Biosensor for detecting miRNA through label-free rolling circle amplification self-assembly MNAzyme coupled fluorescent RNA aptamer

Abstract

The invention belongs to the technical field of biological analysis, and particularly relates to nicking rolling circle amplification, self-loading multi-component nuclease (MNAzyme), a fluorescent RNA aptamer and application of the fluorescent RNA aptamer in miRNA detection. The invention provides an unmarked sensitive sensing platform, which is characterized in that cutting rolling circle amplification is integrated into a self-assembled multi-component nuclease (MNAzyme) and a fluorescent RNA (Ribonucleic Acid) aptamer, and the platform is used for monitoring miR-21. By utilizing the super-sensitivity, programmability and operability of the biosensing environment detection platform, the expression of miRNA in different breast cancer cells can be quantitatively detected, normal cells and different cancer cells can be distinguished, and the biosensing environment detection platform has huge potential in the aspects of biomedical research and clinical diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis, and specifically relates to nicking rolling circle amplification, self-assembling multi-component nuclease (MNAzyme), fluorescent RNA aptamer and application thereof in miRNA detection. Background Art

[0002] MicroRNA (miRNA) is a class of endogenous, non-coding, short single-stranded RNA that participates in many biochemical processes such as gene expression and cell differentiation. In a complex dialogue, miRNA is a key mediator between cancer cells and their hosts. A large amount of evidence shows that miRNA can act as a gene regulator and has been shown to be an oncogene. Therefore, sensitive detection of miRNA is crucial for early clinical diagnosis, miRNA-targeted therapy, and monitoring tumor prognosis. Although a variety of methods for detecting miRNA have been developed, it is extremely difficult to achieve sensitive, selective, and accurate quantification of miRNA due to the characteristics of miRNA itself, such as small size, high homology, low abundance, and easy degradation. Traditional miRNA detection methods include real-time quantitative polymerase chain reaction (RT-qPCR), Northern blotting, and microarray analysis. However, these methods are usually subject to complex probe design, large sample consumption, sophisticated instruments, and complex data analysis. Therefore, it is imperative to develop new methods with high sensitivity, simplicity, and convenience to reliably detect miRNA for early diagnosis and prognosis of tumors.

[0003] Compared with proteases and ribozymes, DNAzymes have several advantages, including better stability, more flexible functionalization, lower cost, and faster reaction speed. Based on these advantages, they have been used to detect various target molecules, including metal ions, nucleic acids, pathogens, and proteins. In addition, in order to improve the designability of DNAzymes, Mokany et al. introduced another derivative of DNAzyme, namely multicomponent nuclease (MNAzyme). To date, there have been some reports on miRNA analysis methods involving DNAzyme / MNAzyme. However, the sensitivity of DNAzyme / MNAzyme for the detection of low-abundance targets is relatively low and is easily affected by complex environments.

[0004] Isothermal amplification technology combined with DNAzyme / MNAzyme has the potential to alleviate this problem because it is easy to integrate with other components and has high amplification efficiency under constant temperature conditions. Common isothermal amplification strategies include rolling circle amplification, loop-mediated isothermal amplification, hybridization chain reaction, and catalytic hairpin assembly. Among these isothermal amplification strategies, rolling circle amplification is an efficient and mature nucleic acid amplification method that can achieve exponential target amplification in a shorter reaction time. DNAzyme / MNAzyme is highly compatible with RCA technology and can be used to design biosensors for signal amplification and improve detection sensitivity. However, these technologies inevitably require the use of expensive fluorophore / quencher dual-labeled signal probes and time-consuming design procedures. Therefore, it is very necessary to construct new simple and low-cost RCA-MNAzyme combined biosensors to detect miRNA. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention proposes a biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamers to solve the problems of low specificity, low sensitivity and high cost of the existing methods.

[0006] The technical solution of the present invention is as follows:

[0007] A biosensor for detecting miRNA by label-free rolling circle amplification self-assembly MNAzyme coupled with fluorescent RNA aptamer, characterized by comprising the following steps:

[0008] (1) The design of the stem-loop anti-Mango RNA sequence includes the sequence of the complete fluorescent RNA aptamer Mango and the inhibitory sequence of the fluorescent RNA aptamer Mango, as well as the specific cleavage site of the MNAzyme;

[0009] (2) The design of the hairpin padlock probe includes three functional fragments, namely, the complementary region of the target, the complementary region of Partzyme A, and the complementary region of Partzyme B. The recognition site of Nt.BstNBI endonuclease is inserted between the three functional fragments, respectively, to split the complementary chain of Partzyme A into two, and to make the 5' end phosphorylated, wherein the 5' and 3' ends of the hairpin padlock probe can be complementary to the primer base;

[0010] (3) adding primers to the hairpin padlock probe obtained in step (2), and after hybridization, ligating the 5' and 3' ends of the hairpin padlock under the action of T4 DNA ligase to obtain a closed dumbbell-shaped probe, and purifying the dumbbell-shaped probe using exonuclease I and exonuclease III;

[0011] (4) adding miRNA-21 to the dumbbell-shaped probe obtained in step (3), circularizing the dumbbell-shaped probe through toe chain displacement specific binding, and initiating a nicking rolling circle amplification (RCA) reaction in the presence of Bst DNA polymerase; with the help of Nt.BstNBI endonuclease, the RCA product can be nicked on the dumbbell-shaped probe and shifted during the subsequent replication process, thereby generating a large number of nicked fragments;

[0012] (5) adding a reaction buffer to the gap fragment obtained in step (4), the gap fragment self-assembles into MNAzyme through hybridization, and then the MNAzyme can specifically recognize and cleave the cleavage site on the substrate stem-loop anti-Mango RNA sequence in step (1), releasing the fluorescent RNA aptamer Mango;

[0013] (6) The fluorescent RNA aptamer Mango obtained in step (5) is combined with TO1-Biotin fluorescent dye, and the fluorescent signal is detected using F4600.

[0014] Preferably, the method is characterized in that the target is bound to the dumbbell-shaped probe by a toe-mediated strand displacement reaction.

[0015] Preferably, it is characterized in that Partzyme A, Partzyme B, and the target are amplified simultaneously.

[0016] Preferably, it is characterized in that the amplification products of nicking rolling circle amplification are recycled and further drive the assembly of MNAzyme.

[0017] Preferably, it is characterized in that the MNAzyme can repeatedly cycle through new substrates, resulting in signal amplification.

[0018] Preferably, it is characterized in that the application is selected from any one of the following applications:

[0019] (1) Differentiating miRNA-21 from its homologous analogs

[0020] (2) Identification and detection of miRNA-21 in different cells;

[0021] (3) Application of identification and detection of miRNA-21 in serum.

[0022] Furthermore, the label-free rolling circle amplification self-assembled MNAzyme coupled fluorescent RNA aptamer biosensor for detecting miRNA is used in miRNA detection in the field of bioanalysis technology.

[0023] Compared with the prior art, the biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer of the present invention has the following advantages:

[0024] The present invention provides a novel and direct fluorescent sensor for detecting miRNA. The present invention uses toe-mediated strand displacement to perform hybridization reaction between target miRNA and dumbbell-shaped probe, so that this strategy has significant specificity and single-base mismatch recognition ability. The combination of nicking rolling circle amplification and self-assembling MNAzyme in the present invention can effectively reduce signal interference caused by the formation of unnecessary intermediate complexes. The amplification mode of nicking rolling circle amplification in the present invention ensures the optimal reaction process, and the gradual activation of the MNAzyme system ensures accurate and sensitive detection of low-concentration miRNA. The method is easily implemented in a label-free and separation-free manner, and has the characteristics of simplicity, economy, and easy operation. In addition, the present invention also verifies the practicality of the sensor by detecting miRNA-21 in cell lysate and human serum, and the performance of the sensor is minimally affected. The present invention is expected to provide great potential for tumor diagnosis, prognosis and treatment in terms of ultra-sensitive detection of miRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the basic principle diagram of the present invention.

[0026] Figure 2 This is a graph of experimental optimization results of the present invention.

[0027] Figure 3 The figure is a non-denaturing polyacrylamide gel electrophoresis diagram for verifying the feasibility of the present invention.

[0028] Figure 4 This is a fluorescence signal intensity diagram for preliminary verification of the feasibility of the present invention.

[0029] Figure 5 This is a result diagram of the sensitivity of the method of detecting targets of different concentrations in the present invention.

[0030] Figure 6 It is a linear relationship diagram fitted according to the fluorescence signals of targets with different concentrations in the present invention.

[0031] Figure 7 This is a result diagram for verifying the specificity of base mutation in the present invention.

[0032] Figure 8 This is a result diagram of verifying the specificity of homologs in the present invention.

[0033] Fig. 9 This is a comparison chart of the relative expression detection results of miRNA-21 in different cells by the present invention and RT-qPCR.

[0034] Fig.10 This is a comparison chart of the detection results of miRNA-21 concentration in serum samples by the present invention and RT-qPCR. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all experimental materials, reagents and equipment in the examples can be obtained through conventional purchasing channels.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0040] Example 1: Construction of a biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamers

[0041] Sequence design

[0042]

[0043] (1) The design of the stem-loop anti-Mango RNA sequence contains the complete fluorescent RNA aptamer Mango sequence and the inhibitory sequence of the fluorescent RNA aptamer Mango, as well as the specific cleavage site of the MNAzyme; it is transcribed by T7 RNA polymerase;

[0044] (2) The design of the hairpin padlock probe includes three functional fragments, namely, the complementary region of the target, the complementary region of Partzyme A, and the complementary region of Partzyme B. The recognition site of Nt.BstNBI endonuclease is inserted between the three functional fragments, respectively, to split the complementary chain of Partzyme A into two, and to make the 5' end phosphorylated, wherein the 5' and 3' ends of the hairpin padlock probe can be complementary to the primer base;

[0045] The basic principle of the biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer is as follows Figure 1 As shown, the following steps are included:

[0046] (1) In vitro transcription and purification

[0047] According to the instruction manual of AmpliScribe T7 High Yield Transcription Kit, the anti-Mango RNA sequence for in vitro detection was obtained by in vitro transcription using the linearized pUC57 plasmid (with T7 promoter) and the complementary sequence of the anti-Mango RNA sequence as template. Typically, 20 μL of transcription reaction was prepared by mixing RNase-Free water (10 μL), linearized plasmid template (2 μL 0.5 μg / μL), 10XAmpliScribe T7 reaction buffer (2 μL), rNTP (1.5 μL 100 mM), DTT (2 μL 100 mM), RNase inhibitor (0.5 μL) and T7 RNA polymerase (2 μL). The reaction mixture was then precipitated with ammonium acetate (20 μL 5 M), incubated on ice for 15 min, and centrifuged at 12,000 rpm for 15 min at 4 °C. The precipitate was washed with 70% ethanol, resuspended in RNase-Free H2O, and stored at -20 °C for future use.

[0048] (2) Synthesis of dumbbell-shaped probe

[0049] To synthesize dumbbell-shaped probes, primers were mixed with 5'-phosphorylated hairpin padlock probes, and specific ligation and circularization of the padlock were induced by T4 DNA ligase. Ligation of the hairpin padlock strands was performed in a 10 μL reaction system, with the addition of reaction buffer (1 μL 10X), T4 DNA ligase (1 μL 10U / μL), primers (1 μL 2μM), hairpin padlock (1 μL 2μM), and nuclease-free water (6 μL). The reaction was incubated at 37°C for 30 min, followed by incubation with exonuclease I (1 μL 20U / μL) and exonuclease III (1 μL 20U / μL) at 37°C for 30 min and denaturation at 80°C for 20 min.

[0050] (3) Sensor preparation and selectivity determination

[0051] The target and dumbbell probe were reacted with another 20 μL mixture containing Thermopol buffer (10X), MgSO4 (100 mM), dNTP mixture (250 mM), dumbbell probe (200 nM), different concentrations of miRNA-21, Bst DNA polymerase (0.4 U / μL) and Nt.BstNBI endonuclease (0.5 U / μL) at 55°C for 45 min, and then inactivated at 80°C for 20 min. Typically, the RCA product (100 nM) was mixed with anti-Mango RNA sequence (200 nM) and reaction buffer (40 mM Tris-HCl, 100 mM KCl, 50 mM MgCl2, pH = 7.4) and reacted at 37°C for 60 min.

[0052] To evaluate its selectivity, the sensor was also used to detect other homologous sequences, including miR-155 (200 nM), miR-141 (200 nM), miR-122 (200 nM), Let-7a (200 nM), and miR-18a (200 nM). In addition, the sensor was also used to detect mismatched sequences at the same concentration. The above RNAs were added to the platform and reacted at 37 °C.

[0053] (4) Gel electrophoresis analysis

[0054] Utilize 10% non-denaturing polyacrylamide gel electrophoresis (PAGE) to analyze different samples.10 μ L sample and loading buffer (1.1 μ L 10X) are added in the hole, in 1X TBE buffer (45mM Tris, 45mM boric acid, 10mM EDTA, pH=8.0), carry out 10% non-denaturing polyacrylamide gel electrophoresis analysis, at room temperature, constant voltage 110V is carried out for 60min.Then the gel is washed three times, and dyed with ethidium bromide reagent (diluted with 1X TBE).Use 365nm ultraviolet gel imaging operating system to image the gel.

[0055] (5) Spectral measurement and analysis

[0056] Different concentrations of miRNA were added to the platform and reacted at 37°C for 60 min. The reaction was carried out in a buffer (40mM Tris-HCl, 100mM KCl, 50mM MgCl2, PH=7.6, 100nM TO1-Biotin). Subsequently, 100nM of RCA product was mixed with 200nM anti-Mango RNA sequence and reaction buffer (40mM Tris-HCl, 100mM KCl, 50mMMgCl2, PH=7.4) and reacted at 37°C for 60 min. After the reaction, the TO1-Biotin fluorescence was recorded using an F-4600 fluorescence spectrophotometer with an excitation wavelength of 500nm and an emission wavelength of 540nm.

[0057] (6) Cell culture

[0058] Human breast cancer cell lines (MDA-MB-231 cells and MCF-7 cells), human cervical cancer cell lines (HeLa cells) and human normal liver cell lines (L-02) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin. The cells were cultured on sterilized glass coverslips, placed in culture plates with 35mmX14mm wells, and cultured in the corresponding culture medium to 50-70% abundance, at a temperature of 37°C and a humidity of 5% CO2.

[0059] (7) Total miRNA extraction

[0060] Total miRNA was extracted from four cells (MDA-MB-231, MCF-7, HeLa and L-02) or 0.5mL serum using SanPrep Column microRNA Extraction Kit. First, cells or serum were placed in a centrifuge tube, 1mL miRNA Extractor was added, and lysis was performed for 5-10min. Then, 0.2mL CHCl3 was added to the centrifuge tube, shaken vigorously for 30s, and allowed to stand for 3min. Then, the centrifuge tube was centrifuged at 12000rpm and 4℃ for 10min, the upper aqueous phase was aspirated and transferred to a new centrifuge tube. An equal volume of isopropanol was added to the above centrifuge tube, mixed evenly, left at room temperature overnight, centrifuged at 12000rpm and 4℃ for 10min, and the supernatant was discarded. 1mL 75% ethanol was added to wash the precipitate, centrifuged at 12000rpm and 4℃ for 3min, the supernatant was discarded, dried at room temperature for 10min, and finally 30uL nuclease-free water was added to completely dissolve the miRNA for subsequent experiments.

[0061] (8) RT-qPCR analysis

[0062] After obtaining total miRNA from different cell lines and clinical serum samples, the cDNA template was prepared according to the instructions of the miRNA First Strand cDNA Synthesis Kit (Stem-Loop). The basic steps of real-time fluorescence quantitative PCR are as follows: ChamQ UniversalSYBR qPCR Master Mix (10 μL), forward primer (0.4 μL 10 μM), reverse primer (0.4 μL 10 μM), reverse transcription reaction product (1 μL) and DEPC-treated water (8.2 μL) were added to 20 μL of the total reaction system solution. The miRNA was detected using the QuantStudio3RealTime system according to the following steps: 95°C (5 min); 95°C (15 min), 60°C (30 s), 40 cycles; 95°C (15 s), 60°C (60 s), 95°C (15 s). U6 was used as the internal control and 2 -ΔΔCT The relative expression of miRNA-21 was analyzed by 2 -ΔΔCT The results of the analysis.

[0063] Example 2: Optimization of experimental conditions for label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer biosensor for detecting miRNA

[0064] (1) Optimization of dNTPs concentration

[0065] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal when the concentration of dNTPs is 100, 200, 300, 400, and 500 nM that when the concentration of dNTPs is 500 nM, the signal-to-noise ratio is the highest. Figure 2 As shown in A.

[0066] (2) Dumbbell probe concentration optimization

[0067] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal when the concentration of the dumbbell probe is 100, 150, 200, 250, and 300 nM that when the concentration of the dumbbell probe is 200 nM, the signal-to-noise ratio is the highest. Figure 2 As shown in B.

[0068] (3) Optimization of the dosage of Bst DNA polymerase and Nt.BstNBI

[0069] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal of different dosages of Bst DNA polymerase that when the dosage of Bst DNA polymerase is 5U, the signal-to-noise ratio is the highest. Figure 2 As shown in C.

[0070] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal of different dosages of Nt.BstNBI that when the dosage of Nt.BstNBI is 8U, the signal-to-noise ratio is the highest. Figure 2 As shown in C.

[0071] (4) Time optimization of RCA reaction and MNAzyme reaction

[0072] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal at different RCA reaction times that when the RCA reaction time is 45 minutes, the signal-to-noise ratio is the highest. Figure 2 As shown in D.

[0073] Using the construction method in Example 1, it can be seen from the intensity of the fluorescence signal of MNAzyme at different reaction times that when the RCA reaction time is 60 min, the signal-to-noise ratio is the highest. Figure 2 As shown in D.

[0074] Example 3: Feasibility test of biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer

[0075] (1) Using the construction method in Example 1, the electrophoresis results showed that miRNA-21 successfully hybridized with the dumbbell probe, and a large number of gap fragments were amplified after adding Bst DNA polymerase and Nt.BstNBI, and the gap fragments were able to self-assemble into MNAzymes, which was equivalent to the band position of the MNAzyme assembled by trigger, Partzyme A and Partzyme B, indicating that the sensor is feasible. Figure 3 shown.

[0076] (2) Using the construction method in Example 1, the fluorescence results showed that we observed a significant increase in fluorescence intensity compared with the control group, while when Nt.BstNBI, Bst DNA polymerase or Dumbbell probe was not present, the fluorescence intensity obtained did not increase significantly compared with the control group, indicating that they all play a key role in the miRNA detection system. In the presence of the target, the fluorescence intensity of the sensor is significantly enhanced, which shows that the sensor is feasible, such as Figure 4 shown.

[0077] Example 4: Sensitivity detection of biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer

[0078] (1) Using the construction method in Example 1, sample solutions with final concentrations of miRNA-21 were prepared at 0, 1fM, 5fM, 10fM, 50fM, 100fM, 500fM, 1pM, and 2pM, respectively. Different concentrations of miRNA-21 were added to the reaction system. The intensity of the fluorescence signal showed that the fluorescence signal gradually increased with the increase of the miRNA-21 concentration, indicating that the method has high sensitivity. Figure 5 shown.

[0079] (2) Figure 7 The fluorescence values ​​corresponding to different miRNA-21 concentrations were linearly fitted to obtain the quantitative working curve of this embodiment. The working curve showed a good linear relationship in the range of 1 to 100 fM, and the related equation was F = 320.51 + 151.24 lg C miRNA-21 (R 2 =0.9941), and the LOD calculated according to the 3σ rule is 113.2aM, indicating that the method has high sensitivity. Figure 6 shown.

[0080] Example 5: Specific detection of miRNA biosensor by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer

[0081] (1) Using the construction method in Example 1, the fluorescence results showed that the fluorescence intensity of the Mis-1 sequence was significantly lower than that of the miRNA-21 target sequence. In contrast, compared with the miRNA-21 target sequence, the fluorescence intensities of the Mis-2 sequence and the Mis-3 sequence were negligible, indicating that the sensor was highly specific. Figure 7 shown.

[0082] (2) Using the construction method in Example 1, the fluorescence results showed that in the presence of miR-155, miR-141, miR-122, let-7a and miR-18a, the fluorescence intensity was undetectable, while the target miRNA-21 had obvious hybridization with the dumbbellprobe, resulting in a significant change in the fluorescence in the system, which shows that the sensor has high specificity, such as Figure 8 shown.

[0083] Example 6: Accuracy detection of biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer

[0084] Table 1

[0085]

[0086] Using the construction method in Example 1, different concentrations of miRNA-21 were added to 10% human serum, and the recovery rate was determined. The measured recovery rates were 99.21-111.8%, and the RSD was 1.22-4.86%. It has the ability to detect miRNA-21 in complex biological matrices, which shows that the sensor has high accuracy, as shown in Table 1.

[0087] Example 7: Physical detection of biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer

[0088] (1) The construction method in Example 1 was used to compare the relative expression results of miRNA-21 in different cells detected by the sensor and RT-qPCR. The expression level of miRNA-21 in MDA-MB-231 cells was the highest, the expression level of miRNA-21 in MCF-7 cells was second only to that in MDA-MB-231 cells, and the expression level of miRNA-21 in HeLa cells was relatively low. The expression level of miRNA-21 in L-02 cells was the lowest. The relative expression results of the sensor and RT-qPCR were consistent, which showed that the sensor had very good practicality. Fig. 9 shown.

[0089] (2) The construction method in Example 1 was used to compare the results of the sensor with those of the RT-qPCR method for determining the concentration of miRNA-21 in clinical serum samples. The miRNA-21 content in the serum of 7 breast cancer patients and 7 healthy subjects was detected. The results showed that the average content of miRNA-21 in the 7 breast cancer patients was much higher than that in the 7 healthy subjects, which was consistent with the previous research results on the upregulation of miRNA-21 expression in breast cancer patients. The relative expression results of the sensor and RT-qPCR were consistent, indicating that the sensor has very good practicality. Fig.10 shown.

[0090] Note: Serum samples of breast cancer patients and normal subjects were obtained from Changsha Maternal and Child Health Hospital and the Hunan Provincial Key Laboratory for Regional Hereditary Birth Defect Prevention and Control of Changsha Maternal and Child Health Hospital Affiliated to Hunan Normal University, which has been approved by the Ethics Committee of Changsha Maternal and Child Health Hospital Affiliated to Hunan Normal University (No.: EC-20240828-09).

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer, characterized in that: The following steps are involved: (1) The design of the stem-loop anti-Mango RNA sequence contains the complete fluorescent RNA aptamer Mango sequence and the inhibitory sequence of the fluorescent RNA aptamer Mango, as well as the specific cleavage site of the MNAzyme; it is transcribed by T7 RNA polymerase; (2) The design of the hairpin padlock probe includes three functional fragments, namely, the complementary region of the target, the complementary region of Partzyme A, and the complementary region of Partzyme B. The recognition site of Nt.BstNBI endonuclease is inserted between the three functional fragments, respectively, to split the complementary chain of Partzyme A into two, and to make the 5' end phosphorylated, wherein the 5' and 3' ends of the hairpin padlock probe can be complementary to the primer base; (3) adding primers to the hairpin padlock probe obtained in step (2), and after hybridization, ligating the 5' and 3' ends of the hairpin padlock under the action of T4 DNA ligase to obtain a closed dumbbell-shaped probe, and purifying the dumbbell-shaped DNA probe using exonuclease I and exonuclease III; (4) adding miRNA-21 to the dumbbell-shaped probe obtained in step (3), circularizing the dumbbell-shaped probe through toe chain displacement specific binding, and initiating a nicking rolling circle amplification (RCA) reaction in the presence of Bst DNA polymerase; with the help of Nt.BstNBI endonuclease, the RCA product can be nicked on the dumbbell-shaped probe and displaced during the subsequent replication process, thereby generating a large number of nicked fragments; (5) adding a reaction buffer to the cleaved fragments obtained in step (4), and the cleaved fragments are self-assembled into MNAzyme through hybridization, and then the MNAzyme can specifically recognize and cleave the cleavage site on the substrate stem-loop anti-Mango RNA sequence in step (1), releasing the fluorescent RNA aptamer Mango; (6) The fluorescent RNA aptamer Mango obtained in step (5) is combined with TO1-Biotin fluorescent dye, and the fluorescent signal is detected using F4600.

2. The biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to claim 1, characterized in that: The target is bound to the dumbbell-shaped probe through a toehold-mediated strand displacement reaction.

3. The biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to claims 1-2, characterized in that: Partzyme A, Partzyme B, and the target are amplified simultaneously.

4. The biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to claims 1-3, characterized in that: The amplification products of the nicking RCA are recycled and further drive the assembly of the MNAzyme.

5. The biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to claims 1-4, characterized in that: MNAzymes can repeatedly cycle to cleave new substrates, resulting in signal amplification.

6. The biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to claims 1-5, characterized in that: The application is selected from any one of the following applications: (1) Differentiating miRNA-21 from its homologous analogs (2) Identification and detection of miRNA-21 in different cells; (3) Application of identification and detection of miRNA-21 in serum.

7. Application of the biosensor for detecting miRNA by label-free rolling circle amplification self-assembled MNAzyme coupled with fluorescent RNA aptamer according to any one of claims 1 to 6 in miRNA detection in the field of bioanalysis technology.

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