DNA-RNA triple helix probe-based crisper / cas13a system combined with rolling circle amplification for detecting double miRNA and application thereof

Abstract

This invention relates to a detection system and its application for detecting dual miRNAs using a CRISPR / Cas13a system based on DNA-RNA triple-helix probes combined with rolling circle amplification (rolling circle amplification), belonging to the field of molecular diagnostics and biosensing technology. The detection system includes a DNA-RNA triple-helix molecular beacon 1, a DNA-RNA triple-helix molecular beacon 2, padlock probes P1 and P2, a pre-activated CRISPR / Cas13a system, and a rolling circle amplification reaction system. This invention applies the CRISPR / Cas13a system based on DNA-RNA triple-helix probes combined with rolling circle amplification to detect dual miRNAs for the joint detection of miRNA155 and miRNA21 in the plasma of lung cancer patients. The results are highly consistent with RT-qPCR (RT-qPCR). 2 The combined diagnostic AUC of 0.975 for the dual biomarkers was >0.95, providing a reliable new platform for early lung cancer screening.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and biosensing technology, specifically relating to a detection system and application of dual miRNA detection based on a CRISPR / Cas13a system combined with rolling circle amplification using a DNA-RNA triple helix probe. Background Technology

[0002] Lung cancer is mainly divided into non-small cell lung cancer and small cell lung cancer. Despite continuous advancements in early diagnosis, prognostic assessment, and targeted therapy, the overall mortality rate of lung cancer remains high. From 2019 to 2021, the survival rate of lung cancer patients in my country was less than 30%, placing enormous pressure on clinical prevention and treatment. Therefore, exploring highly sensitive and specific early diagnostic biomarkers for lung cancer and constructing precise risk stratification models has significant clinical and public health value for improving patient prognosis and reducing the disease burden.

[0003] MicroRNAs (miRNAs) are a class of endogenous single-stranded non-coding small RNA molecules that play important regulatory roles in cellular transcription. Studies have shown that miRNAs, due to their specific abnormal expression patterns in various malignant tumors, including non-small cell lung cancer, have become novel potential biomarkers for early tumor detection and screening. In the development and metastasis of lung cancer, miRNA155, as a key regulatory molecule, exhibits abnormal expression across multiple pathological stages, including tumorigenesis, invasion and metastasis, chemotherapy resistance, and immune escape, making it an important biomarker for early screening, diagnosis, and prognostic assessment of lung cancer. The expression profile of miRNA155 is closely related to the survival rate of patients with specific types of lung cancer; high expression usually indicates a poor prognosis and significantly shortened survival. miRNA155 is significantly upregulated in lung cancer and is closely related to tumor stage, metastasis, and prognosis. Abnormal expression of miRNA21 is involved in tumorigenesis, metastasis, drug resistance, and immune escape. Furthermore, studies using integrated transcriptome analysis have confirmed that both miRNA155 and miRNA21 are significantly upregulated in lung adenocarcinoma, jointly participating in the key miRNA transcription factor regulatory network in tumors, and can serve as important combined biomarkers for the diagnosis and prognostic assessment of lung adenocarcinoma. miRNA21 and miRNA155 are abnormally highly expressed in non-small cell lung cancer, making them important biomarkers for lung cancer screening, diagnosis, and prognostic assessment. Compared to single-indicator detection, combined detection of these two targets can improve detection sensitivity and specificity, and establishing a highly sensitive and specific detection method has significant clinical value for the early diagnosis and precision treatment of lung cancer.

[0004] In recent years, biosensing technology has provided a new technical approach for miRNA detection. In biosensor research, miRNA detection strategies based on signal amplification have developed rapidly. By integrating nucleic acid amplification technology, signal amplification is achieved stepwise, significantly improving detection sensitivity. Compared to traditional PCR, isothermal nucleic acid amplification requires no sophisticated instruments, uses simple primers, and has a mild reaction, making it more suitable for rapid, low-cost, on-site detection. Rolling circle amplification (Rolling Circle Amplification) is an isothermal nucleic acid amplification technique that relies on target miRNA to mediate template circularization, followed by polymerase catalysis to generate long-chain DNA containing numerous repetitive sequences. This achieves efficient amplification and signal amplification, combining high sensitivity and specificity in miRNA detection, making it an ideal method for constructing highly sensitive biosensors.

[0005] Nucleic acids, as important biological macromolecules, play a central role in the storage, transmission, and expression of genetic information, making them a key area of ​​research in modern life sciences. Triple-helical nucleic acids consist of three strands linked by Watson-Crick pairing and Hoogsteen hydrogen bonds, with the third strand binding to the major groove of the double helix, forming a triple-helix conformation. In functional nucleic acid probe design, single-stranded nucleic acids can form intramolecular hairpin structures, antiparallel complementary strands can form double-stranded probes, and introducing a third strand can construct a triple-helix probe system. These structure-specific probes are highly sensitive to environmental changes, undergoing reversible conformational transitions under the influence of ionic strength, pH, and biomolecules, enabling precise "on-off" control and generating detectable signals. The reversible conversion between triple-helix and double-helix structures provides an ideal molecular switching mechanism for intelligent responsive biosensors. Triple-helix DNA probes, with their reversible structure and molecular recognition characteristics, have broad application prospects in nucleic acid signal amplification and biological detection.

[0006] The CRISPR-Cas system originates from the innate immune mechanisms of bacteria and archaea. CRISPR / Cas13a, in particular, is widely used in gene editing and bioassay research due to its high specificity, high efficiency, and ease of operation. When Cas13a bound to crRNA pairs with activator RNA bases, its nuclease domain is activated and performs non-specific trans-cleavage of the surrounding RNA single strand. This property can be used to cleave ssRNA probes carrying fluorescent reporter molecules to achieve signal output. CRISPR-Cas-based detection methods offer advantages such as high sensitivity, high specificity, and rapid, simple operation. The demand for simultaneous detection of multiple biomarkers in in vitro diagnostics has driven the development of multiplexed detection systems, currently mainly achieved by using multiple Cas proteins or spatially separating different targets. Existing Cas protein multiplex detection methods suffer from complexity and high cost due to the non-specificity of trans-cleavage; therefore, there is an urgent need for new multi-target detection strategies that do not rely on multiple crRNAs. Summary of the Invention

[0007] The first objective of this invention is to provide a detection system based on a DNA-RNA triple helix probe using a CRISPR / Cas13a system combined with rolling circle amplification to detect dual miRNAs. This detection system enables the simultaneous differentiation and detection of miRNA155 and miRNA21 using a single Cas protein and the same crRNA.

[0008] The second objective of this invention is to provide an application of a detection system based on a DNA-RNA triple helix probe using a CRISPR / Cas13a system combined with rolling circle amplification to detect dual miRNAs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A detection system for dual miRNAs based on a CRISPR / Cas13a system using DNA-RNA triple-helix probes combined with rolling circle amplification includes: DNA-RNA triple helix molecular beacon 1, which includes a DNA hairpin H1 and an RNA probe 1. The stem of the DNA hairpin H1 is bound to the RNA probe 1 by hydrogen bonds. The loop of the DNA hairpin H1 specifically recognizes the amplification product of the first target miRNA. The two ends of the RNA probe 1 are labeled with a fluorescent group 1 and a quencher 1, respectively. DNA-RNA triple helix molecular beacon 2, which includes a DNA hairpin H2 and an RNA probe 2. The stem of the DNA hairpin H2 is bound to the RNA probe 2 by hydrogen bonds. The loop of the DNA hairpin H2 specifically recognizes the amplification product of a second target miRNA. The two ends of the RNA probe 2 are labeled with a fluorescent group 2 and a quencher 2, respectively. The stem sequences of DNA hairpin H1 and DNA hairpin H2 are identical, and the sequences of RNA probe 1 and RNA probe 2 are identical so that a single Cas13a can trans-cleave RNA probe 1 and RNA probe 2. The padlock probe P1 is specifically complementary to the first target miRNA so that in the presence of the first target miRNA, the padlock probe P1 is circularized into a circular DNA template by the action of ligase. The padlock probe P2 is specifically complementary to the second target miRNA so that, in the presence of the second target miRNA, the padlock probe P2 is circularized into a circular DNA template by the ligase. The pre-activated CRISPR / Cas13a system includes Cas13a, crRNA, and acRNA; The rolling circle amplification reaction system includes phi29 DNA polymerase and dNTPs.

[0010] Furthermore, the sequence of the DNA hairpin H1 is shown in SEQ ID NO:1, the sequence of the RNA probe 1 is shown in SEQ ID NO:2, the fluorescent group 1 is FAM, and the quencher 1 is BHQ1.

[0011] Furthermore, the sequence of the DNA hairpin H2 is shown in SEQ ID NO:3, the sequence of the RNA probe 2 is shown in SEQ ID NO:4, the fluorescent group 2 is Cy5, and the quencher 2 is BHQ2.

[0012] Furthermore, the sequence of the padlock probe P1 is shown in SEQ ID NO:5, and the sequence of the padlock probe P2 is shown in SEQ ID NO:6.

[0013] Furthermore, the pre-activated CRISPR / Cas13a system is prepared by mixing Cas13a, crRNA, and buffer solution and incubating to obtain the Cas13a / crRNA complex, followed by adding acRNA and incubating to obtain the final product.

[0014] Furthermore, the final concentration of Cas13a is 0.5–1.5 μmol / L, the final concentration ratio of Cas13a to crRNA is 0.5:1–1:1, and the final concentration of acRNA is 0.5–1.5 μmol / L; the sequence of crRNA is shown in SEQ ID NO:7, and the sequence of acRNA is shown in SEQ ID NO:8; the buffer solution is a Tris-HCl buffer solution.

[0015] Furthermore, the ligase is a T4 ligase, with a final concentration of 0.02–0.1 U / μL; the final concentration of the phi29 DNA polymerase is 0.04–0.2 U / μL; the final concentration of the DNA-RNA triple helix beacon one is 125–1000 nmol / L; the final concentration of the DNA-RNA triple helix beacon two is 125–1000 nmol / L; the final concentration of the padlock probe P1 is 1.5–2.5 nmol / L; the final concentration of the padlock probe P2 is 1.5–2.5 nmol / L; the final concentration of the pre-activated CRISPR / Cas13a system is 20–60 nmol / L; and the final concentration of the dNTPs is 400–600 nmol / L.

[0016] Furthermore, the preparation method of the DNA-RNA triple helix molecular beacon 1 includes: mixing DNA hairpin H1 with buffer solution II, annealing and incubating, then adding RNA probe 1, and incubating for 0.5–2.5 h to obtain the final concentration ratio of DNA hairpin H1 to RNA probe 1 is 1:1; the preparation method of the DNA-RNA triple helix molecular beacon 2 includes: mixing DNA hairpin H2 with buffer solution II, annealing and incubating, then adding RNA probe 2, and incubating for 0.5–2.5 h to obtain the final concentration ratio of DNA hairpin H2 to RNA probe 2 is 1:1.

[0017] Furthermore, the second buffer solution is a Tris-HCl buffer solution with a pH of 5.5–9; the Tris-HCl buffer solution comprises: 5–25 mmol / L Tris-HCl and 5–100 mmol / L Na+. + and 5–25 mmol of Mg 2+ .

[0018] Application of a detection system based on DNA-RNA triple helix probes, CRISPR / Cas13a combined with rolling circle amplification to detect dual miRNAs, in the preparation of lung cancer diagnostic and / or auxiliary diagnostic products.

[0019] The beneficial effects of this invention are: This invention combines the signal amplification advantages of the CRISPR / Cas13a system with the specific recognition capabilities of nucleic acid hybridization technology to construct a highly sensitive dual-miRNA detection method. By designing DNA-RNA triple-helix molecular beacons (DR-THMB)1 and DR-RNA triple-helix molecular beacons 2, and regulating their complementary pairing patterns with miRNA155 and miRNA21, a two-component analysis can be achieved to simultaneously distinguish and detect miRNA155 and miRNA21 under the presence of a single Cas protein and the same crRNA.

[0020] This invention utilizes a CRISPR / Cas13a system based on DNA-RNA triple-helix probes, combined with rolling circle amplification (RoBAC) for the joint detection of miRNAs 155 and 21 in the plasma of lung cancer patients. The results are highly consistent with RT-qPCR. 2 The combined diagnostic AUC of 0.975 for the dual biomarkers was >0.95, providing a reliable new platform for early lung cancer screening.

[0021] This invention employs DNA-RNA triple helix molecular beacons coupled with RCA and Cas13a cleavage to construct a cascaded signal amplification, enabling simultaneous detection of miRNA155 and miRNA21 in a single tube with dual channels, achieving picomolar sensitivity and eliminating cross-interference. Attached Figure Description

[0022] Figure 1 This is a fluorescence verification image of DR-THMB formation; Figure 2 The diagram shows the optimization results for DR-THMB formation conditions, where A represents the optimized buffer solution, B represents the optimized buffer solution pH, C represents the optimized Tris-HCl concentration in the buffer solution, and D represents the optimized Na+ concentration in the buffer solution. + The concentration optimization results are shown in the figure. E represents the concentration of Mg in the buffer solution. 2+ The concentration optimization results are shown in Figure F, where F represents the DR-THMB incubation time optimization results. Figure 3 This is a PAGE diagram showing the generation results of the RCA product in Experiment Example 2. Lane 1: miRNA155; Lane 2: miRNA21; Lane 3: Padlock probe P1; Lane 4: Padlock probe P2; Lane 5: miRNA155 + Padlock probe P1 + Padlock probe P2 + T4 ligase; Lane 6: miRNA21 + Padlock probe P1 + Padlock probe P2 + T4 ligase; Lane 7: miRNA155 + miRNA21 + Padlock probe P1 + Padlock probe P2 + T4 ligase; Lane 8: miRNA155 + Padlock probe P1 + Padlock probe P2 + T4 ligase + phi29 DNA polymerase; Lane 9: miRNA21 + Padlock probe P1 + Padlock probe P2 + T4 ligase + phi29 DNA polymerase; Lane 10: miRNA155 + miRNA21 + Padlock probe P1 + Padlock probe P2 + T4 ligase + phi29 DNA polymerase; Lane 11: Padlock probe P1 + Padlock probe P2 + T4 ligase; Figure 4 The image shows the fluorescence verification results of miRNA155 and miRNA21 detected in Experiment Example 2. Figure 5 Figure showing the optimized concentration of T4 ligase; Figure 6 Figure showing the optimized concentration of phi29 DNA polymerase; Figure 7 Cas13a concentration optimization diagram; Figure 8 Optimization plot of DR-THMB1 and DR-THMB2 concentrations; Figure 9 Figure showing the optimized ratio of Cas13a to crRNA; Figure 10 Figure showing the optimized reaction time of T4 ligase; Figure 11 A graph showing the optimization of RCA amplification time; Figure 12 An optimized diagram of the Cas13a cleavage reaction time; Figure 13 Fluorescence spectra of miRNA155 in a single-component detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for the detection of dual miRNAs; Figure 14 Standard curve for single-component detection of low concentration miRNA155 in a detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for detecting dual miRNAs; Figure 15 Standard curve for single-component detection of high concentration of miRNA155 in a detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for detecting dual miRNAs; Figure 16 The fluorescence spectrum of a single-component detection system for miRNA21 is shown in the CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification for the detection of dual miRNAs. Figure 17 Standard curve for single-component detection of low concentration miRNA21 in a detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for detecting dual miRNAs; Figure 18 Standard curve for single-component detection of high concentration of miRNA21 in a detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for the detection of dual miRNAs; Figure 19 Fluorescence spectra of miRNA21 and miRNA155 in a two-component detection system based on DNA-RNA triple helix probe CRISPR / Cas13a combined with rolling circle amplification for the detection of dual miRNAs; Figure 20 Standard curve for the two-component detection system of CRISPR / Cas13a system based on DNA-RNA triple helix probe combined with rolling circle amplification for the detection of dual miRNAs, and for the detection of low concentrations of miRNA155. Figure 21 Standard curve for the two-component detection system of CRISPR / Cas13a system based on DNA-RNA triple helix probe combined with rolling circle amplification to detect high concentration of miRNA155. Figure 22 Standard curve for the two-component detection system of CRISPR / Cas13a system based on DNA-RNA triple helix probe combined with rolling circle amplification for the detection of dual miRNAs, showing the detection of low concentrations of miRNA21. Figure 23 Standard curve for the two-component detection system of CRISPR / Cas13a system based on DNA-RNA triple helix probe combined with rolling circle amplification for the detection of dual miRNAs, showing the detection of high concentrations of miRNA21. Figure 24 The fluorescence spectra of miRNA155 and miRNA21 were obtained by combining the CRISPR / Cas13a system based on DNA-RNA triple helix probes with rolling circle amplification for the detection of dual miRNAs. Figure 25 This image shows the fluorescence intensity of a CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification for detecting dual miRNAs, specifically miRNA155. *** represents the fluorescence intensity compared to the miRNA155 group. P <0.001; Figure 26 This image shows the fluorescence intensity of a CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification for detecting dual miRNAs, specifically miRNA21. *** represents the fluorescence intensity compared to the miRNA21 group. P <0.001; Figure 27 The image shows a detection system for detecting miRNA155 in clinical plasma samples using a CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification. * indicates... P <0.05; Figure 28 Figure 21 illustrates a detection system for detecting miRNAs in clinical plasma samples using a CRISPR / Cas13a system based on DNA-RNA triple-helix probes combined with rolling circle amplification (RoBAM). * indicates... P <0.05; Figure 29 Figure 1 shows the detection of miRNA21 in clinical plasma samples by RT-qPCR. * indicates... P <0.05; Figure 30 The image shows the detection of miRNA155 in clinical plasma samples by RT-qPCR. * indicates... P <0.05; Figure 31 ROC curves for single miRNA and dual miRNA detection in the early diagnosis of lung cancer; Figure 32This diagram illustrates the detection principle of a CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification for the detection of dual miRNAs. In the diagram, A shows the circularization of miRNA155, miRNA21 with padlock probes P1 and P2 under the action of T4 ligase; B is a schematic diagram of DR-THMB1 and DR-THMB2; and C is a schematic diagram of the detection principle of miRNA155 and miRNA21.

[0023] Figures 5-12 , Figure 14 , Figure 15 , Figure 17 , Figure 18 , Figures 20-23 The error bar represents the standard deviation of three independent experiments. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0025] The nucleic acid sequences used in this invention are shown in Table 1.

[0026] Table 1. Nucleic acid names and base sequences

[0027] Example 1 The detection system for dual miRNAs based on the CRISPR / Cas13a system combined with rolling circle amplification (DR-THMB / Cas13a-RCA) using DNA-RNA triple helix probes in Example 1 includes: DR-THMB1 at a final concentration of 750 nmol / L, DR-THMB2 at a final concentration of 750 nmol / L, padlock probe P1 at a final concentration of 2 nmol / L, padlock probe P2 at a final concentration of 2 nmol / L, T4 ligase at a final concentration of 0.08 U / μL, phi29 DNA polymerase at a final concentration of 0.16 U / μL, dNTPs at a final concentration of 500 nmol / L, and a pre-activated CRISPR / Cas13a system at a final concentration of 50 nmol / L.

[0028] The preparation method of DR-THMB1 includes the following steps: DNA hairpin H1 with a final concentration of 2.5 μmol / L is annealed in a Tris-HCl buffer solution at 95 °C for 5 min, followed by incubation at room temperature for 1 h. Then, RNA probe 1 with a final concentration of 5 μmol / L is added, and the mixture is reacted in a thermostatic metal shaker at 25 °C and 500 r / min for 1.5 h. The hairpin loop region of DNA hairpin H1 is 24 nt long, and the stem of DNA hairpin H1 and RNA probe 1 are both 12 nt long. RNA probe 1 and the stem of DNA hairpin H1 fold together via Hoogsteen hydrogen bonds to form a hybrid triple helix structure.

[0029] The preparation method of DR-THMB2 includes the following steps: DNA hairpin H2 with a final concentration of 2.5 μmol / L is annealed in a Tris-HCl buffer solution at 95 °C for 5 min, followed by incubation at room temperature for 1 h. Then, RNA probe 2 with a final concentration of 5 μmol / L is added, and the mixture is reacted in a thermostatic metal shaker at 25 °C and 500 r / min for 1.5 h. The hairpin loop region of DNA hairpin H2 is 24 nt long, and the stem of DNA hairpin H2 and RNA probe 2 are both 12 nt long. RNA probe 2 and the stem of DNA hairpin H2 fold together via Hoogsteen hydrogen bonds to form a hybrid triple helix structure.

[0030] DNA hairpins H1 and H2 were simulated using NUPACK software. The ΔG for DNA hairpin H1 was -11.17 kcal / mol, and the ΔG for DNA hairpin H2 was -8.70 kcal / mol, indicating that DNA hairpins H1 and H2 can form stable hairpin structures at 25 °C. To verify that the RNA probe can form a stable triple helix structure with the DNA hairpin, Cy5 was modified at the 5' end of H2, resulting in a hairpin HP. Figure 1 It can be seen that when the hairpin probe HP is present alone, the fluorescence signal of Cy5 can be detected. When RNA probe 2 and HP are present at the same time, they combine to form a triple helix structure. Due to the fluorescence resonance energy transfer of the BHQ2 labeled at the 3' end of RNA probe 2 near Cy5 on HP, the fluorescence signal disappears. This confirms that the DR-THMB triple helix probe was successfully assembled.

[0031] The pre-activated CRISPR / Cas13a system was prepared as follows: Cas13a (final concentration 1 μmol / L), crRNA (final concentration 1 μmol / L), and Tris-HCl buffer were incubated at 37 °C for 30 min to synthesize the Cas13a / crRNA complex. Before use, acRNA (final concentration 1 μmol / L) was added to the Cas13a / crRNA complex and pre-incubated to bind it to the pre-activated Cas13a protease, thus obtaining the pre-activated CRISPR / Cas13a system.

[0032] The Tris-HCl buffer solution contains 15 mmol / L Tris-HCl, 10 mmol / L MgCl2 and 50 mmol / L NaCl, pH 7.0.

[0033] Application Example 1 The steps for detecting miRNA155 and miRNA21 using a CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification are as follows: S1: Prepare DR-THMB1 and DR-THMB2 according to the steps in Example 1; S2: Take a 200 μL EP tube and add padlock probe P1 (final concentration 2 nmol / L), padlock probe P2 (final concentration 2 nmol / L), T4 ligase (final concentration 0.08 U / μL), 1 μL of 10×T4 buffer (0.2×), and ddH2O to form a 10 μL reaction system. Place the tube in a constant temperature metal shaker and react for 2 h at 22 ℃ and 300 r / min. S3: Add phi29 DNA polymerase to the reaction system of S2 at a final concentration of 0.16 U / μL, 2 μL of 10×phi29 buffer (0.4×), 2 mg / L of BSA, 500 nmol / L of dNTP and ddH2O to form a 20 μL reaction system. Place the system in a constant temperature metal shaker and react for 2 h at 30 ℃ and 500 r / min. S4: Prepare a pre-activated CRISPR / Cas13a system according to the steps in Example 1; S5: Add DR-THMB1 (final concentration 750 nmol / L), DR-THMB2 (final concentration 750 nmol / L), pre-activated CRISPR / Cas13a system (final concentration 50 nmol / L), and Tris-HCl buffer solution to the reaction system in S3 to form a 50 μL reaction system. Incubate the system in a constant temperature metal shaker at 37 ℃ and 500 r / min for 40 min. After the reaction, transfer the 50 μL reaction system to a 96 black ELISA plate and detect the fluorescence signal using a multi-functional microplate reader. FAM excitation wavelength: 465 nm, emission wavelength: 490-600 nm; Cy5 excitation wavelength: 619 nm, emission wavelength: 645-750 nm.

[0034] The detection principle of miRNA155 and miRNA21 is as follows: The loop portion of DNA hairpin H1 specifically recognizes rolling circle amplification products induced by miRNA155. In the presence of miRNA155, the padlock probe P1 circularizes under the action of T4 ligase to form a circular DNA template. Under the mediated rolling amplification of phi29 DNA polymerase, a large amount of long single-stranded DNA amplification products are generated. These amplification products bind to the loop portion of DNA hairpin H1, causing DR-THMB1 to disintegrate and releasing labeled RNA probe 1. The pre-incubated activated Cas13a protein trans-cleaves the released RNA probe 1, causing the fluorophore to separate from the quencher, thereby generating a fluorescent signal.

[0035] The loop portion of DNA hairpin H2 specifically recognizes rolling circle amplification products induced by miRNA21. In the presence of miRNA21, the padlock probe P2 circularizes under the action of T4 ligase to form a circular DNA template. Under the mediated rolling amplification of phi29 DNA polymerase, a large amount of long single-stranded DNA amplification products are generated. These amplification products bind to DNA hairpin H2, causing DR-THMB probe 2 to disintegrate and release RNA probe 2. The pre-incubated activated Cas13a protein trans-cleaves the released RNA probe 2, causing the fluorophore to separate from the quencher, thereby generating a fluorescent signal.

[0036] When miRNA155 and miRNA21 are present simultaneously, the two recognition and amplification processes occur in parallel, releasing RNA probe 2 and RNA probe 1 respectively. After trans-cleavage by Cas13a, a dual fluorescent signal response is generated. If only a single target miRNA is present, only the corresponding single fluorescent signal is generated, thereby achieving simultaneous and differential detection of the two miRNAs.

[0037] Experimental Example 1 1. The 5' end of the DNA hairpin H2 was labeled with a Cy5 fluorescent group to verify the formation and condition optimization of DR-THMB: From Figure 2 As can be seen from A and B, DR-THMB forms optimally in a Tris-HCl buffer solution at pH 7.0. From... Figure 2 From C, D, and E, it can be seen that the concentration of Tris-HCl in the Tris-HCl buffer solution is 15 mmol / L, and the Na... + Concentration of 50 mmol / L, Mg 2+ DR-THMB formation is optimal at a concentration of 10 mmol / L. Figure 2 As can be seen from F, the optimal formation of DR-THMB is achieved when the incubation time is 1.5 h.

[0038] 2. Optimize enzyme concentration according to the steps in Application Example 1: From Figure 5 , Figure 6 and Figure 7 As can be seen, the fluorescence values ​​of FAM and Cy5 both increased with increasing concentrations of T4 ligase, phi29 DNA polymerase, and Cas13a. When the concentrations of T4 ligase, phi29 DNA polymerase, and Cas13a were 0.08 U / μL, 0.16 U / μL, and 50 nmol / L, the difference between the fluorescence signals of FAM and Cy5 tended to plateau. Therefore, the concentrations of T4 ligase (0.08 U / μL), phi29 DNA polymerase (0.16 U / μL), and Cas13a (50 nmol / L) were selected.

[0039] 3. After forming the triple-helix probes DR-THMB1 and DR-THMB2 according to the steps in Application Example 1, their concentrations were optimized. Figure 8 It can be seen that when the concentrations of DR-THMB1 and DR-THMB2 are 750 nmol / L, ΔF FAM With ΔF Cy5 All values ​​reached their maximum.

[0040] 4. Optimize the Cas13a to crRNA ratio following the steps in Application Example 1. Figure 9 It can be seen that when the ratio of Cas13a to crRNA is 1:1, ΔF FAM With ΔF Cy5 All values ​​reached their maximum.

[0041] 5. Optimize the T4 ligase reaction time, RCA amplification time, and Cas13a cleavage reaction time following the steps in Application Example 1: From Figure 10 It can be seen that the optimal reaction time for T4 ligase is 2 hours. From... Figure 11It can be seen that the optimal amplification time for RCA is 2 hours. From... Figure 12 It can be seen that the optimal reaction time for Cas13a is 40 min.

[0042] Experimental Example 2 Feasibility analysis The padlock probes P1 and P2 designed in this invention have their two ends complementary to miRNA155 and miRNA21, respectively. The middle sequences of padlock probes P1 and P2 are completely identical to the hairpin loop and stem portions (4 bases) of H1 and H2, respectively. This ensures that when there are no target miRNAs 155 and 21, padlock probes P1 and P2 exist in single-stranded form. When the corresponding target is present, padlock probes P1 and P2 can efficiently pair with the corresponding target and undergo circularization.

[0043] The minimum free energy structures of padlock probes P1 and P2 were simulated using NUPACK software. The simulated ΔG for padlock probe P1 was -2.85 kcal / mol, indicating that it forms only 2-3 intramolecular base pairs and is predominantly a single-stranded linear conformation. The simulated ΔG for P2 was -2.58 kcal / mol, also existing in a stable single-stranded linear state. Furthermore, no significant non-specific hybridization was observed between the four strands of padlock probes P1 and P2, and DNA hairpins H1 and H2.

[0044] The rolling circle amplification system was validated using polyacrylamide gel electrophoresis (PAGE), and the results are as follows: Figure 3As shown, lanes 1 and 2, with only miRNA155 and miRNA21 added respectively, both showed single corresponding bands; lanes 3 and 4, with only padlock probes P1 and P2 added respectively, were used to determine the probe band positions; lane 5, with miRNA155, padlock probes P1 and P2 added, along with T4 ligase, showed new bands, with the lower band being the padlock probe P2 band and the upper band being the circular complex formed by miRNA155 and padlock probe P1 under the action of T4 ligase; lane 6, with miRNA21 added, padlock probes P1 and P2 added, showed new bands. Lane 2 and T4 ligase also showed new bands, with the lower band being the padlock probe P1 band and the upper band being the circular complex formed by miRNA21 and padlock probe P2, indicating that padlock probes P1 and P2 only specifically bind to their matched targets. Lane 7, with the addition of miRNA155, miRNA21, padlock probes P1 and P2, and T4 ligase, showed characteristic bands consistent with those in lanes 5 and 6, indicating that both padlock probes P1 and P2 can be triggered and circularized by their corresponding targets. Lane 8, based on the system in lane 5, added phi29. DNA polymerase, due to the large molecular weight and structural entanglement of the rolling circle amplification product, caused the product to remain in the sample well. Lane 9, with the addition of phi29 DNA polymerase to the system in lane 6, also showed a product retention band at the well opening, indicating successful rolling circle amplification. Lane 10, with the addition of miRNA155, miRNA21, padlock probes P1, P2, T4 ligase, and phi29 DNA polymerase, still showed a clear amplification product band at the well opening, confirming that both miRNA155 and miRNA21 targets can trigger efficient amplification. Lane 11, with the addition of only padlock probes P1, P2, and T4 ligase, did not show a circular DNA band in the absence of targets, indicating that padlock probes P1 and P2 remain stable in the absence of targets and do not undergo non-specific circularization.

[0045] from Figure 4 It can be seen that when neither miRNA155 nor miRNA21 is present, the FAM and Cy5 fluorescence signals are at low levels; when only miRNA155 is present, the FAM fluorescence signal increases significantly, while the Cy5 signal remains at a low level; when only miRNA21 is present, the Cy5 fluorescence signal is significantly enhanced, while the FAM signal shows no significant change; when both miRNA155 and miRNA21 are present, both FAM and Cy5 fluorescence signals increase significantly. This confirms that the detection system of dual miRNAs based on the DNA-RNA triple helix probe CRISPR / Cas13a system combined with rolling circle amplification has good feasibility and specificity.

[0046] Experimental Example 3 Standard curve Following the steps in Application Example 1, single-component detection of miRNA155 was performed. Figure 13As can be seen, the fluorescence intensity increases with the gradual increase of miRNA155 concentration from 0 to 10 nmol / L. Figure 14 It can be seen that from 5 pmol / L to 500 pmol / L, the linear equation for the first stage is ΔF. FAM =5129441.48C miRNA155 +734912.11 (R) 2 =0.992). From Figure 15 It can be seen that from 500 pmol / L to 10 nmol / L, the linear equation for the second stage is ΔF. FAM =638761.80C miRNA155 +2994180.82 (R) 2 =0.993).

[0047] Following the steps in Application Example 1, single-component detection of miRNA21 was performed. Figure 16 It can be seen that the fluorescence intensity increases as the concentration of miRNA21 gradually increases from 0 to 10 nmol / L. From... Figure 17 It can be seen that from 5 pmol / L to 500 pmol / L, the linear equation for the first stage is ΔF. Cy5 =7290194.39C miRNA21 +662768.21 (R) 2 =0.996). From Figure 18 It can be seen that from 500 pmol / L to 10 nmol / L, the linear equation for the second stage is ΔF. Cy5 =508973.69C miRNA21 +4438487.78 (R) 2 =0.995).

[0048] Following the steps in Application Example 1, a two-component detection of miRNA155 and miRNA21 was performed. Figure 19 It can be seen that the fluorescence intensity increases as the concentrations of miRNA155 and miRNA21 gradually increase from 0 to 10 nmol / L. From... Figure 20 It can be seen that the linear equation for the first stage, from miRNA155 concentration of 5 pmol / L to 500 pmol / L, is ΔF. FAM =5127757.36C miRNA155 +430753.49 (R) 2 =0.992). From Figure 21 It can be seen that when the miRNA155 concentration decreased from 500 pmol / L to 10 nmol / L, the linear equation for the second stage was ΔF. FAM =604011.70C miRNA155+2891169.43 (R) 2 =0.991). From Figure 22 It can be seen that the linear equation for the first stage, from miRNA21 concentration of 5 pmol / L to 500 pmol / L, is ΔF. Cy5 =7570931.05C miRNA21 +235421.69 (R) 2 =0.994). From Figure 23 It can be seen that the linear equation for the second stage, from miRNA21 concentration of 500 pmol / L to 10 nmol / L, is ΔF. Cy5 =530990.90C miRNA21 +4030429.51 (R) 2 =0.995).

[0049] △F FAM =F-F0 represents the difference between the fluorescence value of the experimental group and the fluorescence value of the blank group. Where ΔF FAM F0 represents the fluorescence intensity of the FAM signal in the positive sample, and C represents the fluorescence intensity of the FAM signal in the blank sample. miRNA155 This represents the concentration of miRNA155. △F Cy5 =F-F0 represents the difference between the fluorescence value of the experimental group and the fluorescence value of the blank group, ΔF Cy5 F0 represents the Cy5 signal fluorescence intensity of the positive sample, and C represents the Cy5 signal fluorescence intensity of the blank sample. miRNA21 This represents the concentration of miRNA21.

[0050] Test Example 4 Methodological evaluation 1. Testing line Single-component detection of miRNA155 was performed following the steps in Application Example 1: The fluorescence intensity of 9 blank solutions was detected following the steps in Application Example 1, and the average FAM fluorescence intensity of the blank solutions was calculated. Standard deviation SD =245998.97, Substituting the calculated value into the standard curve, the detection line for miRNA155 can be calculated. DL The concentration was 0.60 pmol / L.

[0051] Following the steps in Application Example 1, single-component miRNA detection was performed: The fluorescence intensity of nine blank solutions was detected using the methods described in Application Example 1, and the average fluorescence intensity of Cy5 in the blank solutions was calculated. Standard deviation SD =222225.14, Substituting the desired value into the standard curve, the detection rate of miRNA21 can be calculated. DL It is 0.54 pmol / L.

[0052] Following the steps in Application Example 1, a two-component detection of miRNA21 and miRNA155 was performed. The fluorescence intensity of nine blank solutions was also detected following the steps in Application Example 1, and the average FAM fluorescence intensity of the blank solutions was calculated. Standard deviation SD =144806.94, Substituting the desired value into the standard curve allows for the calculation of the effect on miRNA155 detection. DL The concentration was 0.72 pmol / L; the average fluorescence intensity of Cy5 in the blank solution was calculated. Standard deviation SD =80130.01, Substituting into the standard curve, the detection efficacy of miRNA21 was calculated. DL It is 0.66 pmol / L.

[0053] 2. Spiking recovery rate and precision Following the steps in Application Example 1, miRNA155 and miRNA21 in 10% human plasma were detected. Three replicates were set for each group. The fluorescence intensity was substituted into the standard curve to calculate the corresponding concentration. The results for miRNA155 are shown in Table 2: the recoveries for low, medium, and high concentrations ranged from 94.35% to 102.73%. RSD The recovery rate ranged from 2.91% to 3.81%, indicating good precision of the method. The results for miRNA21 are shown in Table 3: the recoveries for low, medium, and high concentrations ranged from 96.42% to 99.73%. RSD The range is 2.23% to 3.04%, indicating that the method has good precision.

[0054] Table 2. Precision and spiked recovery of miRNA155 (n=3)

[0055] Table 3. Precision and spiked recovery of miRNA21 (n=3) 50 48.21±1.34 96.42 2.78 500 493.3±11.00 98.65 2.23 5000 4986.5±151.59 99.73 3.04 Experimental Example 5 Specific detection The study detected miRNA141, miRNA122, miRNA10b, and mixtures of these three substances with miRNA155 and miRNA21, as well as blank samples, and compared them with either the miRNA155 group or the miRNA21 group. Figure 25As can be seen from the FAM spectrum, the fluorescence signals of the miRNA155 group and the mix group were significantly increased, while the fluorescence values ​​of miRNA141, miRNA122, and miRNA10b were lower; from Figure 26 It can be seen that in the Cy5 spectrum, the fluorescence signals of the miRNA21 group and the mix group are significantly increased, while the fluorescence values ​​of miRNA141, miRNA122, and miRNA10b are lower. Figure 25 The t-test was used for comparison. P <0.05 indicates that this method has good specificity for miRNA155; Figure 26 The t-test was used for comparison. P <0.05 indicates that this method has good specificity for miRNA21.

[0056] Experimental Example 6 Actual sample testing Human blood samples were collected by the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and approved by the Clinical Trial Ethics Committee (Ethics Approval No.: ZZURIBG2R2021-126). This study included subjects aged ≥18 years, with 8 healthy individuals serving as negative controls and 10 patients diagnosed with lung cancer serving as positive controls, for methodological evaluation and clinical sample validation. Small RNA was extracted from plasma as the test sample. 5 μL of the small RNA extract was used as the detection target and the detection method described in Example 1 was applied. The results are as follows: Figure 27 , Figure 28 , Figure 29 , Figure 30 As shown, the expression levels of miRNA155 and miRNA21 in lung cancer patient samples were significantly higher than those in healthy controls, and the RT-qPCR results showed the same trend, confirming the robustness and diagnostic reliability of the CRISPR / Cas13a system based on DNA-RNA triple helix probes combined with rolling circle amplification for detecting dual miRNAs in the detection of miRNA155 and miRNA21. Furthermore, from Figure 31 As can be seen, the AUC value of the CRISPR / Cas13a system based on DNA-RNA triple helix probe combined with rolling circle amplification for detecting dual miRNAs in this invention reaches 0.975, which is significantly higher than the AUC value of single miRNA detection of miRNA155 and miRNA21, indicating that the dual-target combined detection strategy has better diagnostic efficacy in the early diagnosis of lung cancer. The AUC values ​​of single miRNA detection of miRNA155 are 0.913 and miRNA21 is 0.850.

[0057] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, characterized in that, include: DNA-RNA triple helix molecular beacon 1, which includes a DNA hairpin H1 and an RNA probe 1. The stem of the DNA hairpin H1 is bound to the RNA probe 1 by hydrogen bonds. The loop of the DNA hairpin H1 specifically recognizes the amplification product of the first target miRNA. The two ends of the RNA probe 1 are labeled with a fluorescent group 1 and a quencher 1, respectively. DNA-RNA triple helix molecular beacon 2, which includes a DNA hairpin H2 and an RNA probe 2. The stem of the DNA hairpin H2 is bound to the RNA probe 2 by hydrogen bonds. The loop of the DNA hairpin H2 specifically recognizes the amplification product of a second target miRNA. The two ends of the RNA probe 2 are labeled with a fluorescent group 2 and a quencher 2, respectively. The stem sequences of DNA hairpin H1 and DNA hairpin H2 are identical, and the sequences of RNA probe 1 and RNA probe 2 are identical. The padlock probe P1 is specifically complementary to the first target miRNA so that in the presence of the first target miRNA, the padlock probe P1 is circularized into a circular DNA template by the action of ligase. The padlock probe P2 is specifically complementary to the second target miRNA so that, in the presence of the second target miRNA, the padlock probe P2 is circularized into a circular DNA template by the ligase. The pre-activated CRISPR / Cas13a system includes Cas13a, crRNA, and acRNA; The rolling circle amplification reaction system includes phi29 DNA polymerase and dNTPs.

2. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The sequence of the DNA hairpin H1 is shown in SEQ ID NO:1, the sequence of the RNA probe 1 is shown in SEQ ID NO:2, the fluorescent group 1 is FAM, and the quencher 1 is BHQ1.

3. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The sequence of the DNA hairpin H2 is shown in SEQ ID NO:3, the sequence of the RNA probe 2 is shown in SEQ ID NO:4, the fluorescent group 2 is Cy5, and the quencher 2 is BHQ2.

4. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The sequence of the padlock probe P1 is shown in SEQ ID NO:5, and the sequence of the padlock probe P2 is shown in SEQ ID NO:

6.

5. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The pre-activated CRISPR / Cas13a system is prepared by mixing Cas13a, crRNA, and buffer solution and incubating to obtain the Cas13a / crRNA complex, followed by adding acRNA and incubating to obtain the final product.

6. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 5, is characterized in that... The final concentration of Cas13a is 0.5–1.5 μmol / L, the final concentration of Cas13a to crRNA is 0.5:1 to 1:1, and the final concentration of acRNA is 0.5–1.5 μmol / L; the sequence of crRNA is shown in SEQ ID NO:7, and the sequence of acRNA is shown in SEQ ID NO:8; the buffer solution is a Tris-HCl buffer solution.

7. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The ligase is T4 ligase, with a final concentration of 0.02–0.1 U / μL; the final concentration of the phi29 DNA polymerase is 0.04–0.2 U / μL; the final concentration of DNA-RNA triple helix beacon one is 125–1000 nmol / L; the final concentration of DNA-RNA triple helix beacon two is 125–1000 nmol / L; the final concentration of padlock probe P1 is 1.5–2.5 nmol / L; the final concentration of padlock probe P2 is 1.5–2.5 nmol / L; the final concentration of the pre-activated CRISPR / Cas13a system is 20–60 nmol / L; and the final concentration of the dNTPs is 400–600 nmol / L.

8. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 1, is characterized in that... The preparation method of the DNA-RNA triple helix molecular beacon 1 includes: mixing DNA hairpin H1 with buffer solution II, annealing and incubating, then adding RNA probe 1, and incubating for 0.5-2.5 h to obtain the final concentration ratio of DNA hairpin H1 to RNA probe 1 is 1:1; the preparation method of the DNA-RNA triple helix molecular beacon 2 includes: mixing DNA hairpin H2 with buffer solution II, annealing and incubating, then adding RNA probe 2, and incubating for 0.5-2.5 h to obtain the final concentration ratio of DNA hairpin H2 to RNA probe 2 is 1:

1.

9. The detection system for dual miRNAs based on a CRISPR / Cas13a system using a DNA-RNA triple-helix probe combined with rolling circle amplification, as described in claim 8, is characterized in that... The second buffer solution is a Tris-HCl buffer solution with a pH of 5.5–9; The Tris-HCl buffer solution comprises: 5–25 mmol / L Tris-HCl and 5–100 mmol / L Na+. + and 5-25 mmol of Mg 2+ .

10. The application of the detection system based on DNA-RNA triple helix probes, CRISPR / Cas13a system combined with rolling circle amplification for detecting dual miRNAs, as described in any one of claims 1 to 9, in the preparation of lung cancer diagnostic and / or auxiliary diagnostic products.

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