DNA walker for MicroRNA detection and application thereof

Abstract

The invention relates to a DNA walker for MicroRNA detection and application thereof, the walker comprises an S1 / S2 at AuNPs three-dimensional motion carrier, a DNAzyme system and a connection probe, the S1 / S2 at AuNPs three-dimensional motion carrier comprises AuNPs, an S1 chain and an S2 chain, and the S2 chain is connected with a fluorophore; the DNAzyme system comprises DNAzyme and a first hairpin structure, the DNAzyme is used for cutting an S2 chain and exciting fluorescence, and the first hairpin structure is complementarily paired with a base of the connecting probe and comprises a first region complementarily paired with a base of microRNA to be detected; the connection probe comprises a second region which is complementarily paired with the base of the first hairpin structure, and a second hairpin structure which is connected with one end of the second region and is complementarily paired with the base of the S1 chain. Compared with the prior art, the method has the advantages that the MicroRNA detection process is simplified, and the detection sensitivity and efficiency are greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a DNA walker for MicroRNA detection and its application. Background Art

[0002] MicroRNA (miRNA) is a single-stranded short non-coding RNA with a length of about 20 nucleotides, which is a key component of various biological processes and a reliable disease marker. The abnormal expression of miRNA is closely related to infectious diseases such as pneumonia. Specifically, previous studies have confirmed that certain miRNAs are widely involved in the pathological development of bacterial pneumonia by regulating inflammatory responses and immune regulatory processes. The identification of these miRNAs is crucial for exploring related signal transduction pathways and enhancing the understanding of the pathological mechanisms of bacterial pneumonia. Therefore, the development of efficient miRNA detection tools is of great significance for the early diagnosis, treatment, and development of diseases. However, due to the characteristics of low abundance of miRNA in total RNA samples, high homology among family members, and short length, the development of effective miRNA detection strategies remains a major challenge.

[0003] Traditional miRNA detection methods include northern blotting technology and DNA microarray. Their applications are hindered to a certain extent due to the need for large amounts of samples and limited detection sensitivity. The gold standard for miRNA detection is RT-PCR, but it requires expensive temperature control instruments and cumbersome operations. To improve the effectiveness of miRNA detection, isothermal nucleic acid amplification technologies have been widely developed, such as rolling circle amplification (RCA), isothermal exponential amplification reaction (EXPAR), and strand displacement amplification (SDA). Most of these methods achieve sensitive and rapid miRNA analysis by combining the efficient activities of endonucleases and polymerases. However, some studies have shown that the combination of endonucleases and polymerases can lead to a large number of non-specific amplifications, which may result in false positives and thus affect the detection results. Therefore, creating new strategies based on endonucleases or polymerases to complete high-precision and rapid miRNA analysis remains a major challenge.

[0004] As a molecular machine based on DNA nanotechnology, the DNA Walker utilizes the programmability of DNA sequences and molecular recognition ability to amplify detection signals through target-driven walking behavior, thereby achieving highly sensitive detection of miRNA. The high processing ability and fast walking kinetics exhibited by the DNA Walker have attracted great attention in many fields. Standard DNA Walkers usually include a track element, a walking strand element, and a driving element. It can independently and systematically follow a predetermined path to rapidly release payloads through pH changes, strand exchange events, and DNA cleavage, facilitated by DNAzymes or proteases. Due to its rapid signal amplification, flexible programmability, and adaptable design, the DNA Walker has become a potential technology for miRNA detection, including simple strand hybridization or enzymatic cleavage reactions.

[0005] The commonly used DNAzymes for DNA Walkers mainly rely on metal ions and serve as a DNA amplification strategy without proteases (usually replaced by metal ions). However, DNAzyme-driven DNA Walkers require highly specific recognition sequences, and non-specific sequences may trigger conformational changes in DNAzymes, leading to false positive results. Therefore, there is an urgent need for innovative alternative methods and technologies to improve the performance and stability of DNA Walkers for extremely sensitive, selective, and rapid nucleic acid detection. Summary of the Invention

[0006] The purpose of the present invention is to provide a DNA Walker for MicroRNA detection and its application to overcome the problems of low sensitivity and poor selectivity in existing MicroRNA detection technologies.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] One of the technical solutions of the present invention is to provide a DNA Walker for MicroRNA detection, including an S1 / S2@AuNPs three-dimensional motion carrier, a DNAzyme system, and a linking probe.

[0009] Among them, the S1 / S2@AuNPs three-dimensional motion carrier includes AuNPs and S1 and S2 strands connected to the surface of the AuNPs, and the S2 strand is connected with a fluorescent group.

[0010] The DNAzyme system includes a DNAzyme and a first hairpin structure connected to one end of the DNAzyme. The DNAzyme is used to cleave the S2 strand and excite fluorescence. The first hairpin structure is base complementary to the linking probe and includes a first region that is base complementary to the microRNA to be detected.

[0011] The connecting probe includes a second region that is base complementary paired with the first hairpin structure, and a second hairpin structure that is connected to one end of the second region and is base complementary paired with the S1 strand.

[0012] In the present invention, the present invention is a dynamic self-assembled DNA nanomechanical system, and a dynamic molecular machine with the synergistic action of three components is creatively developed: ① S1 / S2@AuNPs three-dimensional motion carrier: The high specific surface area of AuNPs greatly improves the binding efficiency of DNAzyme and substrate, enhances the fluorescence signal, and at the same time increases the local concentration of the connecting probe; ② DNAzyme system: Using a hairpin structure design to achieve target-triggered conformational switching and self-activation; ③ Connecting probe (LP): Through "fulcrum-hinge" type dynamic hybridization, precisely control the assembly and release processes. The synergistic action of these three realizes the programmed assembly, precise regulation, and specific recognition of the nanomechanical system.

[0013] In some specific embodiments, one end of the S1 strand and the S2 strand fixed on the surface of AuNPs is provided with a thiol group, and the other end of the S2 strand is provided with a FAM fluorophore.

[0014] In some specific embodiments, the nucleotide sequence of the S1 strand is as shown in SEQ ID NO.1, the nucleotide sequence of the S2 strand is as shown in SEQ ID NO.2, the nucleotide sequence of the DNAzyme system is as shown in SEQ ID.NO.3, the nucleotide sequence of the connecting probe is as shown in SEQ ID NO.4, and the microRNA to be detected is miRNA-155 with a nucleotide sequence as shown in SEQ ID NO.5.

[0015] In some specific embodiments, the diameter of the AuNPs is 13 ± 2 nm, and the molar ratio of the S1 strand and the S2 strand modified on the surface of the AuNPs is 1:1, and the density is 2.5×10 3 DNA strands / gold nanoparticles.

[0016] The second technical solution of the present invention is to provide an application of the DNA walker as described in the above technical solution in detecting microRNA for non-diagnostic and therapeutic purposes.

[0017] In some specific embodiments, the method for the DNA walker to detect microRNA includes the following steps:

[0018] (1) Mix the S1 strand and the S2 strand according to the molar ratio, add them to the AuNPs solution, incubate at room temperature, centrifuge, and collect the precipitate and resuspend it in PBS buffer containing NaCl to obtain the S1 / S2@AuNPs three-dimensional motion carrier;

[0019] (2) Mix the S1 / S2@AuNP three-dimensional motion carrier, DNAzyme system, linking probe, and microRNA solution to be detected obtained in step (1) in a PBS buffer containing Mn 2+ and react. Measure the fluorescence intensity of the microRNA solution to be detected.

[0020] In some specific embodiments, in step (1), the final concentration ratio of the S1 strand, S2 strand, and AuNP solution is 1:1:10 -3 ;

[0021] The concentration of the PBS buffer containing NaCl is 10 mM, the concentration of NaCl is 0.1 M, and the pH is 7.4.

[0022] In some specific embodiments, in step (1), the incubation time at room temperature is at least 24 h.

[0023] In the present invention, the surface modification of AuNPs requires strict control of the salt concentration (0.1 M NaCl) to balance the DNA adsorption efficiency and stability.

[0024] In some specific embodiments, in step (2), the final concentration ratio of the three-dimensional motion carrier, DNAzyme, linking probe, and microRNA to be detected is 10:10:(1 - 20):(10 -6 - 1);

[0025] The concentration of Mn 2+ in the PBS buffer containing Mn 2+ is (500 - 1300) μM.

[0026] In some specific embodiments, in step (2), the reaction temperature is 37 °C and the reaction time is 10 - 30 min.

[0027] In the present invention, to ensure that the hybridization time of microRNA and the DNAzyme system is ≥10 minutes during the activation stage of the DNAzyme system triggered by the target microRNA, non-specific binding is avoided.

[0028] The principle of detecting microRNA by the DNA walker of the present invention is: Binding Figure 1, in the presence of the target microRNA, the target microRNA base-pairs with the first region (illustrated as the "1" region of the hairpin structure) on the DNAzyme system, thereby opening the hairpin structure (illustrated as the "1" + "2" region) on the DNAzyme system to form a miRNA / DNAzyme complex. Subsequently, the second region on the linker probe LP strand base-pairs with the hairpin structure on the DNAzyme system to form an LP / DNAzyme unit (illustrated as the base-pairing between the "2*" region on the LP strand and the "2" region on the DNAzyme system), and the "1*" region on the linker probe LP strand competes with the target microRNA to base-pair with the "1" region on the DNAzyme system, prompting the release of the target microRNA. The released target microRNA activates the unfolding of the hairpin structure of the DNAzyme system again in a cycle. At the same time, the hairpin structure (illustrated as the "4" region) on the LP strand unfolds, and the LP / DNAzyme unit base-pairs with the S1 strand through the unfolded "4" region on the LP strand to form a stable S1-LP-DNAzyme unit complex (DNAzyme motor). The binding part of the LP strand and the S1 strand can serve as a fulcrum for releasing DNAzyme and as the long arm of the DNAzyme motor. Catalyzed by metal ions, DNAzyme exhibits its cleavage activity, and the activated DNAzyme motor performs autonomous progressive movement along the AuNPs surface. The S2 strand substrate molecule on the AuNPs surface base-pairs with DNAzyme (illustrated as the "3" region), and the FAM-labeled fragment is cleaved and released. Due to the unstable hybridization between the S2 strand and DNAzyme, after DNAzyme cleaves the S2 strand and releases the FAM-labeled fragment, it dissociates from one S2 strand and transfers to another S2 strand, successfully performing autonomous and progressive movement along the three-dimensional path based on AuNPs. One FAM fragment is released with each step, resulting in an increase in the fluorescence signal, enabling efficient monitoring of the DNA walker movement.

[0029] Through the three-segment cooperative recognition of "Region 1 (the binding region on the DNAzyme system for the target microRNA) + Region 2 (the binding region on the DNAzyme system for the LP strand) + Region 4 (the anchoring site for the S1 strand on the LP strand)", the binding efficiency and accuracy are significantly improved. The hairpin structure of the DNAzyme system is in a closed state when not bound to the target microRNA. Non-target microRNAs cannot trigger the unfolding of the hairpin structure on the DNAzyme system due to mismatch sequences and cannot activate the DNA walker through simple hybridization. Only the target microRNA can achieve specific activation of the DNA walker and enhancement of the fluorescence signal.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention innovatively constructs a dual-loop amplification mechanism mediated by target microRNA:

[0032] The first secondary loop: The target microRNA triggers the continuous reconstruction of the DNAzyme system through the dynamic cycle process of "binding - releasing - rebinding", realizing the activation of multiple DNAzyme systems by single-molecule microRNA, and having high selectivity for the target microRNA. MicroRNA with a single-base mismatch is difficult to trigger the continuous reconstruction of the DNAzyme system;

[0033] The second secondary loop: The DNAzyme motor can perform autonomous progressive movement on the surface of three-dimensional gold nanoparticles, and through the continuous enzymatic cleavage reaction of "cutting - dissociation - rebinding", realizes the output of multiple fluorescence signals generated by a single activation.

[0034] This design breaks through the limitations of traditional single-loop signal amplification strategies and significantly improves the detection sensitivity.

[0035] (2) The present invention realizes the integration of enzymatic autonomous movement and signal transduction, simplifying the process and cost of detecting microRNA: ① Innovatively integrates the catalytic function of DNAzyme and the movement function of molecular walker; ② Realizes continuous substrate cleavage without exogenous enzymes through the self-catalytic property of DNAzyme activated by metal ions; ③ Based on the design of the three-dimensional movement trajectory of gold nanoparticles, compared with the traditional two-dimensional surface system, greatly improves the stepping efficiency of the DNAzyme motor; ④ The fluorescence signal release is synchronized with the movement of the DNA walker in real time, realizing the visual monitoring of the detection process.

[0036] (3) The present invention is applicable to clinical use, can directly detect serum samples without RNA extraction and RNA reverse transcription steps, and subsequent bedside detection (POCT) can be realized through naked-eye interpretation (in the form of test strips) or a mobile phone camera + portable fluorescence instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the principle of the DNA walker of the present invention.

[0038] Figure 2 To verify the effect of S1 / S2@AuNPs, Figure 2 B is to verify the activation of the DNAzyme system triggered by the target microRNA, Figure 2 C is the fluorescence intensity of the DNA walker detecting the target microRNA.

[0039] Figure 3 A is the sensitivity fluorescence response graph of different microRNA concentrations,Figure 3 B and D are the linear range and detection limit of the microRNA concentration, Figure 3 C is the specific fluorescence response diagram of different interferents and the target. Specific implementation manners

[0040] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0041] In the following embodiments, if there is no special description of raw materials or processing techniques, it means that they are all conventional commercially available raw material products or conventional processing techniques in the art.

[0042] Example 1: Preparation and verification of S1 / S2@AuNPs

[0043] 1 Materials:

[0044] (1) The diameter of the selected AuNPs is 13 nm, and OD520 = 3.0.

[0045] (2) The nucleotide sequence of the S1 strand is shown in SEQ ID NO.1, specifically: 5′-Cy3-SH-TTT TTT TTT TTTTTT CTC TAT GAT GTT GTC AAG TTT TTG GCT CCCAG-SH-3′.

[0046] (3) The nucleotide sequence of the S2 strand is shown in SEQ ID NO.2, specifically: 5′-FAM-SH-TTT TTT TTT TTTTTA CTA TrAGG AAG TAGT-SH-3′, where r is A or G.

[0047] (4) Buffer: 10 mM PBS solution containing 0.1 M NaCl, pH 7.4.

[0048] 2 Preparation process:

[0049] (1) Mix the S1 strand and the S2 strand at a molar ratio of 1:1, with a final concentration of 1 μM each, and add the AuNPs solution (final concentration 1 nM).

[0050] (2) Incubate at room temperature for 24 hours, and fix the S1 strand and the S2 strand on the surface of AuNPs through Au-S bonds respectively.

[0051] (3) Centrifuge at 12,000 rpm for 15 minutes, remove the unbound DNA strands, wash 3 times repeatedly, and resuspend in the buffer to obtain S1 / S2@AuNPs.

[0052] 3 Functional verification:

[0053] Use a fluorescence spectrophotometer to detect the fluorescence intensity of the solution before and after the S1 chain and S2 chain modify AuNPs, as Figure 2 shown in A:

[0054] S1 / S2 mixture: Both the Cy3 (excitation wavelength is 552 nm, emission wavelength is 570 nm) and FAM (excitation wavelength is 494 nm, emission wavelength is 520 nm) signals are significant.

[0055] After S1 / S2 binds to AuNPs: The Cy3 and FAM signals decrease, proving that the S1 chain and S2 chain are successfully immobilized on the surface of AuNPs and the fluorescent groups are quenched by the surface plasmon resonance of AuNPs.

[0056] Example 2: Verify the activation of the DNAzyme system triggered by the target microRNA

[0057] 1 Materials:

[0058] (1) Target microRNA: miRNA-155, whose nucleotide sequence is as shown in SEQ ID NO.5, specifically: 5′-UUAAUG GUA AUC GUG AUA GGG GU-3′.

[0059] (2) The nucleotide sequence of the DNAzyme system is as shown in SEQ ID NO.3, specifically: 5′-ACC ATT AAG TACACA TGG TTA ATG GTA ATC GTG ATC CTCA AGG AGC TGG AAG GCT GGG AGC CAT ATG TGTACT CCA GCT CCT TGA CTA CTT CTC CGA GCC GGT CGA AAT AGT-3′, and a Cy3 group is added at the 5' end.

[0060] (3) The nucleotide sequence of the ligation probe (LP) is as shown in SEQ ID NO.4, specifically: 5′-ACC ATT AAGTAC ACA TGG TTA ATG GTA ATC GTG ATC CAT GTG TAC-3′.

[0061] (4) S1 / S2@AuNPs prepared in Example 1.

[0062] 2 System construction

[0063] Experimental group: S1 / S2@AuNPs (10 nM), DNAzyme system (10 nM), miRNA-155 (1 pM), and LP (10 nM) were mixed in PBS buffer (containing 0.75 mM Mn 2+ ) and reacted at 37 °C for 30 min.

[0064] Control group: (1) Target microRNA was missing; (2) LP was missing; (3) Target microRNA and LP were missing; (4) Only S1 / S2@AuNPs; (5) Only DNAzyme system.

[0065] 3 Fluorescence detection

[0066] The detection conditions were as follows: the excitation wavelength of the Cy3 group was 552 nm, and the emission wavelength was 570 nm.

[0067] The close proximity of the S1 strand to the DNAzyme system was tested by labeling the end of the DNAzyme system with Cy3. As Figure 2 shown in B, columns 1-5 represent the control groups: DNAzyme system, S1 / S2@AuNPs, S1 / S2@AuNPs + DNAzyme system, S1 / S2@AuNPs + DNAzyme system + LP, LP, and column 6 represents the experimental group S1 / S2@AuNPs + DNAzyme system + LP + microRNA.

[0068] When only S1 / S2@AuNPs was mixed with the Cy3-labeled DNAzyme system or only S1 / S2@AuNPs, LP was mixed with the Cy3-labeled DNAzyme system, the recorded fluorescence signal remained at a high level, indicating that the DNAzyme was not bound to the S1 strand, see Figure 2 columns 3 and 4 of B. When the target microRNA and LP were added, the recorded Cy3 signal decreased significantly, which means that the S1 strand base-paired with the LP strand, that is, the S1 strand was close to the DNAzyme system, and the Cy3 signal on the DNAzyme system was quenched by the AuNP, see Figure 2 column 6 of B.

[0069] The above results indicate that miRNA-155 (target microRNA) can trigger the unfolding of the hairpin structure of the DNAzyme system to form a miRNA / DNAzyme complex; the LP strand hybridizes with the hairpin structure of the DNAzyme system and transfers the LP / DNAzyme unit to the vicinity of the S1 strand, and the Cy3-labeled DNAzyme system is close to the S1 strand on the AuNP surface through the LP strand, resulting in Cy3 fluorescence quenching.

[0070] Example 3: Application of DNA Walker in Detecting microRNA

[0071] 1 Materials:

[0072] (1) Target microRNA: miRNA-155, whose nucleotide sequence is shown in SEQ ID NO.5, specifically: 5′-UUAAUG GUA AUC GUG AUA GGG GU-3′.

[0073] (2) The nucleotide sequence of the DNAzyme system is shown in SEQ ID NO.3, specifically: 5′-ACC ATT AAG TACACA TGG TTA ATG GTA ATC GTG ATC CTCA AGG AGC TGG AAG GCT GGG AGC CAT ATG TGTACT CCA GCT CCT TGA CTA CTT CTC CGA GCC GGT CGA AAT AGT-3′, and a Cy3 group is added at the 5′ end.

[0074] (3) The nucleotide sequence of the ligation probe (LP) is shown in SEQ ID NO.4, specifically: 5′-ACC ATT AAGTAC ACA TGG TTA ATG GTA ATC GTG ATC CAT GTG TAC-3′.

[0075] (4) S1 / S2@AuNPs prepared in Example 1.

[0076] 2 Experimental groups: AuNPs (10 nM) with S1 strand and S2 strand immobilized on the surface, DNAzyme system (10 nM), target microRNA (miRNA-155, 1 pM), and LP (10 nM) were mixed in PBS buffer (containing 0.75 mM Mn 2+ ) and reacted at 37 °C for 30 min.

[0077] Control groups: ① miRNA-155 was absent; ② miRNA-155 and LP were absent.

[0078] 3 Fluorescence kinetics monitoring

[0079] The fluorescence signal was read using a real-time fluorescence monitor to capture the dynamic movement process of the DNAzyme motor. The detection conditions were: the excitation wavelength of the Cy3 group was 552 nm, and the emission wavelength was 570 nm; the excitation wavelength of the FAM group was 494 nm, and the emission wavelength was 520 nm.

[0080] As Figure 2As shown in C, the FAM signal of the experimental group system (legend: S1 / S2@AuNPs+miR+LP) was significantly enhanced.

[0081] The FAM signal of control group ① (legend: S1 / S2@AuNPs+LP) was equivalent to the background value of control group ② (legend: S1 / S2@AuNPs), indicating that miRNA-155 (the target microRNA) is an essential factor triggering signal amplification.

[0082] The above results show that the enhancement of the FAM signal is due to the cyclic cleavage movement of the DNAzyme motor. In each step of the movement, the DNAzyme cleaves the S2 strand, releasing the FAM-labeled fragment (the FAM group of the S2 strand was initially quenched by AuNPs and becomes free in the solution after cleavage, and the fluorescence is restored).

[0083] Example 4: Sensitivity and selectivity of DNA walker

[0084] 4.1 microRNA concentration gradient analysis

[0085] 1 Materials

[0086] (1) Target microRNA: miRNA-155, whose nucleotide sequence is shown in SEQ ID NO.5, specifically: 5′-UUAAUG GUA AUC GUG AUA GGG GU-3′.

[0087] (2) The nucleotide sequence of the DNAzyme system is shown in SEQ ID NO.3, specifically: 5′-ACC ATT AAG TACACA TGG TTA ATG GTA ATC GTG ATC CTCA AGG AGC TGG AAG GCT GGG AGC CAT ATG TGTACT CCA GCT CCT TGA CTA CTT CTC CGA GCC GGT CGA AAT AGT-3′, and a Cy3 group is added at the 5' end.

[0088] (3) The nucleotide sequence of the ligation probe (LP) is shown in SEQ ID NO.4, specifically: 5′-ACC ATT AAGTAC ACA TGG TTA ATG GTA ATC GTG ATC CAT GTG TAC-3′.

[0089] (4) S1 / S2@AuNPs prepared in Example 1.

[0090] 2 Experimental system: Mix S1 / S2@AuNPs (10 nM), DNAzyme system (10 nM), target microRNA (miRNA-155, from 0.01 fM to 10 nM), and LP (10 nM) in PBS buffer (containing 0.75 mM Mn 2+ ) and react at 37 °C for 30 min. Detect the change in FAM fluorescence signal. The excitation wavelength of the FAM group is 494 nm, and the emission wavelength is 520 nm.

[0091] As Figure 3 shown in the results of A: As the concentration of miRNA-155 increases (10 fM → 1 nM), the FAM fluorescence intensity increases step by step, indicating that the signal amplification ability of the DNAzyme walker is positively correlated with the concentration of the target microRNA.

[0092] 4.2 Detection limit analysis

[0093] Based on the experimental system for sensitivity analysis, when the concentration of miRNA-155 is in the range of 10 fM to 1 nM, there is a significant linear relationship between the fluorescence intensity (Y) and the logarithm of the miRNA concentration (lgC): Y = 1146×lgC + 89.24, R 2 = 0.9925, as Figure 3 shown in B and D.

[0094] 4.3 Selectivity analysis

[0095] 1 Materials

[0096] (1) Target microRNA group: miRNA-155, whose nucleotide sequence is as shown in SEQ ID NO.5, specifically: 5′-UUA AUG GUA AUC GUG AUA GGG GU-3′.

[0097] Interfering microRNA group: miRNA-149, miRNA-21 (homologous sequences), and single-base mismatch sequences M1-155 (1-base mismatch), M2-155 (2-base mismatch), M3-155 (3-base mismatch);

[0098] The nucleotide sequence of M1-155 is as shown in SEQ ID NO.6, specifically: 5′-UUA AUG GUA AUC GUG AUAG A G GU-3′;

[0099] The nucleotide sequence of M2-155 is as shown in SEQ ID NO.7, specifically: 5′-UUA AUG GUA A G C GUG AUAG A G GU-3′;

[0100] The nucleotide sequence of M3-155 is shown in SEQ ID NO.8, specifically: 5′-UUA A C G GUA A G C GUG AUAG A G GU-3′;

[0101] The underlined part is the mismatched base.

[0102] (2) The nucleotide sequence of the DNAzyme system is shown in SEQ ID NO.3, specifically: 5′-ACC ATT AAG TACACA TGG TTA ATG GTA ATC GTG ATC CTCA AGG AGC TGG AAG GCT GGG AGC CAT ATG TGTACT CCA GCT CCT TGA CTA CTT CTC CGA GCC GGT CGA AAT AGT-3′, and a Cy3 group is added at the 5′ end.

[0103] (3) The nucleotide sequence of the ligation probe (LP) is shown in SEQ ID NO.4, specifically: 5′-ACC ATT AAGTAC ACA TGG TTA ATG GTA ATC GTG ATC CAT GTG TAC-3′

[0104] (4) S1 / S2@AuNPs prepared in Example 1.

[0105] 2 Experimental system: Mix S1 / S2@AuNPs (10 nM), DNAzyme system (10 nM), microRNA, and LP (10 nM) in PBS buffer (containing 0.75 mM Mn 2+ ), and react at 37 °C for 30 min. Detect the change in FAM fluorescence signal. The excitation wavelength of the FAM group is 494 nm, and the emission wavelength is 520 nm.

[0106] The concentrations of the target microRNA group and the interfering microRNA group are both divided into 1 pM and 10 pM.

[0107] As Figure 3 shown in C, at concentrations of 1 pM and 10 pM, only the target miRNA-155 caused a significant fluorescence enhancement.

[0108] The fluorescence signals of homologous miRNAs (-149, -21) or mismatched sequences (M1~3-155) had no statistical difference from the blank group (p>0.05).

[0109] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A DNA walker for MicroRNA detection, characterized in that, It includes an S1 / S2@AuNPs three-dimensional motion carrier, a DNAzyme system, and a linking probe. Among them, the S1 / S2@AuNPs three-dimensional motion carrier includes AuNPs, and an S1 strand and an S2 strand connected to the surface of the AuNPs. The S2 strand is also connected to a fluorescent group. The DNAzyme system includes a DNAzyme and a first hairpin structure connected to one end of the DNAzyme. The DNAzyme is used to cleave the S2 strand and excite fluorescence. The first hairpin structure is base complementary paired with the linking probe and includes a first region that is base complementary paired with the microRNA to be detected. The linking probe includes a second region that is base complementary paired with the first hairpin structure, and a second hairpin structure connected to one end of the second region and base complementary paired with the S1 strand.

2. The DNA walker for MicroRNA detection according to claim 1, wherein One end of the S1 strand and the S2 strand fixed on the surface of AuNPs is provided with a thiol group, and the other end of the S2 strand is provided with a FAM fluorescent group.

3. The DNA walker for MicroRNA detection according to claim 1, wherein The nucleotide sequence of the S1 strand is as shown in SEQ ID NO.1, the nucleotide sequence of the S2 strand is as shown in SEQ ID NO.2, the nucleotide sequence of the DNAzyme system is as shown in SEQ ID.NO.3, the nucleotide sequence of the linking probe is as shown in SEQ ID NO.4, and the microRNA to be detected is miRNA-155 with a nucleotide sequence as shown in SEQ ID NO.

5.

4. The DNA walker for MicroRNA detection according to claim 1, wherein The diameter of the AuNPs is 13 ± 2 nm, and the molar ratio of the S1 strand and the S2 strand modified on the surface of the AuNPs is 1:1, with a density of 2.5×10 3 DNA strands / gold nanoparticles.

5. Application of a DNA walker as described in any one of claims 1 to 4 in detecting microRNA for non-diagnostic and non-therapeutic purposes.

6. The application according to claim 5, wherein The method for the DNA walker to detect microRNA includes the following steps: (1) Mix the S1 strand and the S2 strand according to the molar ratio, add them to the AuNPs solution, incubate at room temperature, centrifuge, and collect the precipitate and resuspend it in a PBS buffer solution containing NaCl to obtain the S1 / S2@AuNPs three-dimensional motion carrier. (2) Mix the S1 / S2@AuNPs three-dimensional motion carrier, DNAzyme system, linking probe, and the microRNA solution to be detected obtained in step (1) in a PBS buffer containing Mn 2+ and react, and measure the fluorescence intensity of the microRNA solution to be detected.

7. The application according to claim 6, characterized in that, In step (1), the final concentration ratio of S1 chain, S2 chain, and AuNPs solution is 1:1:10 -3 ; The concentration of the PBS buffer solution containing NaCl is 10 mM, the concentration of NaCl is 0.1 M, and the pH is 7.

4.

8. The application according to claim 6, wherein In step (1), the incubation time at room temperature is at least 24 h.

9. The application according to claim 6, wherein In step (2), the final concentration ratio of the S1 / S2@AuNPs three-dimensional motion carrier, the DNAzyme system, the linking probe, and the microRNA solution to be detected is 10:10:(1-20):(10 -6 ~1); The PBS buffer solution containing Mn 2+ has a Mn 2+ concentration of (500 - 1300) μM.

10. The application according to claim 6, wherein In step (2), the reaction temperature is 37 °C, and the reaction time is 10 - 30 min.

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