C. and the reaction time is 1 h.

[0017] A preferred technical solution, in step S3, the reaction mixture includes 1×NEBuffer r2.1, 50nM LbaCas12a, 25nM dsDNA, 50nM FQ Reporter; the reaction temperature is 37°C; and the reaction time is 40min.

[0018] According to a preferred technical solution, in step S4, the fluorescence detection setting parameters are: excitation wavelength 480 nm, emission wavelength 500 nm, and slit width 5 nm.

[0019] The third aspect of the present invention provides an application of the allosteric ribozyme driven crRNA switch sensor as described in the first aspect in the detection of miR-155.

[0020] In a preferred technical solution, the samples tested include cancer cells, normal cells, serum of breast cancer patients and normal serum.

[0021] Compared with the prior art, the method of detecting breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor has the following beneficial effects:

[0022] (1) The present invention provides a method for detecting breast cancer miR-155 marker using an allosteric ribozyme-driven crRNA switch sensor, which is easy to operate and reduces the involvement of primers, thereby preventing nonspecific recognition caused by off-target amplification.

[0023] (2) The present invention provides a method for detecting the breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor, which has low background interference and the special modification of crRNA effectively avoids the risk of background signal leakage.

[0024] (3) The present invention provides a method for detecting the breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor. The method has strong specificity. The ribozyme specifically cuts RNA and initiates CRISPR, further improving the accuracy of gene editing.

[0025] (4) The present invention provides a method for detecting the breast cancer miR-155 marker using an allosteric ribozyme-driven crRNA switch sensor, which is programmable and can detect other biomarkers by flexibly replacing the sequence of the recognition element. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to further enhance the understanding of the present invention, the following is a brief introduction to the drawings required for the specific implementation method. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. In the drawings:

[0027] Figure 1 This is a schematic diagram of the principle of the allosteric ribozyme-driven crRNA switch sensor of the present invention for detecting the breast cancer miR-155 marker.

[0028] Figure 2 This is a graph showing the effect of different blocking lengths of Locked crRNA on inhibiting fluorescence signals.

[0029] Figure 3 This is a graph showing the results of an experiment to optimize the length of the flanking arms of the hammerhead ribozyme.

[0030] Figure 4 This is a diagram of the experimental results for optimizing the blocking region of the allosteric ribozyme.

[0031] Figure 5 The figure is a non-denaturing polyacrylamide gel electrophoresis diagram for verifying the feasibility of the method in the present invention.

[0032] Figure 6 This is a fluorescence spectrum diagram for verifying the feasibility of the method in the present invention.

[0033] Figure 7 This is a diagram showing the effects of potassium ion concentration and magnesium ion concentration on the sensor detection of miR-155.

[0034] Figure 8 is the ribozyme cleavage reaction time, temperature and BR miR-155 Graph comparing the concentration of Locked crRNA to the effect of sensor detection of miR-155.

[0035] Fig. 9 This is a sensitivity analysis diagram for detecting different concentrations of miR-155 in the present invention.

[0036] Fig.10 It is a nonlinear and linear relationship diagram fitted according to the fluorescence signal of miR-155 at different concentrations in the present invention.

[0037] Fig.11 It is a radar chart for verifying the specificity of the method in the present invention.

[0038] Fig.12 Real-time fluorescence response and linear relationship diagram of RT-qPCR analysis of different concentrations of miR-155 standards.

[0039] Fig.13 A is a real-time fluorescence response diagram of RT-qPCR analysis of different cell lines; B is a relative expression bar graph of miR-155 detection in different cell lines by the method of the present invention and RT-qPCR.

[0040] Fig.14 A is a heat map of the detection of miR-155 in the serum of breast cancer patients and normal people by the method of the present invention and RT-qPCR; B is a box plot of the detection of miR-155 in the serum of breast cancer patients and normal people by the method of the present invention and RT-qPCR. DETAILED DESCRIPTION

[0041] Sequence Listing:

[0042]

[0043]

[0044] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0046] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0047] The experimental methods in the following examples are all conventional methods unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0048] Example 1

[0049] A method for detecting breast cancer miR-155 marker by using an allosteric ribozyme-driven crRNA switch sensor, wherein the specific synthesis steps are as follows:

[0050] S1、miR-155-BR miR-155 Complex formation: 1.2 μL 200 μM BR miR-155 Mix with 1 μL of miR-155 solution of different concentrations, add 2 μL 100mM Tris-HCl (pH 7.6), 2 μL 100mM MgCl2, 2 μL 100mM KCl and 10.8 μL RNase-free ddH2O to the mixture. Incubate the mixture at 37°C for 30 minutes, and obtain miR-155-BR by hybridization reaction. miR-155 Complex;

[0051] S2. Acquisition of crRNA cleavage products: MiR-155-BR miR-155 The complex was mixed with 1 μL of 200 μM Locked crRNA and incubated at 25°C for 1 h to obtain the crRNA cleavage product obtained by the cleavage of the substrate by the allosteric hammerhead ribozyme;

[0052] S3, CRISPR / Cas12a cleavage reaction: 1 μL of the above cleavage mixture was mixed with 2 μL 10×NEBuffer r2.1, 1 μL 1 μM Lba Cas12a, 2 μL 250 nM dsDNA, 2 μL 500 nM FQ Reporter and 12 μL RNase-free ddH2O to a total volume of 20 μL. The reaction system was incubated at 37°C for 40 min.

[0053] S4. Collecting fluorescence signals: Collect the fluorescence spectrum of the mixture on a F7000 spectrophotometer. The excitation wavelength is 480 nm, the emission wavelength is 500 nm to 600 nm, and the slit width is 5 nm. The data analysis uses the FAM fluorescence intensity at 530 nm.

[0054] Example 2

[0055] Design and screening of allosteric ribozyme-driven crRNA switch sensor for detecting miR-155 markers in breast cancer.

[0056] (1) Test the degree to which locked crRNA with different blocking lengths inhibits fluorescence signals.

[0057] First, locked crRNAs with different stem lengths (5, 7, 9, 11, 13, and 15 nt) were prepared to verify the inhibition of Cas12a activity by crRNA switch. The preparation method was the same as steps S3 and S4 of Example 1. A blank sample was used as a control to calculate the fluorescence intensity ratio F / F0. The results are shown in Figure 2 B. Replace FQ Reporter with ssDNA and analyze using polyacrylamide gel electrophoresis. The results are shown in Figure 2 C.

[0058] from Figure 2 As shown in B, as the stem length increases from 5nt to 15nt, the fluorescence intensity F / F0 gradually decreases. When the stem length is greater than 13nt, the signal-to-noise ratio of Locked crRNA is the lowest. Figure 2 As can be seen in C, as the length of Locked crRNA decreases, the ssDNA substrate cleavage fragments gradually increase. Therefore, it is concluded that Locked crRNA (13nt) and Locked crRNA (15nt) can inhibit the activity of Cas12a. In order to avoid the stem being too stable due to the long blocking length, affecting the binding efficiency of the subsequent ribozyme flanking arm with the substrate, Locked crRNA (13nt) was finally determined as the cleavage substrate sequence of the ribozyme.

[0059] (2) Investigate the effects of hammerhead ribozymes with different flanking arm lengths on substrate cleavage.

[0060] Five hammerhead ribozymes with different lengths of flanking arms (3+7, 3+8, 5+7, 5+8, 7+7) were designed to cut Locked crRNA (13nt). 1.2μL 200μM HHR with different lengths of flanking arms was mixed with 1μL 200μM Locked crRNA solution, and 2μL 100mM Tris-HCl (pH 7.6), 2μL 100mM MgCl2, 2μL 100mM KCl and 11.8μL RNase-free ddH2O were added to the mixture. The mixed system was incubated at 25℃ for 1h, and the fluorescence signal was collected using F7000, and the fluorescence intensity ratio F / F0 was calculated. The results are shown in Figure 3 .

[0061] from Figure 3 As can be seen, HHR(7+7) has the highest signal-to-noise ratio, so HHR(7+7) was selected as the basic sequence for the design of allosteric ribozymes.

[0062] (3) Allosteric ribozyme structure optimization.

[0063] A series of allosteric ribozyme sequences containing different numbers of toes (0, 1, 2, 4, 6) were synthesized to cleave Locked crRNA (13nt). The preparation method was the same as steps S1-S3 of Example 1, and the real-time fluorescence response signal was collected using an ELISA instrument. The results are shown in Figure 4 .

[0064] from Figure 4 As can be seen from the figure, as the number of toes increases, the fluorescence intensity gradually increases, so BR is selected. miR-155 -5 sequence was used for subsequent detection of miR-155.

[0065] Example 3

[0066] Feasibility analysis of allosteric ribozyme-driven crRNA switch sensor for detecting miR-155 marker in breast cancer.

[0067] In order to investigate the feasibility of the method of the present invention for detecting miR-155, the results of polyacrylamide gel electrophoresis were analyzed. Figure 5 . Then the fluorescence spectrum technology was used for analysis, and the results are shown in Figure 6 .

[0068] from Figure 5 As can be seen in Figure 2, when the target miR-155 and BR miR-155 During incubation, a low migration band (lane 3) was observed, indicating that BR miR-155 Can recognize and bind miR-155. miR-155The complex was incubated with Locked crRNA, and a clear band (lane 5) with faster electrophoretic migration was obtained. This band corresponds to the cleavage product of Locked crRNA under the ribozyme-catalyzed RNA cleavage activity. The undesigned ribozyme HHR was incubated with Locked crRNA, and a band with a molecular weight similar to the new band in lane 5 was obtained. This preliminary confirmation of BR miR-155 Activated to cut Locked crRNA and release crRNA.

[0069] from Figure 6 As can be seen in the figure, in the absence of miR-155 (b, green area), the observed fluorescence signal is negligible compared to the HHR cleavage Locked crRNA output signal (d, purple area). In contrast, the introduction of miR-155 can drive the CRISPR / Cas12a response, and the signal (c, blue area) is restored by about 87.9% compared to group d. This confirms that crRNA switching can be activated by BR miR-155 to regulate the conformational transition.

[0070] Example 4

[0071] The effects of different reaction conditions on the allosteric ribozyme-driven crRNA switch sensor for detecting the miR-155 marker in breast cancer were tested.

[0072] (1) Optimization of potassium ion concentration

[0073] The preparation method of the sensor is the same as that of Example 1, except that the concentration of potassium ions in the buffer solution is changed to 1, 10, 50, 100, and 500 mM. Figure 7 A.

[0074] from Figure 7 As can be seen in A, the fluorescence signal is highest when the potassium ion concentration is 100 mM.

[0075] (2) Optimization of magnesium ion concentration

[0076] The preparation method of the sensor is the same as that of Example 1, except that the concentration of magnesium ions in the buffer solution is changed to 0.1, 0.5, 1, 10, and 100 mM. Figure 7 B.

[0077] from Figure 7 As can be seen in B, the fluorescence signal is highest when the magnesium ion concentration is 10 mM.

[0078] (3) Optimization of ribozyme cleavage reaction time

[0079] The preparation method of the sensor was the same as that of Example 1, except that the ribozyme cleavage reaction time was changed to 5, 15, 30, 45, and 60 min. Figure 8 A.

[0080] from Figure 8 As shown in A, the signal-to-noise ratio was the highest and the fluorescence signal was the best when the ribozyme cleavage reaction time was 60 min.

[0081] (4) Optimization of ribozyme cleavage reaction temperature

[0082] The preparation method of the sensor is the same as that of Example 1, except that the ribozyme cleavage reaction temperature is changed to 15, 25, 37, 45, and 55°C. Figure 8 B.

[0083] from Figure 8 As can be seen in B, the signal-to-noise ratio is the highest and the fluorescence signal is the best when the ribozyme cleavage reaction temperature is 25°C.

[0084] (5)BR miR-155 Optimization of reaction concentration ratio with Locked crRNA

[0085] The preparation method of the sensor is the same as that of Example 1, except that BR miR-155 The reaction concentration ratio with Locked crRNA was 0.5:1, 1:1, 1.2:1, 2:1, and 10:1. The results are shown in Figure 8 C.

[0086] from Figure 8 As can be seen from C, BR miR-155 When the reaction concentration ratio with Locked crRNA was 1.2:1, the signal-to-noise ratio was the highest and the fluorescence signal was the best.

[0087] Example 5

[0088] Performance analysis experiments of allosteric ribozyme-driven crRNA switch sensors.

[0089] (1) Sensitivity test

[0090] The preparation method of the allosteric ribozyme driven crRNA switch sensor is the same as that of Example 1, except that the concentration of the target molecule miR-155 is changed to 0.01, 0.05, 0.1, 0.5, 1.5, 10, 40, and 100 nM. Fig. 9 .

[0091] from Fig. 9 As can be seen from the figure, as the concentration of miR-155 increases, the fluorescence intensity is positively correlated with the concentration of the target molecule.

[0092] (2) Establishment of standard curve

[0093] use Fig. 9 The fluorescence intensity corresponding to different miR-155 concentrations was fitted nonlinearly and linearly. The results are shown in Fig.10 .

[0094] from Fig.10 As can be seen from the figure, from 100 pM to 40 nM, there is a good linear relationship between the fluorescence intensity and the logarithmic value of miR-155 concentration, and the regression equation is F = 426.77LgC-68.18 (R 2 =0.9981), and the limit of detection (LOD) was 16 pM.

[0095] (3) Specificity detection

[0096] The preparation method of the allosteric ribozyme-driven crRNA switch sensor is the same as that in Example 1, except that the target molecule is changed to three cancer-related miRNAs (let-7a, miR-21, miR-16) or miR-155 mutants. Fig.11 .

[0097] from Fig.11 As can be seen from the figure, the fluorescence intensity of the miRNA with three base mutations and other interfering RNA sequences can be ignored like the background signal, indicating that the method of the present invention has excellent specificity.

[0098] Example 6

[0099] Physical application test of allosteric ribozyme-driven crRNA switch sensor to detect breast cancer miR-155 marker.

[0100] (1) Establishment of standard curve by RT-qPCR method

[0101] The miRNA First Strand cDNA Synthesis Kit (Stem-Loop Method) was used to prepare cDNA according to the product protocol. First, 20 μL of reaction solution contained 1 μL of different concentrations of miR-155 standard (0.01, 0.1, 1, 10, 100 nM), 2 μL of HiScript II Enzyme Mix, 2 μL of 10×RT Mix, 1 μL of 2 μM stem-loop primer, and 14 μL of RNase-free ddH2O. Then, the solution was incubated at 25°C for 5 min, 50°C for 15 min, and incubated at 85°C for 5 min to inactivate the reverse transcriptase. RT-qPCR was performed on a QuantStudio 3 real-time fluorescence quantitative PCR instrument using miRNA Universal SYBR qPCR Master Mix according to the protocol. The 20 μL reaction solution contained 1 μL cDNA sample, 10 μL 2×miRNA Universal SYBR qPCR Master Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM) and 8.2 μL ddH2O. The qPCR program was: incubate at 95°C for 5 min, incubate at 95°C for 10 s, incubate at 60°C for 30 s, for a total of 40 cycles. Real-time fluorescence signals were collected and the results are shown in Fig.12 A. According to Fig.12 A linear fit is performed on the CT value corresponding to the intersection of the threshold line A and the fluorescence spectrum. The results are shown in Fig.12 B.

[0102] from Fig.12 As shown in B, from 10 pM to 100 nM, there is a good linear relationship between the CT value and the logarithmic value of miR-155 concentration, and the regression equation is CT = -3.2866LgC + 39.152 (R 2 =0.9995).

[0103] (2) Analysis of different cell lines using allosteric ribozyme-driven crRNA switch sensors and gold standard RT-qPCR

[0104] According to the product manual, Trizol reagent was used to isolate total RNA from different cell lines (MCF-7, HeLa, MCF-10A). RT-qPCR analysis was performed on different cell lines as in Example 6 (1), with U6 as the internal reference for miRNA-155. The results are shown in Fig.13 A. The preparation method of Example 1 was used to detect miR-155 in different cell lines. The two analysis methods were compared. (-ΔΔCt) The relative expression of miR-155 was calculated by Fig.13 B.

[0105] from Fig.13As shown in A, the relative expression levels of MCF-7 cells and HeLa cells were significantly higher than those of MCF-10A cells.

[0106] from Fig.13 As can be seen in B, the relative expression levels of miR-155 measured by the two methods are almost the same, indicating that the method of the present invention can accurately detect miR-155 in cell samples.

[0107] (3) Clinical serum samples were analyzed using allosteric ribozyme-driven crRNA switch sensor and gold standard RT-qPCR.

[0108] Using the same method as in Example 6(2), miR-155 was detected in 5 normal serum samples and 10 breast cancer patient sera using the allosteric ribozyme-driven crRNA switch sensor and the gold standard RT-qPCR, and the standard curve constructed in Example 6(1) was used for quantification. The results are shown in Fig.14 A and 14B.

[0109] from Fig.14 A and Fig.14 As shown in B, the overall expression level of miR-155 in breast cancer patients is significantly higher than that in healthy individual samples. The measurement results show that the method of the present invention is highly consistent with the RT-qPCR results, indicating that the method of the present invention has potential applicability in detecting miR-155 in clinical applications.

[0110] Note: Serum specimens from breast cancer patients and normal subjects were obtained from the Hunan Metabolic Fatty Liver Disease Clinical Research Center, Clinical Research Institute, Nanhua Hospital Affiliated to Hengyang Medical College, University of South China, and approved by the Medical Ethics Committee of Nanhua Hospital Affiliated to University of South China (No. 2023-ky-163).

[0111] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An allosteric ribozyme driven crRNA switch sensor, characterized in that The sensor includes BR miR-155 , substrate Locked crRNA, Lba Cas12a, double-stranded DNA activator and FQ Reporter; wherein the BR miR-155 The allosteric hammerhead ribozyme specifically recognizes miR-155, and its sequence is shown in SEQ ID NO: 1; the substrate Locked crRNA contains a ribozyme cleavage site, and its sequence is shown in SEQ ID NO: 2; and the sequence of the double-stranded DNA activator is shown in SEQ ID NO:

3.

2. The sensor according to claim 1, characterized in that The substrate Locked crRNA contains a ribozyme cleavage site NUH (N is any base, H is A, C or U).

3. The sensor according to claim 1, characterized in that The sequence of the FQ Reporter is as follows: 5'-FAM-TTATT-BHQ-3'.

4. A method for detecting breast cancer miR-155 markers using an allosteric ribozyme-driven crRNA switch sensor, characterized in that: The following steps are involved: S1, the BR miR-155 After mixing with miR-155 solution and incubating, miR-155-BR was formed through hybridization reaction. miR-155 complex, restoring the cleavage activity of the hammerhead ribozyme; S2, miR-155-BR in step S1 miR-155 The complex and the substrate Locked crRNA are mixed evenly, and the hammerhead ribozyme recognizes and cleaves the substrate to obtain the crRNA cleavage product; S3, LbaCas12a is mixed with the hammerhead ribozyme cleavage product crRNA of step S2, and then the reaction mixture is added to continue incubation to perform CRISPR / Cas12a cleavage reaction; S4. The reaction product of step S3 is used to detect the fluorescence signal by F7000 fluorescence spectrophotometer.

5. The method according to claim 4, characterized in that In step S1, the hybridization reaction buffer contains 10 mM Tris-HCl (pH 7.6), 10 mM MgCl2, and 100 mM KCl; the reaction temperature is 37°C; and the reaction time is 30 min.

6. The method according to claim 4, characterized in that In step S2, the ribozyme cleavage reaction temperature is 25° C. and the time is 1 h.

7. The method according to claim 4, characterized in that In step S3, the reaction mixture includes 1×NEBufferr2.1, 50nM Lba Cas12a, 25nM dsDNA, and 50nM FQ Reporter; the reaction temperature is 37°C; and the reaction time is 40min.

8. The method according to claim 4, characterized in that In step S4, the fluorescence detection setting parameters are: excitation wavelength 480nm, emission wavelength 500nm, slit width 5nm.

9. Application of the method for detecting breast cancer miR-155 marker by an allosteric ribozyme driven crRNA switch sensor according to any one of claims 1 to 8 in miR-155 detection.

10. The use according to claim 9, characterized in that: The samples tested included cancer cells, normal cells, serum from breast cancer patients, and normal serum.

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