A sensing probe for detecting miRNA, a detection method and uses thereof

Abstract

The present application relates to a sensing probe for detecting miRNA, its detection method and use. The sensing probe has a handle-loop structure and is formed by hybridization of a circular padlock probe and a hairpin structure H probe. The circular padlock probe includes a DNA polymerase extension sequence region, an endonuclease recognition sequence region, a template sequence region, and a target miRNA binding sequence region, and the bases are complementary to form a circle. The hairpin structure H probe has a stem-loop structure, including a loop part and a stem part with complementary base hybridization. The hairpin structure H probe contains a reporter sequence region, an endonuclease recognition sequence region, a primer sequence region for promoting chain growth, and a template sequence region. After the circular padlock probe binds to the target miRNA, the hairpin structure H probe is released by DNA polymerase chain extension. The sensing probe for detecting miRNA has the advantages of rapidity, simplicity, low cost, high detection sensitivity and specificity.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and particularly relates to a sensing probe for detecting miRNA, a detection method and uses thereof. Background Art

[0002] Single-stranded nucleic acids play a key role in the pathological investigation and early diagnosis of diseases. Specifically, miRNA is a short RNA molecule composed of 21-23 nucleotides and plays an important role in the occurrence and development of tumors. Compared with blood and tissue samples, urine miRNA detection has attracted great interest, mainly because of the non-invasiveness and convenience of urine collection. Therefore, the identification of urine single-stranded nucleic acids can be used as an important method for diagnosing and tracking malignant tumors.

[0003] In the past few decades, miRNA analysis methods for diagnosing tumors, such as polymerase chain reaction (PCR) technology, have been widely used as a conventional method for detecting nucleic acids, especially single-stranded RNA (ssRNA), due to their excellent sensitivity and precision; however, PCR-based detection methods require expensive and precise thermal cycling instruments, which limits their use in resource-limited settings. In recent years, many new miRNA analysis methods have been developed, and isothermal exponential amplification techniques have been introduced into nucleic acid detection. For example, rolling circle amplification (RCA), catalytic hairpin self-assembly technology (CHA), hybridization chain reaction (HCR), exponential amplification reaction (EXPAR), and primer exchange reaction (PER). Among them, entropy-driven CHA and HCR originate from base complementary pairing. These enzyme-free amplification methods are relatively less effective in amplifying DNA and are significantly interfered by background signals. EXPAR and PER are both efficient amplification techniques for detecting nucleic acids. The target molecule has a specific sequence at its 3' end, which serves as a primer to stimulate signal amplification. However, many ssRNA molecules have long nucleotide sequences and ambiguous 3' ends; therefore, ssRNA cannot be directly used as a primer to initiate amplification. Compared with the above technologies, the RCA system utilizes a fast, accurate, and isothermal amplification procedure and does not require specialized equipment, which has convenience. However, RCA-based biosensors still exhibit insufficient sensitivity and are troubled by inevitable non-specific amplification problems.

[0004] The inventors have found the deficiencies of the above technologies, and it is still a very difficult task to develop a convenient, highly specific, accurate, and effective method for measuring ssRNA. Summary of the Invention

[0005] Based on the above technical problems, the technical problem to be solved by the present application is to provide a sensing probe for detecting miRNA, a biosensor for detecting miRNA, and their uses, which are fast, simple, low-cost, highly sensitive, and specific in detection.

[0006] This application is implemented through the following technical solutions:

[0007] The first aspect of this application provides a sensing probe for detecting miRNA.

[0008] A sensing probe for detecting miRNA, the sensing probe is a handle-loop structure, which is formed by hybridization of a circular padlock probe and a hairpin structure H probe; the circular padlock probe includes a DNA polymerase extension sequence region, an endonuclease recognition sequence region, a template sequence region, and a target miRNA binding sequence region, and the bases are complementary to form a circle; the hairpin structure H probe is a stem-loop structure, including a loop part and a stem part with complementary base hybridization, and the hairpin structure H probe contains a reporter sequence region, an endonuclease recognition sequence region, a primer sequence region for promoting strand growth, and a stem-loop template sequence region; after the circular padlock probe binds to the target miRNA, the hairpin structure H probe is released through DNA polymerase chain extension.

[0009] Further, the circular padlock probe sequentially includes a DNA polymerase extension sequence region, a first endonuclease recognition sequence region, a template sequence region, a second endonuclease recognition region, and a target miRNA binding sequence region in a clockwise direction from the molecular extension, and the bases are complementary hybridized to form a circle; when the circular padlock probe binds to the target miRNA, after strand extension and endonuclease recognition and cleavage, a secondary sequence is released, and the secondary sequence is transcribed from the template sequence, and the secondary sequence hybridizes with the hairpin structure H probe.

[0010] The sequence of the circular padlock probe is shown in Table 1 or as SEQ ID NO.1:

[0011]

[0012] The target miRNA The binding sequence region is in italic font, the endonuclease recognition sequence region is in bold font, the template sequence region is in single underlined area "", and the DNA polymerase extension sequence region is in double underlined area Further, the 5' end of the circular padlock probe is phosphorylated.

[0013] Further, the stem of the hairpin structure H probe is two single strands. Among them, the first single strand contains a reporter sequence region and an endonuclease recognition sequence region, and part of the bases of the first single strand are complementary hybridized with the bases of the loop part to form a stem-loop template sequence region. The second single strand contains a primer sequence region for promoting strand extension and is complementary to the terminal bases of the loop part; after the secondary sequence released by the circular padlock probe binds to the hairpin structure H probe, the reporter sequence is released after strand extension and endonuclease cleavage.

[0014] The reporter sequence described in this application contains a G-quadruplex sequence.

[0015] The hairpin structure H probe described in this application is shown in Table 1 or as SEQ ID NO.2.

[0016]

[0017] Among them, the stem-loop template sequence region is underlined once “” 、 The primer sequence region that promotes strand extension is the word The body is in bold, and the reporting sequence region is double underlined. The endonuclease recognition sequence region is in italic font.

[0018] The preparation method of the sensing probe for detecting miRNA described in this application: After mixing the circular padlock probe with the hairpin structure H probe and T4 DNA ligase, heat it to 90°C, and then cool it down to room temperature (5°C / minute) at a gradient.

[0019] Preferably, the preparation method of the sensing probe for detecting miRNA described in this application: After mixing the circular padlock probe with the hairpin structure H probe, T4 DNA ligase, and T4 DNA ligase buffer, heat it to 90°C, and then cool it down to room temperature (5°C / min) at a gradient.

[0020] The room temperature described in this application is 20 - 40°C, preferably 25 - 30°C.

[0021] The second aspect of this application provides a biosensor for detecting miRNA, including a sensing probe for detecting miRNA.

[0022] Preferably, in the biosensor for detecting miRNA, it includes a sensing probe for detecting miRNA, DEPC aqueous solution, the miRNA to be detected, DNA polymerase buffer, DNA polymerase, endonuclease, dNTPs solution, and ThT solution.

[0023] The third aspect of this application provides a method for detecting miRNA, including using the sensing probe for detecting miRNA or the biosensor. The method for detecting miRNA includes the following steps: Mix the sensing probe for detecting miRNA, DEPC aqueous solution, and the miRNA to be detected with DNA polymerase buffer, and perform the first incubation to hybridize the target miRNA with the sensing probe; then add DNA polymerase, endonuclease, dNTPs solution, and ThT solution, and perform the second incubation; after the reaction is complete, use a fluorescence spectrometer to detect the fluorescence signal.

[0024] In the biosensor described in the second aspect of this application and the detection method described in the third aspect, the DNA polymerase is phi29 DNA polymerase.

[0025] Preferably, in the biosensor described in the second aspect of the present application and the detection method described in the third aspect, the DNA polymerase buffer is phi29 DNA polymerase buffer, and 1×phi29 DNA polymerase buffer contains (Tris–HCl pH 7.5, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 and 4 mM DTT). Preferably, the biosensor contains 10×phi29 DNA polymerase buffer.

[0026] In the biosensor described in the second aspect of the present application and the detection method described in the third aspect, the endonuclease is Nb.BbvCI endonuclease.

[0027] Preferably, in the biosensor described in the second aspect of the present application and the detection method described in the third aspect, the concentration of the sensing probe for detecting miRNA in DEPC aqueous solution is 300 - 700 nM, and the concentration of the sensing probe in DEPC aqueous solution can be selected from 300 nM, 400 nM, 500 nM, 600 nM, 700 nM. Preferably, the concentration is 500 nM.

[0028] Preferably, in the biosensor described in the second aspect of the present application and the detection method described in the third aspect, the DNA polymerase concentration is 0.5 - 3.0 U / L. The enzyme concentration of the DNA polymerase can be selected from 0.5 U / L, 0.6 U / L, 0.7 U / L, 0.8 U / L, 0.9 U / L, 1.0 U / L, 1.1 U / L, 1.2 U / L,

[0029] 1.3 U / L, 1.4 U / L, 1.5 U / L, 1.6 U / L, 1.7 U / L, 1.8 U / L, 1.9 U / L, 2.0 U / L,

[0030] 2.2 U / L, 2.4 U / L, 2.6 U / L, 2.8 U / L, 3.0 U / L. Preferably, the enzyme concentration of the DNA polymerase is 1.2 U / L.

[0031] Preferably, in the biosensor described in the second aspect of the present application and the detection method described in the third aspect, the endonuclease concentration is 0.5 - 3.0 U / L. The enzyme concentration of the DNA polymerase can be selected from 0.5 U / L, 0.6 U / L, 0.7 U / L, 0.8 U / L, 0.9 U / L, 1.0 U / L, 1.1 U / L, 1.2 U / L,

[0032] 1.3 U / L, 1.4 U / L, 1.5 U / L, 1.6 U / L, 1.7 U / L, 1.8 U / L, 1.9 U / L, 2.0 U / L,

[0033] 2.2 U / L, 2.4 U / L, 2.6 U / L, 2.8 U / L, 3.0 U / L. Preferably, the enzyme concentration of the DNA polymerase is 2.0 U / L.

[0034] Preferably, in the biosensor described in the second aspect and the detection method described in the third aspect of the present application, the concentration of the dNTPs solution is 0.10 - 0.50 mM. The concentration of the dNTPs solution can be selected from 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, 0.35 mM, 0.40 mM, 0.45 mM, 0.50 mM. Preferably, the concentration of the dNTPs solution is 0.25 mM.

[0035] Preferably, in the biosensor described in the second aspect and the detection method described in the third aspect of the present application, the concentration of the ThT solution is 1 - 10 μM. The concentration of the ThT solution can be selected from 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM. Preferably, the concentration of the ThT solution is 5 μM.

[0036] Preferably, in the sensing probe described in the second aspect and the detection method described in the third aspect of the present application, for the sensing probe for detecting miRNA, the concentration of the DEPC aqueous solution is 300 - 700 nM, the concentration of the DNA polymerase buffer solution is 40 - 60 mM, the concentration of the DNA polymerase is 0.5 - 3.0 U / L, the concentration of the endonuclease is 0.5 - 3.0 U / L, the concentration of the dNTPs solution is 0.1 - 0.5 mM, the concentration of the ThT solution is 1 - 10 μM, and the miRNA to be detected is 5 - 15 μL.

[0037] Preferably, in the biosensor described in the second aspect and the detection method described in the third aspect of the present application, for the sensing probe for detecting miRNA, the concentration of the DEPC aqueous solution is 300 - 700 nM, the concentration of the 10×phi29 DNA polymerase buffer solution is 40 - 60 mM, the concentration of the 10×phi29 DNA polymerase is 0.5 - 3.0 U / L, the concentration of the Nb.BbvCI endonuclease is 0.5 - 3.0 U / L, the concentration of the dNTPs solution is 0.1 - 0.5 mM, the concentration of the ThT solution is 1 - 10 μM, and the miRNA to be detected is 5 - 15 μL.

[0038] Preferably, in the biosensor described in the second aspect of the present application and the detection method described in the third aspect, in the sensing probe for detecting miRNA, there are 15 - 30 μL of DEPC aqueous solution (500 nM), 1 - 10 μL of 10×phi29 DNA polymerase buffer (50 mM), 1 - 10 μL of Nb.BbvCI endonuclease (2.0 U / L), 1 - 5 μL of dNTPs solution (0.25 mM), 1 - 5 μL of ThT solution (5 μM), and 5 - 15 μL of miRNA to be detected. Preferably, the biosensor for detecting miRNA includes 25 μL of DEPC aqueous solution (500 nM) of the sensing probe for detecting miRNA, 5 μL of 10×phi29 DNA polymerase buffer (50 mM), and Nb.BbvCI endonuclease (2.0 U / L) ， 5 μL, 2 μL of dNTPs solution (0.25 mM), 2 μL of ThT solution (5 μM), and 10 μL of miRNA to be detected.

[0039] For the method for detecting miRNA described in the present application, the first incubation time is 40 - 90 min, and the incubation temperature is 20 - 40°C. Preferably, the first incubation time is 60 min, and the incubation temperature is 25 - 30°C.

[0040] For the method for detecting miRNA described in the present application, the second incubation time is 80 - 130 min, and the incubation temperature is 20 - 40°C. Preferably, the second incubation time is 120 min, and the incubation temperature is 25 - 30°C.

[0041] The present application provides the use of the sensing probe for detecting miRNA or the biosensor for detecting miRNA in detecting miRNA.

[0042] Preferably, the use of the sensing probe for detecting miRNA or the biosensor for detecting miRNA in detecting miRNA for disease diagnosis or treatment.

[0043] Beneficial effects

[0044] 1. The sensing probe for detecting miRNA in the present application has a handle - loop structure, which is formed by hybridization of a circular padlock probe and a hairpin - structure H probe, and has functions such as integrating specific target recognition, rolling - circle amplification, and self - absorption - assisted signal recovery. It can accurately identify specific targets and reduce background signals, thereby achieving highly accurate discrimination and detection of single - strand RNA mutations. The biosensor of the present application does not require molecular beacons and adopts a self - absorption - assisted signal recovery strategy, greatly improving the signal amplification efficiency and detection sensitivity.

[0045] 2. In the detection method of the present application, a large number of nucleic acid G-quadruplexes are generated by rolling circle amplification. These nucleic acid G-quadruplexes can be detected by commercial fluorescent dyes such as thioflavin (ThT) without labeling, enabling the detection of single-stranded nucleic acid sequences in a low-cost and label-free manner. Verified by experimental examples, the RCA strategy mediated by the sensing probe for detecting miRNA based on the above can achieve high-sensitivity and high-specificity detection of miRNA, and can even be quickly and accurately detected in complex urine samples.

[0046] 3. The sensing probe with a handle-loop structure and its biosensor of the present application exhibit excellent accuracy, high sensitivity, high selectivity, stability and reproducibility for miRNA -143.

[0047] 4. The sensing probe with a handle-loop structure and its biosensor of the present application have been successfully applied to the detection of miRNA. The method for detecting miRNA is consistent with the classical miRNA detection method (PCR method) and has good accuracy.

[0048] 5. The detection limit of the detection method of the present application is lower than that of traditional methods, and it can detect low-concentration clinical samples, with rapid and convenient analysis and detection. It has great application potential in biomedical analysis and early clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0050] Figure 1 It is the schematic diagram of detecting miRNA mediated by the sensing probe for detecting miRNA based on the above in Example 1 of the present application;

[0051] Figure 2 It is the detection result of the SYBR Green I signal of the H probe during the assembly process of the sensing probe for detecting miRNA described in Experimental Example 1 of the present application;

[0052] Figure 3 It is the detection result of the fluorescence intensity of the FAM-labeled H probe during the recovery process of the H probe of the sensing probe for detecting miRNA described in Experimental Example 1 of the present application;

[0053] Figure 4 It is the detection result of the fluorescence intensity of the reaction products obtained by each of the methods a-f for detecting miRNA described in Experimental Example 3 of the present application;

[0054] Figure 5 Detection results of the miRNA sensing probe described in Experimental Example 1 of the present application for detecting different concentrations of miRNAs;

[0055] Figure 6 Correlation results between the detection signal of the miRNA sensing probe described in Example 1 of the present application and the miRNA concentration;

[0056] Figure 7 Graph showing the detection specificity results of the method for detecting miRNA in Experimental Example 3 of the present application;

[0057] Figure 8 Graph showing the recovery results of the method for detecting miRNA in Experimental Example 3 of the present application;

[0058] Figure 9 Graph showing the correlation results between the target concentrations calculated by the method for detecting miRNA in Experimental Example 3 of the present application and the PCR method. Detailed implementation mode

[0059] The materials and reagents involved in the following examples are all commercially available products, specifically as follows:

[0060] The HPLC-purified oligonucleotides and HPLC-purified miRNAs used were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China), and the relevant sequences are shown in Table 1.

[0061] Table 1 Sequences of HPLC-purified oligonucleotides used

[0062]

[0063] T4 DNA Ligase, phi29 DNA polymerase, Nb.BbvCI endonuclease and thioflavin T (ThT) were provided by Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Deoxynucleotide triphosphates (dNTPs) were purchased from New England Biolabs (NEB, UK). Other chemicals were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), analytical grade. All miRNAs were dissolved in DEPC water to an appropriate concentration before use.

[0064] Example 1 miRNA sensing probe

[0065] A sensing probe for detecting miRNA, the sensing probe is a handle-loop structure, which is formed by hybridization of a circular padlock probe and a hairpin structure H probe; the circular padlock probe includes a DNA polymerase extension sequence region, an endonuclease recognition sequence region, a template sequence region and a target miRNA binding sequence region, and the bases are complementary to form a circle; the hairpin structure H probe is a stem-loop structure, including a loop part and a stem part with complementary base hybridization, and the hairpin structure H probe contains a reporter sequence region, an endonuclease recognition sequence region, a primer sequence region for promoting chain growth and a stem-loop template sequence region; after the circular padlock probe binds to the target miRNA, the hairpin structure H probe is released by DNA polymerase chain extension.

[0066] As Figure 1 shown, the circular padlock probe sequentially includes a DNA polymerase extension sequence region b (green region), a first endonuclease recognition sequence region c 1 (yellow region), a template sequence region a (blue region), a second nuclease recognition region c 2 (yellow region) and a target miRNA binding sequence region f (red region) in a clockwise direction from the molecular extension, and the bases are complementary hybridized to form a circle; when the circular padlock probe binds to the target miRNA, after strand extension and endonuclease recognition and cleavage, a secondary sequence a* transcribed from the template sequence a is released, and the secondary sequence a* hybridizes with the hairpin structure H probe. The sequence of the circular padlock probe is shown in Table 1 or as SEQ ID NO.1.

[0067] The target miRNA The binding sequence region is in italic font, the endonuclease recognition sequence region is in bold font, the template sequence region is in single underlined region “”, and the DNA polymerase extension sequence region is in double underlined region

[0068] Preferably, the 5'-end of the circular padlock probe is phosphorylated.

[0069] As Figure 1 shown, in the hairpin structure H probe, the stem part is two single strands, wherein the first single strand contains a reporter sequence region d ( Purple area ), an endonuclease recognition sequence region c (yellow region), and a part of the bases of the first single strand are complementary hybridized with the bases of the loop part to form a stem-loop template sequence region a (blue region), and the second single strand contains a primer sequence region (orange region) e and e* for promoting strand extension, which are complementary to the terminal bases of the loop part; after the secondary sequence a* released by the circular padlock probe binds to the hairpin structure H probe and undergoes strand extension and endonuclease cleavage, the reporter sequence d is released, and the reporter sequence d is a nucleic acid G-quadruplex sequence. The hairpin structure H probe is shown in Table 1 or as SEQ ID NO.2.

[0070]

[0071] Among them, the stem-loop template sequence region is underlined once “” , the primer sequence region that promotes strand extension is the word The body is in bold, and the reported sequence region is double underlined The endonuclease recognition sequence region is in italic font

[0072] Method for preparing a sensing probe for detecting miRNA: 1 μL of circular padlock probe (100 μM) and 1 μL of hairpin structure H probe (100 μM), 1 μL of T4 DNA ligase (100 U / μL), 2 μL 10×T4 DNA ligation buffer (500 mM Tris-HCI, 100 mM MgCI 2 , 10 mM ATP, 100 mM DTT, pH 7.8), heated to 90 °C and then cooled down to room temperature in a gradient (5 °C / min ) That's it

[0073] The principle of detecting miRNA by the above-mentioned miRNA-sensing probe is as Figure 1 shown below

[0074] In this application, a hybridization of a circular padlock probe and a hairpin structure H probe is used to construct a sensing probe with a handle-loop structure. Taking miRNA-143 as the detection target, the working mechanism of this biosensor is elaborated. When the target miRNA-143 is present, miRNA-143 precisely attaches to the target miRNA binding sequence region of the circular padlock probe through complementary base pairing. Subsequently, the 3'-end of the target miRNA-143 attached to the circular padlock is continuously elongated by phi29 DNA polymerase at a constant temperature, and the hairpin structure H probe is released from the handle-loop structure sensing probe. During the RCA process, a DNA strand with a specific recognition site for the Nb.BbvCI endonuclease is generated. Then, the Nb.BbvCI endonuclease recognizes and cleaves the cleavage site on the DNA strand, releasing the secondary sequence a* transcribed from the template sequence a, while leaving a new replication site for DNA polymerase. Under the continuously repeated strand extension and cleavage reactions, a large number of secondary sequences a* are produced. The secondary sequence a* hybridizes with the stem-loop template sequence "a” segment of the hairpin structure H probe, activating the released hairpin structure H probe, causing the hairpin structure of the hairpin structure H probe to unfold. The "e*" on the hairpin structure H probe hybridizes with the part labeled "e" and serves as a primer to promote the elongation of the strand ("d*" and "e*"), forming a DNA strand body that can be specifically recognized by the Nb.BbvCI endonuclease. The obtained strand body is cleaved by the Nb.BbvCI endonuclease, and a part of the cleaved DNA strand body can be used as a template for the next round of strand extension and cleavage reaction, while the cleaved and released reporter primer d* is obtained. During the detection process, through the synergistic action of DNA polymerase and endonuclease, a large number of reporter sequences d* are generated through an isothermal amplification reaction. The reporter sequence contains a nucleic acid G-quadruplex. The G-quadruplex can be precisely recognized by the fluorescent dye thioflavin T (ThT), and then the fluorescence signal is detected by a fluorescence spectrometer

[0075] Biosensor for Detecting miRNA in Example 2

[0076] A biosensor for detecting miRNA, comprising 25 μL of the sensing probe DEPC aqueous solution (concentration 500 nM) for detecting miRNA in Example 1; 5 μL of 10×phi29 DNA polymerase buffer (concentration 50 mM); 2 μL of phi29 DNA polymerase (concentration 1.2 U / L); 5 μL of Nb.BbvCI endonuclease (concentration 2.0 U / L); 2 μL of dNTPs solution (concentration 0.25 mM); 2 μL of ThT solution (concentration 5 μM); and 10 μL of the miRNA to be detected.

[0077] The composition of 1×phi29 DNA polymerase buffer: Tris–HCl pH 7.5, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 and 4 mM DTT.

[0078] Method for Detecting miRNA in Example 3

[0079] Using the biosensor for detecting miRNA in Example 2 to detect the miRNA to be detected, which includes the following steps:

[0080] Mix 5 μL of 10×phi29 DNA polymerase buffer (concentration 50 mM) and 25 μL of the sensing probe DEPC aqueous solution (concentration 500 nM) for detecting miRNA evenly to obtain a mixture. Add 10 μL of the miRNA to be detected to the above mixture, and incubate for 60 min for the first time at room temperature. Then add 2 μL of phi29 DNA polymerase (concentration 1.2 U / L), 2 μL of dNTPs solution (concentration 0.25 mM), 2 μL of ThT solution (concentration 5 μM), and 2 μL of Nb.BbvCI endonuclease (concentration 2.0 U / L), and incubate for 120 min for the second time at room temperature (30 °C). After the reaction is completed, use a fluorescence spectrometer to detect the fluorescence signal.

[0081] The fluorescence spectrometer Model: Hitachi F-4700.

[0082] Assembly and Verification of Hairpin Structure H Probe in Example 4

[0083] Assembly of hairpin structure H probe: With a concentration of Add 5 μL of 100 nM hairpin structure H probe to 22 μL of DEPC water, heat to 90 °C, incubate for 10 min, and then slowly cool to room temperature.

[0084] Experimental method: SYBR Green I dye was used to confirm the assembly of the H probe. The SYBR Green I probe shows specific binding to double-stranded DNA structures and produces a fluorescent signal.

[0085] Experimental results: As Figure 2 shown, when the H probe is in a linear state, the fluorescent signal of SYBR Green I is significantly lower. When the H probe is heated to 90 °C and then cooled to room temperature, the SYBR Green I signal gradually increases over time, indicating that the H probe has formed a hairpin structure.

[0086] Example 5 Verification experiment on the stability of the hairpin structure H probe in the sensing probe

[0087] Experimental purpose: Fluorescent dye and quenching group are attached to both ends of the H probe respectively. Whether the target can be promoted to expand by phi29 DNA polymerase is used to confirm whether the H probe is released from the sensing probe, so as to determine the stability of the hybridization between the hairpin structure H probe and the circular padlock probe.

[0088] Experimental results: As Figure 3 shown, when the hairpin structure H probe is in a linear form, the fluorescence signal of the H probe linear group is the highest; when the H probe in the sensing probe is in a stem-loop structure, the fluorescence signal of the sensing probe group decreases significantly; when only the target miRNA exists, the fluorescence signal of the sensing probe + target group is slightly higher than that of the sensing probe stem-loop structure group; when both the target and DNA polymerase are present, the fluorescence intensity of the sensing probe + target + DNA polymerase group is the same as that of the sensing probe + target group, which indicates that even if the hairpin structure H probe is released from the sensing probe, the hairpin structure H probe can still maintain its hairpin structure. After adding endonuclease to the biosensor, the endonuclease recognizes the cleavage site on the DNA strand and cleaves it, releasing a large number of secondary sequences a* transcribed from the template sequence, and a significant increase in the detected fluorescence signal is observed, suggesting that the secondary sequence a* transcribed from the template sequence a hybridizes with the stem-loop template sequence of the hairpin structure H probe, activating the released hairpin structure H probe and causing the hairpin structure of the hairpin structure H probe to unfold. Column "a" When both polymerase and endonuclease are present, the hairpin structure H probe is more likely to be released from the sensing probe. This experiment proves that when the sensing probe encounters the target DNA Example 6 Verification of the practicality of the biosensor for detecting miRNA

[0089] Experimental method: The biosensor of Example 2 was used, and the method for detecting miRNA in Example 3 was used to confirm which necessary experimental conditions in the biosensor.

[0090]

[0091] ​Group a: Without target, DNA polymerase, endonuclease, and ThT in the biosensor of Example 2; Group b: Without target in the biosensor of Example 2;

[0092] Group c: Without polymerase in the biosensor of Example 2;

[0093] Group d: Without endonuclease in the biosensor of Example 2;

[0094] Group e: Without ThT in the biosensor of Example 2;

[0095] Group f: The biosensor of Example 2.

[0096] Experimental results: As Figure 4 shown, the absence of any one experimental component, such as target, DNA polymerase, endonuclease, and ThT, results in no significant difference in fluorescence intensity compared to the control group (Group a). Only in Group f can the fluorescence response be significantly improved when all necessary experimental components are included.

[0097] Experimental Example 7 examined the detection sensitivity of the miRNA detection method of the present application.

[0098] Experimental method: Prepare aqueous solution samples of different concentrations of miRNA, with concentrations of 0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM in sequence, and then detect the above samples of different concentrations of miRNA according to the same method in Example 3.

[0099] Experimental results: As Figure 5 shown, it shows that the fluorescence intensity of this method increases with the increase of miRNA concentration, confirming that only the interaction between target miRNA and circular padlock probe can trigger RCA. The higher the concentration of target miRNA added to the reaction, the more RCA cycles and the higher the fluorescence intensity. Figure 6 It is the exponential relationship between ThT fluorescence intensity and target concentration. There is a good linear relationship between ThT fluorescence intensity and the logarithm of miRNA concentration. The linear regression equation is: ThT = 72.54 * lgC + 138.5, R2 = 0.9963, with a linear range of concentrations from 1 fM to 1 nM, and the determined detection limit (LOD) is 376 aM.

[0100] Experimental Example 8 examined the specificity of the miRNA detection method of the present application.

[0101] Due to the high sequence homology, it has always been challenging to distinguish miRNA family members with high specificity. With only 1 - 5 nucleotides difference from each other, it provides an ideal probe or biosensor for evaluating the specificity of miRNA detection methods. Therefore, sequences with single-base mismatch, two-base mismatch, three-base mismatch, and complete non-complementary type compared with the miRNA sequence of this application are selected for specificity analysis.

[0102] MT1 group: single-base mismatch RNA DEPC aqueous solution of 5’-AAU CCG UCG ACC AGA GUU AGG GUU-3’.

[0103] MT2 group: two-base mismatch RNA DEPC aqueous solution of 5’-AAU CCG UCG ACC GGA GUU AGG GUU-3’.

[0104] MT3 group: three-base mismatch RNA DEPC aqueous solution of 5’-AAU CCG UCG ACC GAA GUU AGG GUU-3’.

[0105] NC group: complete non-complementary type RNA DEPC aqueous solution of 5’-AAC AAT CCA CGA TTC AAC GCA TTCAAC-3’.

[0106] Target group: sensor of Example 2, miRNA-143, 5’-AAU CCG UCG ACG AGA GUU AGG GUU-3’.

[0107] Detect the aqueous solution samples of different miRNAs according to the method of Example 3 of this application, where the concentration of each group of samples is 100 nM.

[0108] Experimental results: As Figure 7 shown, the ThT values of non-specific targets (MT1, MT2, MT3, NC) are much lower than the ThT value of the completely matched target miRNA-143. This experiment proves the specificity of the biosensor of this application in accurately detecting the target miRNA in different types of sample applications, and at the same time can minimize false positives. The sensing probe with a handle-loop structure of this application effectively reduces the possibility of interfering molecules opening the H probe and avoiding non-specific reactions. The miRNA detection method of this application has good performance in distinguishing target miRNAs from other non-target miRNAs in the same miRNA family and has great potential for single nucleotide polymorphism analysis.

[0109] Example 9 examined the detection of miRNA in human urine by the miRNA detection method of the present application

[0110] The synthetic miRNA-143 in urine was diluted to the corresponding concentrations to obtain groups S1-S4, and a recovery test was carried out. The results are as Figure 8 shown. The added amount and detected amount of the target miRNA-143 were equal. The recovery rate of the miRNA detection method was 98.88%-103.12%, showing good stability, which proved the feasibility of the miRNA-143 detection method of the present application in the determination of real biological samples.

[0111] Example 10 Comparison between the detection method of the present application and the PCR method

[0112] Group of the present application: miRNA-143 determined by the method of Example 3 Respectively 500 fM, 1750 fM, 2000 fM, 2800 fM, 4000 fM.

[0113] PCR method: miRNA-143 determined by the traditional miRNA immunofluorescence method was 500 fM, 1750 fM, 2000 fM, 2800 fM, 4000 fM, respectively.

[0114] Experimental results: As Figure 9 shown, the miRNA detection method of the present application was highly consistent with the PCR detection results; thus, it was confirmed that the detection method of the present application could replace the PCR method for clinical use.

Claims

1. A sensor probe for detecting miRNA, characterized in that: The sensor probe is a handle ring structure, which is formed by hybridizing a circular padlock probe and a hairpin structure H probe; the circular padlock probe is composed of a DNA polymerase extension sequence region, a first endonuclease recognition sequence region, a template sequence region, a second endonuclease recognition region and a target miRNA binding sequence region in a clockwise direction, and the base complementary hybridization is a circular ring; when the circular padlock probe binds to the target miRNA, after chain extension and endonuclease recognition and cleavage, a secondary sequence transcribed from the template sequence is released, and the secondary sequence is bound to the hairpin structure H probe. The hairpin structure H probe hybridizes; the hairpin structure H probe is a stem-loop structure, the stem portion is two single strands, the first single strand comprises a reporter sequence region and a nuclease recognition sequence region, the bases of the first single strand part are complementary hybridized with the bases of the loop portion to form a stem-loop template sequence region, and the second single strand comprises a primer sequence region for promoting chain extension, which is complementary to the terminal base of the loop portion; the secondary sequence released by the circular padlock probe combines with the hairpin structure H probe, and then releases a reporter sequence after chain extension and nuclease cleavage, and the reporter sequence is a nucleic acid G-quadruplex sequence.

2. The sensing probe according to claim 1, characterized in that: The sequence of the circular padlock probe is shown in SEQ ID NO.1, and the sequence of the hairpin structure H probe is shown in SEQ ID NO.

2.

3. A biosensor for detecting miRNA, characterized in that: The invention comprises a DEPC aqueous solution of the sensor probe for detecting miRNA according to any one of claims 1 to 2, a DNA polymerase buffer, a DNA polymerase, an endonuclease, a dNTPs solution, a ThT solution, and a miRNA to be detected.

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