Cha@sa-crispr / cas12a system and application thereof in tumor marker molecule detection

Abstract

The application provides a kind of CHA@SA-CRISPR / Cas12a system and its application in tumor marker molecular detection, and the CHA@SA-CRISPR / Cas12a system based on catalytic hairpin self-assembly and CRISPR / Cas12a system construction can be used as a self-sustaining isothermal biosensing platform, and the detection of ultra-sensitive biomarker miRNA-155 is realized by combining the CRISPR / Cas12a feedback circuit initiated by catalytic hairpin self-assembly;Through the target-dependent recognition of two split activator promoters, non-specific background is inhibited, and combined with ds-loop DNA reporter design, the reporter gene leakage and inherent signal noise are minimized to achieve amole level sensitivity;And without exogenous primer, while ensuring the recycling of target RNA, the operation is simple.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a CHA@SA-CRISPR / Cas12a system for detecting miRNA and its application in the molecular detection of tumor markers. Background Technology

[0002] MicroRNAs (miRNAs) are small non-coding RNA molecules ranging from 18 to 25 nucleotides in length. miRNAs play a crucial role in post-transcriptional regulation by modulating the translation of numerous genes. Furthermore, miRNAs have been identified as key regulators of various cellular processes, including apoptosis, differentiation, and proliferation. Clinically, as biomarkers, miRNAs are of great value for disease diagnosis, treatment, and prognosis. The discovery of circulating miRNAs in the blood is significant for tumor marker research, particularly for early prevention, treatment, and diagnosis of tumors. Therefore, miRNA detection has significant clinical implications. Current miRNA detection methods include qRT-PCR, Northern blotting, and microarray analysis. However, these methods suffer from high cost, complex operation, and difficulty in distinguishing miRNAs with similar sequences. Therefore, developing a convenient, reliable, and economical miRNA detection method is extremely important.

[0003] On the one hand, catalytic hairpin self-assembly (CHA) is a detection system based on isothermal enzyme-free nucleic acid signal amplification technology. It designs two sets of complementary probes with hairpin structures based on the target fragment to be detected. When the target fragment is absent, both sets of probes exist in a hairpin structure; however, when the target fragment is present in the detection system, the complementary probes sequentially open and bind to the target fragment, ultimately forming a double strand. The target fragment is displaced during the reaction and can re-enter the next round of reaction, thus amplifying the signal. Compared to traditional PCR technology, CHA catalytic hairpin self-assembly technology has the advantages of no temperature cycling, short reaction time, no enzyme digestion (avoiding errors and contamination caused by enzyme digestion), and high detection sensitivity, capable of detecting target fragments down to the fM level.

[0004] On the other hand, CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated) is an acquired immune system widely found in archaea and bacteria, used to recognize and destroy invading nucleic acids. Based on this, CRISPR / Cas technology is a novel gene-editing system composed of CRISPR loci, Cas effector proteins, and guide RNA. Each CRISPR RNA (crRNA) is designed with a specific recognition region, and successful CRISPR / Cas activation requires adjacent prototypical spacer motifs (PAMs) flanking the target that can be recognized by the crRNA. The CRISPR / Cas system is activated when two conditions are met: the presence of a PAM site corresponding to the CRISPR / Cas system used and nucleic acids complementary to the specific recognition region. Subsequently, the CRISPR / Cas system non-specifically cleaves single-stranded DNA. Cas12a belongs to the class II type V CRISPR system effector protein and is an RNA-guided endonuclease that, guided by CRISPR RNA (crRNA), can bind to specific ssDNA or dsDNA. While cleaving bound nucleic acids, it can also cleave any other ssDNA, a phenomenon known as trans-cleavage activity. Based on these properties, Cas12a is widely used in genome editing and molecular diagnostic technologies.

[0005] To address the problems existing in the prior art, this invention provides a method for detecting miRNA based on a catalytic hairpin self-assembly CRISPR / Cas12a system. This method requires no other exogenous primers and can achieve ultrasensitive detection of miRNA-155 in a single step, providing a highly sensitive, selective, and biostable platform for miRNA detection and advancing research on miRNA in tumor diagnosis and potential clinical applications. Summary of the Invention

[0006] In view of this, in order to solve the above problems, the present invention provides a CHA@SA-CRISPR / Cas12a system for detecting miRNA and its application in the molecular detection of tumor markers.

[0007] To achieve the above objectives, the present invention provides a CHA@SA-CRISPR / Cas12a system for detecting miRNA, comprising at least a catalytic hairpin self-assembly and a CRISPR / Cas12a system; the target miRNA triggers the catalytic hairpin self-assembly to form a DNA double strand; the DNA double strand recombines with the miRNA to form two cleavage activators, thereby activating CRISPR / Cas12a, generating a fluorescent signal and releasing the target miRNA again, thereby reactivating the catalytic hairpin assembly, realizing the cycle of the CHA@SA-CRISPR / Cas12a system.

[0008] In a preferred embodiment, the catalytic hairpin self-assembly consists of hairpin probe HP1 and hairpin probe HP2.

[0009] As a preferred implementation, the CRISPR / Cas12a system consists of Cas12a, crRNA, and ds-loop DNA reporter.

[0010] Preferably, the hairpin probe HP1 consists of 38 nucleotides.

[0011] Preferably, the hairpin probe HP2 consists of 33 nucleotides.

[0012] Preferably, the hairpin probe HP1 contains 15 nucleotides in the stem and loop regions that specifically bind to the target miRNA, and the 19 nucleotides at the 3' end are complementary to the 19 nucleotides at the 5' end of the hairpin probe HP2. The hairpin probe HP1 and the hairpin probe HP2 hybridize to form HP1 / HP2 double-stranded DNA.

[0013] The cycling principle of the CHA@SA-CRISPR / Cas12a system includes: PAM sites and PAM complementary clipping are respectively located in the loop regions of hairpin probes HP1 and HP2; when the target miRNA is added, the target miRNA can specifically bind to the stem-loop region of hairpin probe HP1, causing its stem region to become unstable and exposing the 3' end as a single-stranded landing site; the single-stranded landing site hybridizes complementaryly with the 5' end of hairpin probe HP2, triggering a strand displacement reaction to form HP1 / HP2 double-stranded DNA; through catalytic cycling, the target miRNA is displaced from the stem-loop region of hairpin probe HP1 and released completely, thereby enabling multiple rounds of hairpin opening and formation of HP1 / HP2 double-stranded DNA, realizing the cycling of the CHA@SA-CRISPR / Cas12a system.

[0014] Preferably, the first 12 bases of the HP1 / HP2 double-stranded DNA and the target miRNA can be recognized by crRNA to activate CRISPR / Cas12a cleavage activity, thereby trans-cleaving the ds-loop DNA reporter labeled with fluorescent and quenching groups.

[0015] Preferably, the ds-loop DNA reporter includes an ssDNA reporter and a target miRNA. The ssDNA reporter contains 11 nucleotides that are complementary to the target miRNA, with the sequence TTTATTT in the middle of the complementary bases.

[0016] Preferably, the crRNA consists of 45 nucleotides, including a fixed region of 21 nucleotides starting from the 5' end and a recognition region of 24 nucleotides starting from the 3' end.

[0017] In a preferred embodiment, the target miRNA first opens the pre-annealed hairpin probe HP1, and the opened hairpin probe HP1 induces the opening of the pre-annealed hairpin probe HP2, thereby forming a DNA double helix; the DNA double helix recombines with the miRNA to obtain the cleavage activator.

[0018] In a preferred embodiment, the sequence of the hairpin probe HP1 is shown in SEQ. ID NO.1; the sequence of the hairpin probe HP2 is shown in SEQ. ID NO.2; and the sequence of the crRNA is shown in SEQ. ID NO.5.

[0019] The sequence of the ssDNA reporter is shown in SEQ ID NO. 6.

[0020] As one of the objectives of the invention, the present invention also provides an application of the CHA@SA-CRISPR / Cas12a system as a feedback circuit in the molecular detection of tumor markers, especially in the direct detection of miRNA in clinical blood samples.

[0021] As one of the objectives of this invention, this invention also provides a method for detecting tumor marker molecules based on catalytic hairpin assembly and a CRISPR / Cas12a system. The method involves co-incubating tumor marker molecules with the CHA@SA-CRISPR / Cas12a system as described above, followed by quantitative detection of miRNAs. The tumor marker molecules are derived from blood samples.

[0022] Preferably, the crRNA consists of 45 nucleotides, including a fixed region of 21 nucleotides starting from the 5' end and a recognition region of 24 nucleotides starting from the 3' end.

[0023] Preferably, the detection method includes: incubating a first reaction system comprising at least hairpin probe HP1, hairpin probe HP2 and target miRNA-155 once, then adding Cas12a, crRNA and ssDNA reporter to obtain a second reaction system for a second incubation, and adding ddH2O to detect fluorescence intensity to achieve quantitative detection of miRNA.

[0024] Preferably, the molar ratio of the hairpin probe HP1, the hairpin probe HP2, and the target miRNA is 2:2:1.

[0025] Preferably, the hairpin probe HP1, the hairpin probe HP2, and the target miRNA are reacted at 37°C for 1 hour, and then co-incubated with CRISPR / Cas12a, crRNA, and ssDNA reporter at 37°C before quantitative detection of the target miRNA can be performed.

[0026] As a preferred embodiment, the preparation method of the hairpin probe HP1 and / or the hairpin probe HP2 includes: dissolving the single-stranded DNA of hairpin probe HP1 and / or the single-stranded DNA of hairpin probe HP2 in TE buffer to obtain a mixed solution; then heating the mixed solution at 95°C for 5 min, cooling to 60°C for 30 min, cooling to 37°C for 30 min, and finally cooling to 4°C for 30 min to form a hairpin structure, thereby obtaining the hairpin probe HP1 and / or the hairpin probe HP2.

[0027] The beneficial technical effects obtained by this invention are as follows:

[0028] (1) This invention combines the advantages of catalytic hairpin assembly and CRISPR / Cas12a. Catalytic hairpin assembly is initiated using miRNA, thereby generating double-stranded DNA composed of hairpin probes HP1 and HP2. This activates CRISPR / Cas12a to trans-cleave the ds-loop DNA reporter, emitting fluorescence.

[0029] (2) The segmented crRNA of the present invention splits the 24nt recognition sequence in the crRNA in half to identify the double-stranded DNA product generated by catalytic hairpin assembly and the target miRNA. This design enables CRISPR / Cas12a to recognize RNA sequences and enhances the ability to identify target miRNAs.

[0030] (3) This invention enables two-stage screening of target miRNAs by using catalytic hairpin assembly and segmented crRNA, significantly improving the specificity of the detection system. Even under challenging sample conditions, the reliability and accuracy of miRNA detection are ensured.

[0031] (4) The present invention uses a ds-loop DNA reporter, which can ensure that the target is regenerated and recycled in subsequent reactions. Attached Figure Description

[0032] Figure 1a This is a schematic diagram illustrating the working principle of CHA@SA-CRISPR / Cas12a in a typical embodiment of the present invention.

[0033] Figure 1b This is a schematic diagram illustrating the working principle of CHA@CRISPR / Cas12a in a typical embodiment of the present invention.

[0034] Figures 2a-2d This is a fluorescence intensity comparison diagram used to verify the feasibility of detecting miRNA-155 using CHA@SA-CRISPR / Cas12a in a typical embodiment of the present invention.

[0035] Figure 3 This is a schematic diagram and fluorescence response of the CHA@CRISPR / Cas12a and CHA@SA-CRISPR / Cas12a detection systems before and after the addition of target miRNA in a typical embodiment of the present invention.

[0036] Figures 4a-4d This is a schematic diagram comparing the fluorescence intensity and sensitivity of different fluorescent reporter probes in a typical embodiment of the present invention.

[0037] Figures 5a-5d This is a sensitivity measurement graph, linear range, and standard curve graph of the CHA@SA-CRISPR / Cas12a detection system in a typical embodiment of the present invention.

[0038] Figure 6a and Figure 6b This is a specificity analysis diagram of the CHA@SA-CRISPR / Cas12a detection system in a typical embodiment of the present invention.

[0039] Figure 7a and Figure 7b This is a correlation graph of fluorescence intensity versus target concentration for testing the miRNA-155 content in an actual sample using the CHA@SA-CRISPR / Cas12a detection system in a typical embodiment of the present invention.

[0040] Figures 8a-8bThis is a graph showing the influence of incubation time and the molar ratio of HP1 to HP2 on the detection performance of the CHA@SA-CRISPR / Cas12a detection system in a typical embodiment of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously,

[0042] The described embodiments are some, but not all, of the embodiments in this application.

[0043] As one aspect of the technical solution of this invention

[0044] The CHA@SA-CRISPR / Cas12a system involved in the process involves: recognizing the last 15 bases of the target miRNA through the hairpin probe HP1, and then opening the hairpin probe HP2 to form HP1 / HP2 double-stranded DNA. The HP1 / HP2 double-stranded DNA and the first 12 bases of the target miRNA can be recognized by crRNA together, activating the CRISPR / Cas12a trans-cleavage activity to cleave the ds-loop DNA reporter labeled with fluorescent and quenching groups.

[0045] In some preferred embodiments, the double-stranded DNA is formed by hybridization of hairpin probe HP1 and hairpin probe HP2.

[0046] In some preferred embodiments, the hairpin probe HP1 has 15 bases complementary to the target miRNA and 19 bases complementary to the hairpin probe HP2.

[0047] Another aspect of the present invention provides a method for preparing hairpin probe HP1 and / or hairpin probe HP2, comprising: adding hairpin probe HP1 and / or hairpin probe HP2 and TE buffer to the overall reaction system to obtain solution I and / or solution II; then heating the obtained solution I and / or solution II at 95°C for 5 min, cooling to 60°C for 30 min, cooling to 37°C for 30 min, and finally cooling to 4°C for 30 min to obtain hairpin probe HP1 and / or hairpin probe HP2.

[0048] Furthermore, the concentration of the hairpin probe HP1 and / or hairpin probe HP2 is 100 μM.

[0049] Furthermore, the TE buffer comprises 1×TE buffer.

[0050] In some preferred embodiments, the 20 μL solution I mixture contains 1 μL of 100 μM hairpin probe HP1 and 99 μL of 1×TE buffer.

[0051] The 20 μL solution II mixture contains 1 μL of 100 μM hairpin probe HP2 and 99 μL of 1×TE buffer.

[0052] As a second aspect of the technical solution of the present invention, the present invention provides an integrated method for the direct detection of clinical blood samples, which utilizes catalytic hairpin self-assembly, the synergistic effect of CRISPR / Cas12a and ds-loop DNA reporter to analyze miRNA-155.

[0053] As a third aspect of the technical solution of this invention, this invention also provides analysis of miRNA under the synergistic effect of catalytic hairpin self-assembly, CRISPR / Cas12a, and ss DNA reporter, specifically including: mixing hairpin probe HP1, hairpin probe HP2, DEPC-treated water, Tris-HCl buffer, and target miRNA of different concentrations and reacting at 37°C for 1 hour. After the reaction is complete, add reaction solution, DEPC-treated water, NE buffer 2.1, ss DNA reporter, crRNA, and Cas12a, and then react at 37°C for 1 hour before finally terminating the reaction. Finally, add ddHP2O for fluorescence measurement.

[0054] In some preferred embodiments, the mixture contains hairpin probe HP1, hairpin probe HP2, Tris-HCl buffer, different concentrations of miRNA, DEPC-treated water, NE buffer 2.1, ds-loop DNA reporter, gRNA, and Cas12a.

[0055] Furthermore, the Tris-HCl buffer comprises 1 M Tris-HCl buffer.

[0056] Furthermore, the concentrations of the hairpin probes HP1 and HP2 are 1 μM.

[0057] Furthermore, the NE buffer 2.1 comprises 10×NE buffer 2.1.

[0058] Furthermore, the concentration of the ds-loop DNA reporter is 5 μM.

[0059] Furthermore, the concentrations of the crRNA and Cas12a are 1 μM.

[0060] In some preferred embodiments, the 20 μL reaction solution mixture contains 2 μL hairpin probe HP2, 8 μL Tris-HCl buffer, 7 μL DEPC-treated water, 2 μL hairpin probe HP2, and 1 μL miRNA of varying concentrations. The mixture is incubated at 37°C for 1 hour. After the reaction is complete, 1 μL of the reaction solution is taken from the 20 μL reaction mixture and mixed with 13 μL DEPC-treated water, 2 μL 10×NE buffer 2.1, 2 μL ds-loop DNA reporter, 1 μL crRNA, and 1 μL CRISPR / Cas12a, and then incubated at 37°C for 1 hour. 80 μL ddH2O is added to the 20 μL reaction mixture for fluorescence measurement.

[0061] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0062] CRISPR / Cas12a and 10×NE buffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg / ml Recombinant Albumin, pH 7.9) were purchased from New England Biolabs (USA) Ltd. (Beijing, China). DEPC-treated water and 20×PBS buffer (200 mmol / L Na2HPO4, 35 mmol / L KHP2PO4, 2.74 mol / L NaCl, 53 mmol / L KCl, pH 7.2–7.6), 1 M Tris-HCl buffer (1 mol / L Tris-HCl; pH 7.9–8.1), tetramethylethylenediamine (TEMED), 30% acrylamide:bisacrylamide (29:1), 1×triborate-EDTA (TBE) (18 mM Tris, 18 mM boric acid, 2 mM EDTA, pH = 8.0), 6×DNA loading buffer, 25–500 bp DNA markers, and all oligonucleotides were supplied by Sangon Biotech Co., Ltd. (Shanghai, China).

[0063] The ultrapure water with a resistivity of 18.25 MΩ / cm used throughout the experiment was obtained from the Milli-A10 system (Milli-A10, USA).

[0064] Instruments and Meters

[0065] Fluorescence spectra were measured using an F97 Pro fluorescence spectrophotometer (Shanghai Leng116 Optical Technology Co., Ltd.). Real-time fluorescence spectra were measured using an H1600 isothermal amplification fluorescence detector (Hangzhou Baiheng Technology Co., Ltd.). The reaction temperature was kept constant using a Hercules Bio-Rad T100 thermal cycling system (USA).

[0066] The gel electrophoresis results were visualized under ultraviolet light using a gel imaging system (Hangzhou Derui Gene Scientific Instruments Co., Ltd., China). The specific method included: Polyacrylamide gel electrophoresis (PAGE) analysis: 10 μL of samples containing different components, stained with 100×4S Red Plus, were dropped into the wells of a freshly prepared 14% native-PAGE gel. DNA electrophoresis analysis was performed in 1×TBE buffer at a constant potential of 130 V for 60 minutes. Subsequently, the gel was directly exposed to ultraviolet light, and images were captured using the gel imaging system. The technical solution of this invention is further described in detail below through specific embodiments.

[0067] The sequences used in this invention are shown in Table 1.

[0068] Table 1 Sequence List

[0069] Example 1

[0070] This embodiment provides a CHA@SA-CRISPR / Cas12a system for detecting miRNA, which combines catalytic hairpin self-assembly (CHA) with a CRISPR / Cas12a module activated by division, thereby achieving self-amplified detection of miRNA-155.

[0071] The detection of miRNA-155 using the CHA@SA-CRISPR / Cas12a system is divided into two stages, see [link / reference]. Figure 1a The figure shows a schematic diagram illustrating the working principle of the CHA@SA-CRISPR / Cas12a system. As can be seen from the figure, the detection principle of the CHA@SA-CRISPR / Cas12a detection system proposed in this invention for miRNA-155 includes:

[0072] In the first phase, hairpin probes HP1 and HP2 coexisted in a metastable state in the absence of target miRNA-155. Upon addition of target miRNA-155, it specifically binds to the loop region of hairpin probe HP1, causing instability at its stem and exposing the 3' end as a single-stranded landing site. This single-stranded landing site hybridizes complementary to the 5' end of hairpin probe HP2, triggering a strand displacement reaction to form HP1 / HP2 double-stranded DNA. Through catalytic cycling, miRNA-155 is displaced from hairpin probe HP1 and released completely, enabling multiple rounds of hairpin opening and double-strand formation. In the second stage, HP1 / HP2 double-stranded DNA and the released miRNA-155 synergistically act as two cleavage activators (cleavage activators) to activate the CRISPR / Cas12a system. HP1 / HP2 double-stranded DNA contains the PAM proximal seed sequence required for Cas12a recognition, while miRNA-155 occupies the complementary PAM distal region. Upon assembly with crRNA and Cas12a, the cleavage activator initiates strong trans-cleavage activity, selectively degrading the ds-loop DNA embedded in the double-labeled DNA. The single-chain loop sequence in the reporter, through this cleavage action, separates the fluorophore from the quencher, generating a detectable fluorescent signal.

[0073] Importantly, compared to systems relying on a single double-stranded activator, the two-component mechanism ensures that the Cas12a protein is not activated by the nonspecific formation of only one component, thus significantly reducing background noise. Paralogous cleavage of the Cas12a protein not only generates a signal but also releases pre-isolated miRNA-155 from the reporter gene, thereby promoting target regeneration and amplification. The released miRNA-155 restarts catalytic hairpin self-assembly and CHA by binding to the novel hairpin probe HP1, thereby regenerating the hairpin assembly cycle and the cleavage activation substrate of Cas12a. This feedback loop enables exponential signal amplification with minimal background noise, improving detection sensitivity while reducing background noise.

[0074] Feasibility Study of CHA@SA-CRISPR / Cas12a System for Detecting miRNA-155

[0075] First, hairpin probes HP1 and HP2 were added to 1×TE buffer to obtain solutions I and II, respectively. These solutions were annealed at 95°C for 5 minutes, then incubated at 60°C for 30 minutes, 37°C for 30 minutes, and finally at 4°C for 30 minutes to form hairpin structures. Specifically, 20 μL of solution I contained 1 μL of 100 μM hairpin probe HP1 and 99 μL of 1×TE buffer; 20 μL of solution II contained 1 μL of 100 μM hairpin probe HP2 and 99 μL of 1×TE buffer.

[0076] Next, 2 μL of 1 μM HP1, 2 μL of 1 μM HP2, 1 μL of miRNA-155 at different concentrations, 7 μL of DEPC-treated water, and 8 μL of Tris buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 50 mM MgCl2) were mixed and incubated at 37°C for 60 minutes to initiate the catalytic hairpin self-assembly CHA reaction.

[0077] Meanwhile, after annealing at 95°C for 5 minutes, the ssDNA reporter and miRNA-155 react to form a stable structure, yielding the ds-loop DNA reporter.

[0078] Then, 1 μL of the CHA reaction mixture was added to a solution containing 2 μL of 10×NEBuffer 2.1, 13 μL of DEPC-treated water, 2 μL of 5 μM ds-loop DNA reporter molecule, 1 μL of 1 μM gRNA and 1 μL of 1 μM CRISPR / Cas12a, and incubated at 37°C for 60 minutes to activate the transcleavage process of CRISPR / Cas12a.

[0079] In a preferred embodiment, the sequence of the hairpin probe HP1 is shown in SEQ. ID NO.1; the sequence of the hairpin probe HP2 is shown in SEQ. ID NO.2; and the sequence of the crRNA is shown in SEQ. ID NO.5.

[0080] The sequence of the ssDNA reporter is shown in SEQ ID NO. 6.

[0081] To verify the feasibility of the CHA@SA-CRISPR / Cas12a system in detecting miRNA-155, this embodiment verifies the fluorescence intensity of the CHA@SA-CRISPR / Cas12a system after co-incubation with miRNA-155 under different conditions. The sequence of the miRNA-155 used is shown in Seq. ID NO. 7.

[0082] The specific different detection conditions shown include:

[0083] In the first experimental group, the concentrations of each component were as follows: [HP1] = [HP2] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0084] In the second experimental group, the concentrations of each component were: [HP1] = [HP2] = 50 nM; [miRNA-155] = 50 nM.

[0085] In the third experimental group, the concentrations of each component were: [ssDNA reporter] = 100 M; [miRNA-155] = 100 nM.

[0086] In the fourth experimental group, the concentrations of each component were as follows: [HP1] = [HP2] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ss DNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0087] Incubation conditions include: incubation at 37°C for 60 minutes.

[0088] See results Figures 2a-2d .

[0089] Depend on Figure 2a As shown, fluorescence measurements were performed on the structure formed by binding hairpin probe HP1, hairpin probe HP2 and miRNA-155, which showed a strong fluorescence signal compared to the structure without the added target miRNA.

[0090] Figure 2bAs shown, the lanes sequentially include hairpin probe HP1, hairpin probe HP2, (hairpin probe HP1 + hairpin probe HP2 + miRNA), and hairpin probe HP1 + hairpin probe HP2. As can be seen from the figure, HP1 and HP2 each show distinct bands at different positions. When HP1 and HP2 bind, no additional band is observed, confirming the stable metastable conformation of the hairpin probes. However, after the addition of miRNA-155, a new band appears near the 50 bp region, and this band is higher than the individual HP1 or HP2 bands, indicating that HP1-HP2 hybridization forms a complex.

[0091] like Figure 2c Non-denaturing polyacrylamide gel electrophoresis analysis of the ds-loop DNA reporter showed lanes sequentially including miRNA-155 and miRNA-155+ssDNA reporter. As can be seen from the figure, after the single-stranded DNA reporter gene was mixed with miRNA-155, a higher molecular weight band was produced than that of the single-stranded DNA reporter gene alone. This indicates that hybridization occurred and a double-stranded circular DNA reporter gene was formed.

[0092] like Figure 2d The CHA@SA-CRISPR / Cas12a detection system was analyzed by non-denaturing polyacrylamide gel electrophoresis. The lanes were sequentially labeled HP+ / HP2+Cas12a+ crRNA and HP1+HP2+Cas12a+ crRNA+ds-loop DNA reporter. As shown in the figure, when the double-stranded circular DNA reporter gene was introduced into a mixture of HP1, HP2, and Cas12a-crRNA, the bands were different from those of the target DNA reporter. Figure 2c The bands in the previous test showed no change, confirming that CRISPR / Cas12a could not be activated without the target triggering HP1-HP2 complex. Conversely, when miRNA-155 was added to a mixture of HP1, HP2, and Cas12a-crRNA, the band of the double-stranded circular DNA reporter gene disappeared, and a new band matching the position of miRNA-155 appeared. This indicates that the CHA@SA-CRISPR / Cas12a system was successfully activated and subsequently trans-cleaved the reporter gene, releasing the pre-isolated miRNA.

[0093] Comparison of different fluorescent reporter probes

[0094] This embodiment also validated the CHA@SA-CRISPR / Cas12a system using different fluorescent reporter probes, including the ssDNA reporter sequence as shown in SEQ ID NO:6;

[0095] It includes an ssDNA reporter and a target miRNA. The ssDNA reporter contains 11 nucleotides that are complementary to the target miRNA. The complementary bases are connected by the sequence TTTATTT. The sequence of miRNA-155 is shown in Seq. ID NO. 7.

[0096] Specific experimental conditions include:

[0097] [HP1] = [HP2] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNAreporter] = [ds-loop DNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0098] See results Figure 4a The image shows a comparison of the suppression of background fluorescence signals by ssDNA reporter and ds-loop DNA reporter. The quenching effect of the ds-loop contained in the ds-loop DNA reporter makes its suppression effect on background signals significantly higher than that of the ssDNA reporter.

[0099] Figure 4b The fluorescence signal comparison diagrams using ssDNA reporter and ds-loop DNA reporter show that, in the presence of target miRNA, the signal amplification performance is improved, and the ds-loop DNA reporter exhibits higher sensitivity.

[0100] Figure 4c Real-time fluorescence comparison using ssDNA reporter and ds-loop DNA reporter to illustrate the reaction kinetics of CRISPR / Cas12a trans-cleavage, where the ds-loop DNA reporter exhibits faster cleavage rate and higher final fluorescence intensity compared to the ssDNA reporter.

[0101] Figure 4d This diagram shows the relative response of fluorescence signals from ssDNA reporter and ds-loop DNA reporter. The relative response values ​​were obtained by dynamically detecting the fluorescence intensity of ds-loop DNA and ssDNA reporter at 15 min, 30 min, 45 min, and 60 min. The relative response was calculated using the formula F. ssDNA reporter / F ds-loop DNA reporterAs shown in the figure, the relative response increases with time, indicating that the fluorescence intensity of the ssDNA reporter increases at a higher rate than that of the ds-loop DNA reporter.

[0102] Optimization of conditions

[0103] Furthermore, this embodiment also optimizes the above scheme by optimizing the conditions, specifically including the incubation time and the molar ratio of HP1 to HP2.

[0104] The results are as follows Figure 8b As shown, the signal effect is optimal when the concentration ratio is 1:1. The reaction conditions include: the concentration ratio of hairpin probe HP1 and hairpin probe HP2 is set to 0.5:1, 1:1, 1.5:1, and 2:1; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ds-loop DNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0105] Furthermore, such as Figure 8b As shown, the reaction conditions included: [HP1] = [HP2] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNA reporter] = [ds-loop DNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0106] The reaction time of catalytic hairpin self-assembly was analyzed. As shown in the figure, the highest fluorescence intensity was observed at a reaction time of 60 minutes, indicating that the reaction reached its optimal state. Therefore, 60 minutes was determined to be the most favorable amplification time for catalytic hairpin self-assembly.

[0107] Sensitivity analysis

[0108] See Figures 5a-5d This is a sensitivity evaluation diagram of the CHA@SA-CRISPR / Cas12a detection system provided by the present invention. miRNA-155 solutions were prepared at the following concentrations: 0, 250 aM, 2.5 fM, 25 fM, 250 fM, 2.5 pM, 25 pM, 250 pM, and 2.5 nM. The miRNA-155 solutions at each concentration were then added to the CHA@SA-CRISPR / Cas12a detection system for quantitative detection.

[0109] The detection conditions included: [HP1] = [HP2] = 2.5 nM, [Cas12a] = 50 nM, [crRNA] = 50 nM, and [ds-loop DNA reporter] = 250 nM.

[0110] See Figures 5a-5d ,in, Figure 5a The graph shows a comparison of fluorescence spectra of miRNA-155 solutions at different concentrations. As can be seen from the graph, the fluorescence intensity increases with increasing concentration. Figure 5b and Figure 5c The figures show concentration-peak fluorescence intensity plots and correlation heatmaps for miRNA-155 solutions at various concentrations. The figures demonstrate a clear dose-dependent relationship between peak fluorescence intensity and concentration. A dynamic response standard curve for miRNA-155 concentration-peak fluorescence intensity is also plotted. (See [link to relevant documentation]). Figure 5d As shown in the figure, the calibration curve reveals a strong linear relationship between lg(CmiRNA-155 / aM) and the fluorescence signal. The linear response equation is: F = 329.2lg(CmiRNA-155 / aM). miRNA-155 / aM)-381.9 (R²= 0.9929).

[0111] In summary, the fluorescence signal response gradually increased as the concentration of the target miRNA increased from 250 aM to 2.5 nM. Even at a low concentration of 250 aM, the target miRNA could still induce a fluorescence response higher than the background signal.

[0112] Furthermore, the present invention also provides a comparative CHA@CRISPR / Cas12a detection system, see [link to relevant documentation]. Figure 1b Its working principle diagram includes the hairpin probe HP1 * and hairpin probe HP2 * HP1 hairpin probe for integration with CRISPR / Cas12a * and hairpin probe HP2 * Each consists of a single-stranded circular DNA molecule with an 8-nucleotide 5' overhang. (This is mentioned in the context of the hairpin probe HP1.) * In the middle, the 5' end protrudes as the site for the target miRNA, and the loop region contains the CRISPR / Cas12a PAM site. Hairpin probe HP2 * The 5' end protrusion serves as a miRNA / HP1 * The competing sites in the double strands, with the complementary sequence of PAM located in the loop region. In this case, only HP1... * HP2 * The double strand can activate CRISPR / Cas12a activity.

[0113] See Figure 3In section A, the mechanism of action of CHA@CRISPR / Cas12a is as follows: the target miRNA triggers the catalytic hairpin self-assembly to form a DNA double strand; the DNA double strand can activate CRISPR / Cas12a, which cuts the ssDNA reporter to produce fluorescence.

[0114] The signal-to-noise ratio of the two detection systems for quantitative detection of target miRNA-155 was compared and analyzed.

[0115] Specifically, the detection conditions include: [HP1] = [HP2] = [HP1*] = [HP2*] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNA reporter] = [ds-loop DNA reporter] = 250 nM; [miRNA-155] = 2.5 nM.

[0116] Figure 3 The C in the image is the CHA@CRISPR / Cas12a detection system. Fluorescence measurements were performed on the structure formed by the binding of hairpin probe HP1, hairpin probe HP2*, and miRNA-155. The results showed a low fluorescence signal-to-noise ratio of approximately 3.

[0117] See Figure 3 B in the diagram illustrates the working principle of the CHA@SA-CRISPR / Cas12a detection system (see Example 1). D represents the CHA@SA-CRISPR / Cas12a detection system. Fluorescence measurements were performed on the structure formed by the combination of hairpin probe HP1, hairpin probe HP2, and miRNA-155, showing a high fluorescence signal-to-noise ratio of approximately 8.

[0118] Figure 3 In the figure, E represents the quantitative analysis of the amplification capacity between CHA@CRISPR / Cas12a and CHA@SA-CRISPR / Cas12a. F and F0 represent the peak fluorescence intensity of the amplification system in the presence and absence of target miRNA, respectively. By comparing the fluorescence signal signal-to-noise ratio of the CHA@CRISPR / Cas12a detection system and the CHA@SA-CRISPR / Cas12a detection system, it can be observed that the latter's signal-to-noise ratio is more than twice that of the former, indicating that the CHA@SA-CRISPR / Cas12a detection system provided by this invention has higher detection sensitivity.

[0119] Specificity analysis

[0120] The high homology of microRNAs (miRNAs) underscores the importance of specificity in achieving accurate miRNA detection.

[0121] Therefore, this invention provides a CHA@SA-CRISPR / Cas12a detection system for specific analysis of the quantitative detection of target miRNA-155 and non-target miRNAs. The non-target miRNAs include: miRNA-141 (sequence shown in Seq. ID NO. 8), miRNA-21 (sequence shown in Seq. ID NO. 9), miRNA-16 (sequence shown in Seq. ID NO. 10), and let-7d (sequence shown in Seq. ID NO. 11). These were analyzed together with miRNA-155 to determine the specificity of HP1 and HP2 for miRNA-155, at a concentration of 2.5 nM. The sequences of miRNA-141, miRNA-21, miRNA-16, and let-7f are shown in Table 1.

[0122] Experimental conditions: [HP1] = [HP2] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNA reporter] = 250 nM; [miRNA-21] = [miRNA-141] = [miRNA-16] = [let-7f] = [miRNA-155] = 2.5 nM.

[0123] See Figure 6a As shown in the figure, only the target miRNA-155 produced a strong signal response, while the non-target miRNAs (miRNA-141, miRNA-21, miRNA-16, and let-7f) did not cause significant fluorescence changes compared to the blank sample (without added miRNA). Clearly, the CHA@SA-CRISPR / Cas12a detection system has significant specificity for detecting the target miRNA-155. The fluorescence reactions of the four unrelated non-target miRNAs (miRNA-21, miRNA-141, miRNA-16, and Let-7f) indicate that background activation is negligible. These results confirm the high sequence specificity and strong discrimination ability of the CHA@SA-CRISPR / Cas12a detection system. The error bars were obtained from three replicate tests.

[0124] Furthermore, to evaluate the ability of the CHA@SA-CRISPR / Cas12a detection system to distinguish between mutants and fully matched target miRNA-155, this invention also provides five synthetic single-base mismatched miRNA targets, namely single-base mismatch targets MT-1a (sequence shown in Seq. ID NO. 12), MT-1b (sequence shown in Seq. ID NO. 13), MT-1c (sequence shown in Seq. ID NO. 14), MT-1d (sequence shown in Seq. ID NO. 15), and MT-1e (sequence shown in Seq. ID NO. 16).

[0125] In addition, the specificity of the CHA@SA-CRISPR / Cas12a detection system in distinguishing mutants from fully matched target miRNA-155 was compared and analyzed.

[0126] Experimental conditions: [HP1] = [HP2] = [HP1*] = [HP2*] = 5 nM; [Cas12a] = 50 nM; [crRNA] = 50 nM; [ssDNA reporter] = 250 nM; [MT-1a] = [MT-1b] = [MT-1c] = [MT-1d] = [MT-1e] = [miRNA-155] = 2.5 nM.

[0127] Continue reading Figure 6b The CHA@SA-CRISPR / Cas12a detection system showed minimal signal relative fluorescence response to five single-base mutants (MT-1a to MT-1e) of miRNA-155 compared to the fully matched miRNA-155.

[0128] See Figure 6b The CHA@SA-CRISPR / Cas12a detection system was used to measure fluorescence responses after incubation with perfectly matched miRNA-155 and single-base mismatched sequences (MT-1a to MT-1e). The fluorescence intensity of all mismatched targets exceeded 50% of the response of perfectly matched targets, indicating that the specificity of the conventional system is limited.

[0129] Compared to some previously reported studies, the miRNA-155 provided in this embodiment has shown significant improvement in distinguishing base mutations. While not yet completely 100% effective in distinguishing base mutations, in in vitro experiments assessing anti-interference capabilities, signals from miRNA-141, miRNA-21, miRNA-16, and let-7d consistently correlated with background fluorescence levels. These results demonstrate that the CHA@SA-CRISPR / Cas12a method exhibits high selectivity.

[0130] Sample Analysis

[0131] According to reports, miRNA-155 is typically upregulated in blood samples from breast cancer patients compared to healthy individuals.

[0132] To verify the applicability of the CHA@SA-CRISPR / Cas12a detection system in detecting miRNA-155 in actual samples, human whole blood samples were analyzed, including a healthy group (n=6) and a disease group (n=6). The CHA@SA-CRISPR / Cas12a detection system was added to the samples for quantitative analysis of miRNA-155 content.

[0133] Before testing, the samples are treated with a whole blood test kit to reduce the influence of other impurities.

[0134] See Figure 7a and Figure 7b The study demonstrated that when using the CHA@SA-CRISPR / Cas12a detection system, the patient group exhibited higher fluorescence intensity than the healthy group. Since the fluorescence signal is correlated with the target concentration, this observation indicates that the expression level of miRNA-155 in cancer patients is significantly higher than that in the healthy control group.

[0135] The above results are consistent with existing reports on the upregulation of miRNA-155 in blood samples from breast cancer patients, and confirm its potential for practical application.

[0136] in conclusion:

[0137] The CHA@SA-CRISPR / Cas12a detection system provided by this invention is used for the qualitative and quantitative detection of miRNA-155, providing accurate and sensitive detection of low concentrations of miRNA. These characteristics enable DP-HP to exhibit exceptional ultra-high sensitivity, capable of detecting miRNAs down to femtomolar levels or even attomolar levels, and demonstrating excellent specificity in accurately distinguishing target miRNA-155 from closely related sequences. Its successful application in real blood sample analysis reveals its practical value in clinical settings, particularly in promoting early cancer diagnosis. Furthermore, the detection method based on the CHA@SA-CRISPR / Cas12a detection system has shown excellent biostability when screening miRNAs from human serum, providing promising potential for further monitoring miRNAs in living cells. In particular, the technical solution used in this invention does not require the addition of exogenous primers, achieving ultra-sensitive detection of miRNA-155 at the attomolar level in a single step, effectively avoiding false positives during the detection process.

[0138] Overall, the CHA@SA-CRISPR / Cas12a detection system provides a promising platform for developing advanced molecular diagnostic tools, offering higher sensitivity, specificity, and cost-effectiveness, and opening new avenues for enhancing biomedical research and clinical diagnostics.

[0139] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0140] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A CHA@SA-CRISPR / Cas12a system for detecting miRNA, characterized in that, At least including catalytic hairpin self-assembly and CRISPR / Cas12a system; The target miRNA triggers the catalytic hairpin self-assembly to form a DNA double strand; after recombination with the target miRNA, the DNA double strand forms two cleavage activators that activate CRISPR / Cas12a, cleaving the ds-loop DNA reporter to generate a fluorescent signal and release the target miRNA again. The DNA double strand and the target miRNA, as two cleavage activators, work together to reactivate the CRISPR / Cas12a system, realizing the cycle of the CHA@SA-CRISPR / Cas12a system, which can be used for the self-amplification detection of miRNA; The catalytic hairpin self-assembly consists of hairpin probe HP1 and hairpin probe HP2; The hairpin probe HP1 consists of 38 nucleotides; the stem and loop regions of the hairpin probe HP1 contain 15 nucleotides that specifically bind to the target miRNA, and the 19 nucleotides at the 3' end are complementary to the 19 nucleotides at the 5' end of the hairpin probe HP2. The hairpin probe HP2 consists of 33 nucleotides; The target miRNA specifically binds to the stem-loop region of the hairpin probe HP1 and exposes the 3' end of the hairpin probe HP1 as a single-stranded landing site; the single-stranded landing site hybridizes with the 5' end of the hairpin probe HP2, triggering a strand substitution reaction to form HP1 / HP2 double-stranded DNA, which is the DNA double strand; The CRISPR / Cas12a system consists of Cas12a, crRNA, and ds-loop DNA reporter; crRNA consists of 45 nucleotides, including a fixed region of 21 nucleotides starting from the 5' end and a recognition region of 24 nucleotides starting from the 3' end. The ds-loop DNA reporter consists of an ssDNA reporter and a target miRNA. The ssDNA reporter contains 11 bases complementary to the target miRNA, and the sequence between the complementary bases is TTTATTT. The first 12 bases of the HP1 / HP2 double-stranded DNA and the target miRNA can be recognized by crRNA to activate CRISPR / Cas12a cleavage activity, which trans-cleaves the ds-loop DNA reporter labeled with fluorescent and quenching groups. The sequence of the hairpin probe HP1 is shown in SEQ. ID NO.1; The sequence of the hairpin probe HP2 is shown in SEQ. ID NO.2; The sequence of crRNA is shown in SEQ ID NO. 5; The sequence of the ssDNA reporter is shown in SEQ ID NO. 6; The target miRNA is miRNA-155.

2. The CHA@SA-CRISPR / Cas12a system according to claim 1, characterized in that, The target miRNA first opens the pre-annealed hairpin probe HP1, and the opened hairpin probe HP1 induces the opening of the pre-annealed hairpin probe HP2, thereby forming a DNA double helix; the DNA double helix recombines with the target miRNA to obtain the cleavage activator.

3. The CHA@SA-CRISPR / Cas12a system according to claim 1, characterized in that, The preparation method of the hairpin probe HP1 and / or the hairpin probe HP2 includes: dissolving the single strand of DNA of hairpin probe HP1 and / or the single strand of DNA of hairpin probe HP2 in TE buffer to obtain a mixed solution; then heating the mixed solution at 95°C for 5 min, cooling to 60°C for 30 min, cooling to 37°C for 30 min, and finally cooling to 4°C for 30 min to form a hairpin structure, thereby obtaining the hairpin probe HP1 and / or the hairpin probe HP2.

Images