Methytransferase sensor based on DNA walker and hyperbranched rolling circle amplification and application thereof

Abstract

This invention discloses a methyltransferase sensor based on a DNA walker and hyperbranched rolling circle amplification, and its application, relating to the field of biosensors. The methyltransferase sensor includes a recognition probe, a DNA walker, and a hyperbranched rolling circle amplification system; the recognition probe includes H... M Identification probes and / or H D Identification probe, the H M The nucleotide sequence of the recognition probe is shown in SEQ ID NO.1, wherein the H D The nucleotide sequence of the recognition probe is shown in SEQ ID NO. 2. This invention employs a dual amplification strategy of DNA walker and HRCA to achieve highly sensitive detection of methyltransferases, reducing the blank signal in the detection. Simultaneous detection of two different methyltransferases can be achieved through simple recognition probe design. The detection experiment does not require complex temperature cycling or expensive instruments; it can be completed in a single centrifuge tube, resulting in low system cost.

Description

Technical Field

[0001] This invention relates to the field of biosensors, and in particular to a methyltransferase sensor based on a DNA walker and hyperbranched rolling circle amplification, and its applications. Background Technology

[0002] DNA methylation involves the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to the C-5 position of cytosine or the N6 position of adenine, playing a crucial role in genomic imprinting, gene expression, and cell development in eukaryotes and prokaryotes. DNA methylation modification, as an important mechanism for regulating gene expression, is catalyzed by DNA methyltransferases and plays a vital role in bacterial growth morphology and physiological metabolism, thereby increasing bacterial survival in variable environments. Therefore, developing a sensitive and accurate method for detecting DNA methyltransferase activity is essential for studying bacterial growth morphology and drug resistance.

[0003] To date, various methods have been developed for DNA methyltransferase assays, such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), microarrays, colorimetric detection, fluorescence detection, electrochemical detection, electrochemiluminescence, and the determination of radioactively labeled [3H]-adenosylmethionine. Among these, fluorescence detection offers advantages such as low cost, relatively simple operation, fast response, and good repeatability and consistency, making it highly valuable for DNA methyltransferase assays. However, these fluorescence sensors lack sufficient sensitivity, and accurate DNA methyltransferase activity assays may require more sensitive amplification strategies. Therefore, many signal amplification strategies have been applied in methyltransferase assays to achieve higher detection sensitivity, including real-time quantitative PCR (qPCR), hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), and rolling circle amplification (RCA). Among these amplification strategies, qPCR is considered the most successful due to its high availability and versatility. However, PCR requires complex thermal cycling steps, sophisticated equipment, and long operation times. Compared to PCR, HCR and CHA are representative isothermal non-enzymatic DNA amplification methods, offering faster and more convenient operation. However, due to the tendency of multiple hairpin structures to hybridize, HCR and CHA biosensors are susceptible to high background signals. RCA, as a next-generation isothermal DNA amplification technique, has been used to construct biosensors due to its high efficiency and simplicity. However, the amplification efficiency of RCA is limited, and the sensitivity of RCA-based sensors is lower than that of PCR. To improve the sensitivity of RCA, linear RCA (LRCA) can be converted to hyperbranched rolling coil amplification (HRCA) by introducing a second primer complementary to the RCA product. This primer has a much higher amplification efficiency (up to 10) than LRCA. 9This method has been successfully applied to construct biosensors targeting various different objectives. For example, Lin Zhenyu's team at Fuzhou University developed a label-free Dam methyltransferase biosensor (detection limit = 1.8 U / mL) using methylation-sensitive lysis primers and HRCA. This sensor has a high detection limit, but its sensitivity is significantly lower than that of enzyme-free sensors.

[0004] DNA walkers are novel molecular machines that can travel along predefined DNA tracks. They have been widely used in biosensors, drug delivery, and biocomputing. DNA walkers can rapidly release substrates in a short time, thereby achieving efficient and significant signal enhancement. Summary of the Invention

[0005] The purpose of this invention is to provide a methyltransferase sensor based on DNA walker and hyperbranched rolling circle amplification and its application, in order to solve the problems existing in the prior art. This invention achieves high-sensitivity detection of methyltransferase by using a DNA walker-HRCA strategy.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a methyltransferase sensor based on a DNA walker and hyperbranched rolling circle amplification, the methyltransferase sensor comprising a recognition probe, a DNA walker, and a hyperbranched rolling circle amplification system; the recognition probe includes H M Identification probes and / or H D Identification probe, the H M The nucleotide sequence of the recognition probe is shown in SEQ ID NO.1, wherein the H D The nucleotide sequence of the recognition probe is shown in SEQ ID NO.2.

[0008] Furthermore, the hyperbranched rolling circle amplification system includes phi29 DNA polymerase, primer 1, primer 2, and dNTPs; the nucleotide sequence of primer 1 is shown in SEQ ID NO.9, and the nucleotide sequence of primer 2 is shown in SEQ ID NO.10.

[0009] The present invention also provides the application of the above-described methyltransferase sensor in the preparation of a kit for detecting methyltransferases.

[0010] The present invention also provides a kit for detecting methyltransferases, comprising the above-described methyltransferase sensor.

[0011] Furthermore, the kit also includes a Padlock probe, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0012] Furthermore, the kit also includes functionalized magnetic beads, the preparation method of which includes: mixing biotin-modified DNAzyme and an inhibitor in Tris-HCl buffer, forming a DNAzyme inhibitor double strand after reaction; then dispersing streptavidin magnetic beads in Tris-HCl buffer, followed by adding the DNAzyme inhibitor double strand, and obtaining the functionalized magnetic beads after reaction.

[0013] The nucleotide sequence of the DNAzyme is shown in SEQ ID NO.4; the nucleotide sequence of the blocking agent is shown in SEQ ID NO.5.

[0014] The present invention also provides the application of the above-described methyltransferase sensor or kit in methyltransferase detection.

[0015] The present invention also provides a method for detecting methyltransferase, comprising the steps of detecting methyltransferase using the methyltransferase sensor or kit described above.

[0016] Further, when the methyltransferase is M.SssI methyltransferase, the method includes: using the H in the methyltransferase sensor described above. M The identification probe triggers DNA walking and hyperbranched rolling circle amplification reactions through the interaction of M.SssI methyltransferase and HpaII endonuclease, and the content of M.SssI methyltransferase is detected by quantitative fluorescence signal intensity.

[0017] Further, when the methyltransferase is Dam methyltransferase, the method includes: using the H in the methyltransferase sensor described above. D The recognition probe triggers DNA walking and hyperbranched rolling circle amplification reactions through the interaction of Dam methyltransferase and DpnI endonuclease, and the content of Dam methyltransferase is detected by quantitative fluorescence signal intensity.

[0018] The present invention discloses the following technical effects:

[0019] This invention innovatively incorporates a DNA walker into an HRCA sensing system to construct a novel M.SssI methyltransferase biosensor. This biosensor integrates a DNA walker and HRCA, thereby avoiding the use of multiple hairpin self-assemblies and the resulting low background signal. In the absence of M.SssI methyltransferase, the hairpin H... M It is cleaved by the HpaII endonuclease, producing a DNA fragment called T-DNA. T-DNA can trigger a strand displacement reaction, releasing Pb. 2+The DNA walker then gradually moves across the magnetic bead surface by cleaving the substrate, generating a large number of ssDNA probes (c-DNA). The released c-DNA then triggers a subsequent HRCA reaction, producing a large number of dsDNA probes of varying lengths. SYBR Green I is embedded in the grooves of the dsDNA, producing a significant fluorescent signal. In the presence of M.SssI methyltransferase, hairpin H... M Methylated, it cannot be cleaved by endonucleases. The subsequent DNA walker-HRCA reaction is not initiated, and the fluorescence signal remains at a low level. Due to the dual amplification strategy of DNA walker and HRCA, the biosensor of this invention has high sensitivity, with a detection limit of 0.0002 U / mL. Because M.SssI methyltransferase is sensitive to hairpin H... M This biosensor exhibits excellent selectivity for site-specific recognition. Furthermore, the biosensor proposed in this invention can be used to detect another methyltransferase (Dam methyltransferase). The results of this invention demonstrate that the DNA walker-HRCA strategy has great application potential in DNA methylation-related detection.

[0020] The advantages of the biosensor based on DNA walker and hyperbranched rolling circle amplification of the present invention are as follows: (1) High sensitivity: Combining two-layer amplification reaction greatly improves the detection sensitivity compared with existing technologies. (2) Low background signal: The present invention avoids the strategy of self-assembly of multiple hairpin structures commonly used in isothermal amplification, thus reducing the blank signal of detection. (3) Simultaneous detection of two different methyltransferases can be achieved through simple recognition probe design. (4) The experiment does not require complex temperature cycling and expensive instruments; it can be completed in a single centrifuge tube, resulting in low system cost. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The sensing strategy of the M.SssI methyltransferase sensor based on DNA walker and HRCA amplification is given; where A represents the case where M.SssI methyltransferase is absent in the system, and B represents the case where M.SssI methyltransferase is present in the system.

[0023] Figure 2This is an example of the determination of M.SssI methyltransferase using a DNA walker-HRCA system; where A is the fluorescence spectrum of the M.SssI methyltransferase sensor in the DNA walker-HRCA reaction, corresponding to different concentrations of M.SssI methyltransferase; B is the fluorescence intensity at 530 nm for different concentrations of M.SssI methyltransferase (range = 0-1 U / mL); C is a calibration curve of the change in fluorescence intensity at 530 nm for different concentrations of M.SssI methyltransferase (range = 0.0002-0.1 U / mL) against the logarithm of methyltransferase concentration.

[0024] Figure 3 The sensing strategy for a Dam methyltransferase sensor based on a DNA walker and HRCA amplification is described; where A represents the case where Dam methyltransferase is absent in the system; and B represents the case where Dam methyltransferase is present in the system.

[0025] Figure 4 This is an example of the determination of Dam methyltransferase using a DNA walker-HRCA system. A represents the fluorescence spectrum of the Dam methyltransferase sensor in the DNA walker-HRCA reaction, corresponding to different concentrations of Dam methyltransferase. B represents the fluorescence intensity at 530 nm for different concentrations of Dam methyltransferase (range = 0-1 U / mL). The inset in B is a calibration curve of the change in fluorescence intensity at 530 nm for different concentrations of Dam methyltransferase (range = 0.001-0.2 U / mL) against the logarithm of methyltransferase concentration. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] The DNA sequences used in the following examples are shown in Table 1:

[0032] Table 1

[0033]

[0034] The preparation method of the functionalized magnetic beads used in the following embodiments is as follows:

[0035] A mixture of biotin-modified DNAzyme (1 μM) and a blocking agent (1 μM, i.e., Blocker in Table 1) in Tris-HCl buffer (25 mM Tris-HCl, 200 mM NaAc, pH 7) was heated at 90 °C for 10 min, then cooled to 25 °C to form a double-stranded DNAzyme blocking agent. Subsequently, 10 μL of streptavidin magnetic beads (SMB 1 mg / mL) were washed three times with Tris-HCl buffer and dispersed in Tris-HCl buffer. The double-stranded DNAzyme blocking agent and the biotin-modified substrate (i.e., Substrate in Table 1) were mixed at a molar ratio of 1:20 and added to the streptavidin magnetic beads. The mixture was then shaken at room temperature for 1 h to obtain functionalized magnetic beads, which were stored at 4 °C for subsequent experiments. The functionalized magnetic beads were dispersed after magnetic separation to remove excess DNA strands.

[0036] In the following embodiments, the DNA walker is as follows: Figure 1 As shown, Pb is modified on functionalized magnetic beads. 2+This is a T-dependent DNAzyme that is inhibited by a blocking agent (blocker sequence). In the presence of T-DNA, the blocking agent is removed by hybridization with T-DNA, leading to Pb... 2+ Dependent release of DNAzyme. Add Pb. 2+ The DNAzyme is then activated and hybridizes with the substrate. As a result, the substrate is cleaved, leading to the release of exported DNA (c-DNA). This is accompanied by Pb... 2+ The dangling strand of the DNA-dependent DNAzyme hybridizes with another substrate DNA, triggering the next cleavage. Thus, the autonomous walking of the DNAzyme on the magnetic bead surface involves a cycle of hybridization, cleavage, and release, generating a large number of c-DNA strands. Therefore, a single T-DNA strand can induce the cleavage of the substrate DNA and the release of various c-DNA strands, completing the construction of the DNA walker and signal amplification. The magnetic beads are removed by magnetic separation, and the reaction product (c-DNA) is hybridized with a loop-locked probe for use in the HRCA reaction.

[0037] Example 1: Construction of the DNA walker-HRCA system and determination of M.SssI methyltransferase

[0038] Construction of the DNA walker-HRCA system M.SssI methyltransferase sensor: H M To identify the probe, the interaction between M.SssI methyltransferase and HpaII endonuclease triggers subsequent DNA walking and hyperbranched rolling circle amplification reactions. Ultrasensitive detection of M.SssI methyltransferase is achieved by quantifying the fluorescence signal intensity. The detection principle is described in [link to relevant documentation]. Figure 1 .

[0039] Assay of M.SssI methyltransferase:

[0040] First, add 2μL H M (2 μM) was incubated with 5 μL SAM (1600 μM) and different concentrations of M. SssI methyltransferase at 37 °C for 2 h. The methylation process was carried out in 50 μL NE Buffer 2 (provided by New England Biolabs). Subsequently, 5 μL Cutsmart buffer (10×) and HpaII (20 U / mL) were added to the solution at 37 °C for 2 h. The M. SssI / HpaII-treated product was added to a functionalized magnetic bead solution to initiate Pb at 25 °C. 2+ -DNAzyme-assisted DNA migration process for 0.5 h. Subsequently, 5 μL (1 mM) Pb was used. 2+DNA was activated for 1 hour. Finally, the reaction products were separated by magnetic separation to obtain different concentrations of output DNA (c-DNA) for the HRCA reaction. The obtained c-DNA solution was mixed with 10 nM Padlock probe, 800 U T4 DNA ligase, and 10 μL T4 DNA ligase buffer 10× (provided by New England Biolabs). The total volume of the reaction solution was 100 μL. The ligation reaction was carried out at 20 °C for 1 hour. Unreacted probes were then removed by adding Exo I (20 U) and Exo III (20 U). The mixture was heated to 80 °C and held for 20 minutes to inactivate the exonuclease. The ligation product was added to the HRCA reaction mixture containing phi29 DNA polymerase buffer (provided by New England Biolabs), 40 nM prime 1 and prime 2, 0.5 mM dNTPs, and 20 U phi29 DNAPCR. The HRCA reaction was carried out at 30 °C for 4 hours, and then heated to 65 °C and held for 10 minutes to terminate the polymerization reaction. Then, 4 μL of SYBR GreenI was added to the HRCA product and incubated for 15 minutes before fluorescence measurement. Fluorescence detection of M.SssI methyltransferase was performed using a Hitachi F-4700 fluorescence spectrometer. The results are as follows: Figure 2 As shown, the fluorescence intensity gradually decreases with increasing M.SssI methyltransferase content. This is because H... M The probe gradually becomes methylated, making subsequent restriction enzyme and amplification experiments impossible. This invention allows for the quantification of the concentration of M.SssI methyltransferase using fluorescence intensity.

[0041] This embodiment precisely designs the hair clip H. M Its stem sequence is 5'-CCGG-3', which can be recognized by HpaII endonuclease and M.SssI methyltransferase. Furthermore, T-DNA was engineered to be incorporated into hairpin H... M The ring portion. In the absence of M.SssI methyltransferase, HpaII endonuclease can recognize and cleave hairpin H. M Therefore, hairpin H M It is divided into two parts: T-DNA and double-stranded DNA. T-DNA can activate the DNA walker and HRCA amplifier. The cascaded amplifier of the DNA walker and HRCA consists of a first layer of DNA walker and a second layer of HRCA. In the presence of T-DNA, the blocking DNA is removed by hybridization with T-DNA, leading to Pb. 2+ Release of Pb-dependent DNAzyme. Free DNAzyme hybridized with substrate is released upon addition of Pb. 2+It is subsequently activated. As a result, the substrate DNA is cleaved, triggering the release of export DNA (c-DNA). It carries Pb. 2+ The dangling strand of the DNAzyme hybridizes with another substrate DNA and triggers the next cleavage. Thus, the autonomous movement of the DNAzyme on the magnetic bead surface is accompanied by a cycle of hybridization, cleavage, and release, generating a large number of c-DNA strands, thus completing the first layer of signal amplification. The magnetic beads are removed by magnetic separation, and the reaction product (c-DNA) hybridizes with a circular locking probe for the HRCA reaction. Circularization is performed using T4 DNA ligase, generating a circular circular locking probe. After the addition of Exo I and III, unreacted dsDNA and ssDNA are digested by exonucleases, leaving the circular circular locking probe alone in solution. The HRCA reaction begins with the addition of primers 1 and 2, Phi29 DNA polymerase, and dNTPs. Primer 2 is partially complementary to the circular circular locking probe and extends at the 3' end with the aid of Phi29 DNA polymerase and dNTPs, generating a long single-stranded DNA. This long single-stranded DNA then serves as a template for hybridization with primer 1. Primer 1 is extended by Phi29 DNA polymerase to replace the downstream grown DNA strand, providing multiple binding sites for the rehybridization of primer 2. Therefore, a large number of double-stranded and single-stranded DNAs of varying lengths were formed. The fluorescent signal indicator SYBR Green I inserts into the grooves of the double-stranded DNA with extremely high affinity, producing a strong fluorescent signal. However, when M.SssI methyltransferase is present, H… M The stem-loop portion is methylated and cannot be cleaved by the HpaII endonuclease. Due to Pb 2+ The DNAzyme-dependent pathway is blocked by the blocking probe, preventing subsequent HRCA amplification. Therefore, the DNA walker and the amplifier in the HRCA cascade cannot be activated, and the fluorescence signal remains at a very low level. Thus, this invention allows for the quantification of the concentration of M.SssI methyltransferase by detecting the fluorescence signal.

[0042] Example 2: Construction of the DNA walker-HRCA system and determination of Dam methyltransferase

[0043] Construction of the DNA walker-HRCA system Dam methyltransferase sensor: using H D To identify the probe, the interaction between Dam methyltransferase and DpnI endonuclease triggers subsequent DNA walker and hyperbranched rolling circle amplification (HRCA) reactions, and ultrasensitive detection of Dam methyltransferase is achieved by quantifying the fluorescence signal intensity.

[0044] Assay of Dam methyltransferase:

[0045] 2μL H D(2 μM) was incubated with 5 μL SAM (1600 μM), 40 U / mL DpnI, 5 μL Cutsmart buffer (10×), and different concentrations of Dam methyltransferase at 37 °C for 2 h. Methylation was performed in 50 μL DpnI reaction buffer (provided by New England Biolabs). The Dam / DpnI-treated solution was then added to a functionalized magnetic bead solution to initiate a DNA walker-HRCA reaction similar to the M.SssI MTase assay. The Dam / DpnI-treated product was added to the functionalized magnetic bead solution to initiate Pb at 25 °C. 2+ -DNAzyme-assisted DNA migration process for 0.5 h. Subsequently, 5 μL (1 mM) Pb was used. 2+ DNA was activated for 1 hour. Finally, the reaction products were separated by magnetic separation to obtain different concentrations of output DNA (c-DNA) for the HRCA reaction. The obtained c-DNA solution was mixed with 10 nM Padlock probe, 800 U T4 DNA ligase, and 10 μL T4 DNA ligase buffer 10× (provided by New England Biolabs). The total volume of the reaction solution was 100 μL. The ligation reaction was carried out at 20 °C for 1 hour. Unreacted probes were then removed by adding Exo I (20 U) and Exo III (20 U). The mixture was heated to 80 °C and maintained for 20 minutes to inactivate the exonuclease. The ligation product was added to the HRCA reaction mixture containing phi29 DNA polymerase buffer (provided by New England Biolabs), 40 nM primers 1 and 2, 0.5 mM dNTPs, and 20 U phi29 DNAPCR. The HRCA reaction was carried out at 30 °C for 4 hours, and then heated to 65 °C and maintained for 10 minutes to terminate the polymerization reaction. Then, 4 μL of SYBR Green I was added to the HRCA product and incubated for 15 minutes before fluorescence measurement. The fluorescence of Dam methyltransferase was detected using a Hitachi F-4700 fluorescence spectrometer. The results are shown below. Figure 4 As shown, the fluorescence intensity gradually increases with the increase of Dam methyltransferase content, because H D The probe is gradually methylated, allowing the DpnI endonuclease to cleave H. D The probe triggers subsequent DNA walking and HRCA amplification reactions. Thus, this invention allows for the quantification of Dam methyltransferase concentration using fluorescence intensity.

[0046] By designing a hairpin identification probe H DThis enables the detection of another methyltransferase, Dam. Unlike the M.SssI methyltransferase / HpaII system, DpnI can only cleave the methylation recognition sequence (5'-G-mA-TC-3') with the assistance of Dam methyltransferase, thereby triggering the subsequent DNA walker HRCA reaction (see...). Figure 3 In the absence of Dam methyltransferase, DpnI cannot digest hairpin H. D The DNA walker-HRCA reaction is inhibited, resulting in a weak fluorescence signal. In the presence of Dam methyltransferase and DpnI, the fluorescence signal shows a significant enhancement. Therefore, this invention can simultaneously detect the concentrations of multiple methyltransferases by detecting fluorescence signals.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A methyltransferase sensor based on a DNA walker and hyperbranched rolling circle amplification, characterized in that, The methyltransferase sensor includes a recognition probe, a DNA walker-hyperbranched rolling circle amplification system, a Padlock probe, and functionalized magnetic beads; the recognition probe includes H... M Identification probes and / or H D Identification probe, the H M The nucleotide sequence of the recognition probe is shown in SEQ ID NO.1, wherein the H D The nucleotide sequence of the recognition probe is shown in SEQ ID NO.2; The DNA walker-hyperbranched rolling circle amplification system includes phi29 DNA polymerase, primer 1, primer 2, and dNTPs; the nucleotide sequence of primer 1 is shown in SEQ ID NO.9, and the nucleotide sequence of primer 2 is shown in SEQ ID NO.

10. The nucleotide sequence of the Padlock probe is shown in SEQ ID NO. 8; The preparation method of the functionalized magnetic beads includes: mixing biotin-modified DNAzyme and an inhibitor in Tris-HCl buffer, forming a DNAzyme inhibitor double strand after the reaction; then dispersing streptavidin magnetic beads in Tris-HCl buffer; then adding the DNAzyme inhibitor double strand and biotin-modified substrate; and finally obtaining the functionalized magnetic beads after the reaction. The nucleotide sequence of the DNAzyme is shown in SEQ ID NO.4; the nucleotide sequence of the blocking agent is shown in SEQ ID NO.5; The nucleotide sequence of the substrate is shown in SEQ ID NO.

6.

2. The application of the methyltransferase sensor as described in claim 1 in the preparation of a kit for detecting methyltransferases.

3. A kit for detecting methyltransferases, characterized in that, It includes the methyltransferase sensor of claim 1.

4. The use of a methyltransferase sensor as described in claim 1 or a kit as described in claim 3 in the detection of methyltransferases for non-disease diagnostic purposes.

5. A method for detecting methyltransferases for non-disease diagnostic purposes, characterized in that, The method includes the step of detecting methyltransferase using the methyltransferase sensor of claim 1 or the kit of claim 3.

6. The method according to claim 5, characterized in that, When the methyltransferase is M.SssI methyltransferase, the method includes: using the H in the methyltransferase sensor of claim 1. M The identification probe triggers DNA walking and hyperbranched rolling circle amplification reactions through the interaction of M.SssI methyltransferase and HpaII endonuclease, and the content of M.SssI methyltransferase is detected by quantitative fluorescence signal intensity.

7. The method according to claim 5, characterized in that, When the methyltransferase is Dam methyltransferase, the method includes: using the H in the methyltransferase sensor of claim 1. D The recognition probe triggers DNA walking and hyperbranched rolling circle amplification reactions through the interaction of Dam methyltransferase and DpnI endonuclease, and the content of Dam methyltransferase is detected by quantitative fluorescence signal intensity.

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