Method for detecting breast cancer miR-155 marker by using a conformational nucleic enzyme driven crRNA switch sensor

Abstract

The application provides a method for detecting breast cancer miR-155 markers by using a conformational ribozyme-driven crRNA switch sensor. The sensor is mainly composed of two modules of self-locking inactive Locked crRNA and allosteric hammerhead ribozyme. The Locked crRNA is designed into a hairpin structure with a destroyed repeat region by extending the 3' end of the crRNA. The extended sequence effectively inhibits the assembly of crRNA and Cas12a, thereby inactivating the CRISPR / Cas12a activity. The design of the allosteric hammerhead ribozyme contains an allosteric module for target detection and a hammerhead ribozyme sequence. In the absence of miR-155, the target recognition fragment at the 5' end of the hammerhead ribozyme is partially base-paired with the flanking arm, thereby inhibiting the trans-cleavage activity of the ribozyme; but in the presence of miR-155, the allosteric hammerhead ribozyme is converted into an active conformation, and then catalyzes the cleavage of the Locked crRNA in a sequence-specific manner to release the natural crRNA and activate the CRISPR / Cas12a system to output a detection signal, thereby realizing sensitive detection of breast cancer miR-155 markers. The method has low background interference, strong specificity, easy operability and programmability, and can be applied to quantitative detection of miR-155 markers in cancer cells and breast cancer serum, and has a wide application prospect in on-site detection and biomedical research.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a method for detecting breast cancer miR-155 biomarkers using an allosteric ribozyme-driven crRNA switch sensor. Background Technology

[0002] miR-155 is a multifunctional miRNA that plays a crucial role in various biological processes, including hematopoiesis, immune responses, muscle development, and adipogenesis. In oncology, miR-155 has received particular attention due to its abnormal expression levels in various malignant tumors, including breast cancer, and its close association with tumor development, invasion, and metastasis. In 2024, the Nobel Prize in Physiology or Medicine was awarded to scientists who made outstanding contributions to miRNA research, revealing a novel gene regulation mechanism that provides new strategies for disease diagnosis and treatment. Currently, miRNA detection methods mainly include real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), microarrays, and Northern blotting. While these methods are highly sensitive, they are cumbersome and expensive. Therefore, developing simpler and more cost-effective miRNA detection technologies is an urgent problem to be solved.

[0003] The development of clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) has sparked a research boom in the field of medical diagnostics due to their high specificity and programmability. In particular, class 2 type V CRISPR / Cas12a can bind to guide CRISPR RNA (crRNA) and, after specifically recognizing double-stranded DNA (dsDNA) containing a protospace radjacent motif (PAM), indiscriminately cleaves non-target ssDNA, exhibiting significant reverse cleavage capability. However, the limitation of the CRISPR / Cas12a system is its inability to directly recognize RNA sequences. This leads many biosensors to employ reverse transcription and template amplification techniques to improve detection sensitivity and accuracy, such as recombinase polymerase amplification (RPA), rolling cycle amplification (RCA), and exponential amplification reaction (EXPAR). These isothermal amplification techniques improve 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 analytical results. Furthermore, the CRISPR / Cas12a system relies on signal transduction strategies to detect non-nucleic acid targets, such as small molecules, proteins, and pathogens. However, existing signal transduction strategies are limited to target detection related to DNases and aptamers, and also suffer from insufficient specificity. Therefore, screening for more and more specific molecular recognition tools is crucial for achieving rapid on-site detection in future biomedical and therapeutic applications.

[0004] Hammerhead ribozymes are small RNA molecules capable of catalyzing specific RNA cleavage reactions. They possess a unique secondary structure consisting of a central catalytic core and two arms that bind to the substrate RNA via base pairing and cleave the adjacent sequence NUH↓ (where N is any base and H is A, C, or U). Due to their high sequence specificity and catalytic efficiency, hammerhead ribozymes have shown broad application potential in the field of mRNA therapy, and are being used to develop innovative therapies targeting specific genetic diseases, viral infections, and cancers. Furthermore, hammerhead ribozymes are being combined with CRISPR technology to improve the precision and efficiency of gene editing by cleaving specific RNA molecules. Utilizing the RNA cleavage activity of hammerhead ribozymes, CRISPR-based detection strategies can eliminate the nucleic acid amplification step, enabling the development of rapid, on-site detection methods. Summary of the Invention

[0005] This invention aims to overcome the problems of cumbersome operation steps, high cost, and insufficient specificity and sensitivity in existing miR-155 detection methods, and provides a method for detecting breast cancer miR-155 biomarkers using an allosteric ribozyme-driven crRNA switch sensor. The allosteric ribozyme is carefully designed to couple the target recognition sequence with the 5' flanking arm of a hammerhead ribozyme, forming a hairpin structure through hybridization. The presence of the target molecule induces a conformational change in the allosteric module and disrupts the hairpin structure, restoring the ribozyme's RNA cleavage activity. Then, the activated ribozyme specifically cleaves the restriction site of the locked crRNA hairpin structure, releasing native crRNA and initiating CRISPR / Cas12a function for signal reporting. This achieves sensitive detection of breast cancer miR-155 biomarkers.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides an allosteric ribozyme-driven crRNA switch sensor, the sensor comprising BR miR-155 The substrates include locked crRNA, Lba Cas12a, double-stranded DNA activator, and FQ Reporter; wherein, the BR... miR-155 The 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 containing 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] In a preferred embodiment, the substrate Locked crRNA contains a ribozyme cleavage site NUH (N is any base, and H is A, C, or U).

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

[0010] A second aspect of the present invention provides a method for detecting breast cancer miR-155 biomarkers 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 a hybridization reaction. miR-155 The complex restores the cleavage activity of hammerhead ribozymes;

[0012] S2, Take the miR-155-BR from step S1 miR-155 The complex and the substrate Locked crRNA were mixed and then cleaved by a hammerhead ribozyme to obtain the crRNA cleavage product.

[0013] S3. Mix Lba Cas12a with the crRNA product of hammerhead ribozyme from step S2, then add the reaction mixture and continue incubation to carry out CRISPR / Cas12a cleavage reaction.

[0014] S4. The fluorescence signal of the reaction product from step S3 was detected by an F7000 fluorescence spectrophotometer.

[0015] In a preferred embodiment, 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] In the preferred embodiment, in step S2, the ribozyme cleavage reaction temperature is 25°C and the time is 1 hour.

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

[0018] In the preferred embodiment, in step S4, the fluorescence detection settings are: excitation wavelength 480nm, emission wavelength 500nm, and slit width 5nm.

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

[0020] The preferred technical solution involves testing samples including cancer cells, normal cells, serum from breast cancer patients, and normal serum.

[0021] A method for detecting breast cancer miR-155 biomarkers using an allosteric ribozyme-driven crRNA switch sensor has the following advantages compared to existing technologies:

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

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

[0024] (3) The present invention provides a method for detecting the miR-155 biomarker of breast cancer using an allosteric ribozyme-driven crRNA switch sensor. The method is highly specific, and the ribozyme specifically cuts RNA and initiates CRISPR, which further improves the accuracy of gene editing.

[0025] (4) The present invention provides a method for detecting breast cancer miR-155 biomarkers using an allosteric ribozyme-driven crRNA switch sensor. This method is programmable and can detect other biomarkers by flexibly replacing the sequence of the recognition element. Attached Figure Description

[0026] To enhance understanding of the present invention, the accompanying drawings used in specific implementation methods will be briefly described below. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an improper limitation of the invention. In the drawings:

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

[0028] Figure 2 The effect of different blocking lengths of locked crRNA on the inhibition of fluorescence signal is shown in the figure.

[0029] Figure 3 Figure showing the experimental results for optimizing the flank arm length of the hammerhead ribozyme.

[0030] Figure 4 Figure showing the experimental results for optimizing the blocking region of allosteric ribozymes.

[0031] Figure 5 This is a non-denaturing polyacrylamide gel electrophoresis image used to verify the feasibility of the method in this invention.

[0032] Figure 6 This is a fluorescence spectrum used to verify the feasibility of the method in this invention.

[0033] Figure 7 The graph shows the effects of potassium ion concentration and magnesium ion concentration on the sensor's detection of miR-155.

[0034] Figure 8 For ribozyme lysis reaction time, temperature and BR miR-155 The effect of the concentration ratio of locked crRNA on the sensor detection of miR-155.

[0035] Figure 9 This is a sensitivity analysis chart for detecting different concentrations of miR-155 in this invention.

[0036] Figure 10 This is a graph showing the nonlinear and linear relationships fitted based on the fluorescence signals of miR-155 at different concentrations in this invention.

[0037] Figure 11 This is a radar chart used to verify the specificity of the method in this invention.

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

[0039] Figure 13 In Figure A, the real-time fluorescence response graph of different cell lines analyzed by RT-qPCR is shown; in Figure B, the bar graph shows the relative expression of miR-155 in different cell lines detected by the method of this invention and RT-qPCR.

[0040] Figure 14 A is a heatmap of miR-155 detection in the serum of breast cancer patients and normal serum using the method of the present invention and RT-qPCR; B is a box plot of miR-155 detection in the serum of breast cancer patients and normal serum using the method of the present invention and RT-qPCR. Detailed Implementation

[0041] Sequence List:

[0042]

[0043]

[0044] To make the objectives, 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 some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise defined, 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 pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0046] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were purchased from a regular biochemical reagent store.

[0048] Example 1

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

[0050] S1, miR-155-BR miR-155 Formation of the complex: 1.2 μL of 200 μM BR was added. miR-155 Mix 1 μL of miR-155 solutions of different concentrations, then add 2 μL of 100 mM Tris-HCl (pH 7.6), 2 μL of 100 mM MgCl2, 2 μL of 100 mM KCl, and 10.8 μL of RNase-free ddH2O to the mixture. Incubate the mixture at 37 °C for 30 min, and obtain miR-155-BR through hybridization reaction. miR-155 complex;

[0051] S2, Obtaining crRNA cleavage products: The miR-155-BR from step S1... miR-155 The complex was mixed with 1 μL of 200 μL of locked crRNA and incubated at 25 °C for 1 h to obtain the crRNA cleavage product obtained by allosteric hammerhead ribozyme catalytic cleavage of the substrate.

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

[0053] S4. Acquisition of Fluorescence Signals: The fluorescence spectrum of the mixture was acquired using an F7000 spectrophotometer. The excitation wavelength was 480 nm, the emission wavelength was 500 nm to 600 nm, and the slit width was 5 nm. Data analysis was performed using the FAM fluorescence intensity at 530 nm.

[0054] Example 2

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

[0056] (1) Test the degree to which Locked crRNA of different blocking lengths inhibits fluorescence signal.

[0057] First, locked crRNAs with different stem lengths (5, 7, 9, 11, 13, and 15 nt) were prepared to verify the inhibitory effect of crRNA on Cas12a activity. The preparation method was the same as steps S3 and S4 in Example 1, with a blank sample as a control. The fluorescence intensity ratio F / F0 was calculated, and the results are shown below. Figure 2 B. The FQ Reporter was replaced with ssDNA, and the results were analyzed using polyacrylamide gel electrophoresis. (See attached table). Figure 2 C.

[0058] from Figure 2 As shown in Figure B, the fluorescence intensity F / F0 gradually decreased as the stem region length increased from 5 nt to 15 nt. The signal-to-noise ratio of the locked crRNA was lowest when the stem region length exceeded 13 nt. Figure 2 As shown in Figure C, the ssDNA substrate cleavage fragment gradually increases with decreasing Locked crRNA length. Therefore, it is concluded that Locked crRNA (13nt) and Locked crRNA (15nt) can inhibit Cas12a activity. To avoid the stem becoming too stable due to excessively long blocking length, which could affect the binding efficiency of the subsequent riboprotein flanking arms to the substrate, Locked crRNA (13nt) was ultimately determined as the riboprotein cleavage substrate sequence.

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

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

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

[0062] (3) Optimization of allosteric ribozyme structure.

[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 in Example 1. Real-time fluorescence response signals were acquired using a microplate reader, and the results are shown below. Figure 4 .

[0064] from Figure 4 As can be seen, the fluorescence intensity gradually increases with the increase in the number of toes, therefore BR was selected. miR-155 The -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 breast cancer miR-155 biomarkers.

[0067] To investigate the feasibility of the method of this invention for the detection of miR-155, the results were analyzed using polyacrylamide gel electrophoresis. Figure 5 Further analysis using fluorescence spectroscopy yielded the following results: Figure 6 .

[0068] from Figure 5 It can be seen from this that when the target miR-155 and BR miR-155 During incubation, a low migration band (lane 3) was observed, indicating that BR miR-155 It can identify and bind to miR-155. (The miR-155-BR...) miR-155Incubation of the complex with locked crRNA yielded a clearer band (lane 5) with faster electrophoretic migration. This band corresponds to the cleavage product of locked crRNA under ribozyme-catalyzed RNA cleavage activity. Incubation with an undesigned ribozyme HHR and locked crRNA produced a band with a similar molecular weight to the new lane 5 band. This preliminarily confirms the presence of BR. miR-155 It is activated to cleave locked crRNA and release crRNA.

[0069] from Figure 6 As can be seen, in the absence of miR-155 (b, green region), the observed fluorescence signal is negligible compared to the output signal of HHR-cleaved locked crRNA (d, purple region). Conversely, the introduction of miR-155 drives the CRISPR / Cas12a response, with the signal (c, blue region) recovering by approximately 87.9% compared to group d. This confirms that crRNA switching can be achieved through BR... miR-155 It regulates through conformational changes.

[0070] Example 4

[0071] The effects of different reaction conditions on the detection of breast cancer miR-155 biomarkers by an allosteric ribozyme-driven crRNA switch sensor were tested.

[0072] (1) Potassium ion concentration optimization

[0073] The sensor was prepared using the same method as in Example 1, except that the concentration of potassium ions in the buffer solution was changed to 1, 10, 50, 100, and 500 mM. Results are shown below. Figure 7 A.

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

[0075] (2) Optimization of magnesium ion concentration

[0076] The sensor was prepared using the same method as in Example 1, except that the concentration of magnesium ions in the buffer solution was changed to 0.1, 0.5, 1, 10, and 100 mM. The results are shown below. Figure 7 B.

[0077] from Figure 7 As shown 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 sensor was prepared using the same method as in Example 1, except that the ribozyme lysis reaction time was changed to 5, 15, 30, 45, and 60 min. Results are shown below. Figure 8 A.

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

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

[0082] The sensor was prepared using the same method as in Example 1, except that the ribozyme lysis reaction temperature was changed to 15, 25, 37, 45, and 55°C. The results are shown below. Figure 8 B.

[0083] from Figure 8 As shown in B, the signal-to-noise ratio is highest and the fluorescence signal is best when the ribozyme lysis reaction temperature is 25℃.

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

[0085] The sensor is fabricated using the same method as in Example 1, except that the BR is changed. miR-155 The concentration ratios of the reaction with locked crRNA were 0.5:1, 1:1, 1.2:1, 2:1, and 10:1. Results are shown below. Figure 8 C.

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

[0087] Example 5

[0088] Performance analysis of an allosteric ribozyme-driven crRNA switch sensor.

[0089] (1) Sensitivity detection

[0090] The preparation method of the allosteric ribozyme-driven crRNA switch sensor is the same as in 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. Results are shown in […]. Figure 9 .

[0091] from Figure 9 The results show that 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 Figure 9 Nonlinear and linear fitting were performed on the fluorescence intensities corresponding to different miR-155 concentrations, and the results are shown in [Figure 1]. Figure 10 .

[0094] from Figure 10 The results show that from 100 pM to 40 nM, there is a good linear relationship between fluorescence intensity and the logarithm of miR-155 concentration, with the regression equation being F = 426.77LgC - 68.18(R). 2 =0.9981), and the limit of detection (LOD) is 16 pM.

[0095] (3) Specific detection

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

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

[0098] Example 6

[0099] Application test of physical samples for detecting breast cancer miR-155 biomarkers using an allosteric ribozyme-driven crRNA switch sensor.

[0100] (1) Establishing a standard curve using RT-qPCR method

[0101] cDNA was prepared using a miRNA first-strand cDNA synthesis kit (stem-loop method) according to the product protocol. First, 20 μL of reaction solution contained 1 μL of miR-155 standards at different concentrations (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 primers, and 14 μL of RNase-free ddH2O. The solution was then incubated sequentially at 25°C for 5 min, 50°C for 15 min, and 85°C for 5 min to inactivate reverse transcriptase. RT-qPCR was performed on a QuantStudio 3 real-time 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 qPCRMaster Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), and 8.2 μL ddH2O. The qPCR program was: 95℃ for 5 min, 95℃ for 10 s, and 60℃ for 30 s, for a total of 40 cycles. Real-time fluorescence signals were acquired, and the results are shown below. Figure 12 A. According to Figure 12 Linear fitting was performed on the CT values ​​corresponding to the intersection of the threshold line and the fluorescence spectrum. The results are shown below. Figure 12 B.

[0102] from Figure 12 As shown in Figure B, from 10 pM to 100 nM, there is a good linear relationship between the CT value and the logarithm of the miR-155 concentration, with the regression equation being CT = -3.2866 LgC + 39.152 (R0). 2 =0.9995).

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

[0104] Total RNA was isolated from different cell lines (MCF-7, HeLa, MCF-10A) using Trizolreagent according to the product instructions. RT-qPCR analysis was performed on the different cell lines as in Example 6(1), with U6 used as an internal control for miRNA-155. The results are shown in [Figure 6]. Figure 13 A. Using the same preparation method as in Example 1, miR-155 in different cell lines was detected. The two analytical methods were compared, using 2... (-ΔΔCt) The relative expression level of miR-155 was calculated using the method described above. The results are shown in [link to results]. Figure 13 B.

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

[0106] from Figure 13 As shown 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) Allosteric ribozyme-driven crRNA switch sensor and gold standard RT-qPCR were used to analyze clinical serum samples.

[0108] Following the method in Example 6(2), miR-155 was detected in 5 normal serum samples and 10 breast cancer patients' serum samples using an allosteric ribozyme-driven crRNA switch sensor and gold standard RT-qPCR, respectively. Quantification was performed using the standard curve constructed in Example 6(1). The results are shown in […]. Figure 14 A and 14B.

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

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

[0111] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A mutagenic ribozyme-driven crRNA switch sensor, characterized in that, The sensor includes BR miR-155 The substrates include locked crRNA, Lba Cas12a, double-stranded DNA activator, and FQ Reporter; wherein, the BR... miR-155 The allosteric hammerhead ribozyme that specifically recognizes miR-155 has the sequence 5'-ACCCCUAUCACGAUUAGCAUUAAUCGUGAUCUGAUGAGUCCGGUAGCGGACGAAAUGUUUG-3'; the substrate Locked crRNA contains a ribozyme cleavage site and has the sequence 5'-UAAUUUCUACUAAGUGUAGAUCAAGGAACUGAUUACAAACAUAAUCACGAAUCUACACGGCGUAGA-3'; the double-stranded DNA activator has the sequence 5'-GATCTATTGTCTTGTTTGTAATCAGTTCCTTGTAAATATTGAATTTGATATGACAGAGT-3'.

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

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