High-sensitivity DNA methyltransferase activity determination system and method combined with CRISPR-Cas system

Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system-combined high-sensitivity DNA (deoxyribonucleic acid) methyltransferase activity determination system and a CRISPR-Cas system-combined high-sensitivity DNA methyltransferase activity determination method. DNA tetrahedrons capable of forming dumbbell rings at four corners are designed, when Dam MTase exists, Dam methyltransferase modifies an adenine residue in a GATC sequence, the adenine residue is subjected to Dpn I recognition and shearing and then is combined with a CRISPR / Cas 12a system, and only when a recognition site is methylated, the Dam methyltransferase can be used for detecting the activity of the GATC sequence. Only when the DpnI identifies the site and cuts the site, the exposed single chain can be combined with a CRISPR / Cas system to generate an enzyme digestion effect, the fluorescent single chain is cut and cut to generate fluorescence, and the concentration of Dam MTase can be known according to the size of the fluorescence. Certain clinical significance is realized when the kit is applied to early diagnosis of diseases such as cancers.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to a highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system, and also relates to a method for detecting DNA methyltransferase activity using this system. Background Art

[0002] DNA methylation is a widespread epigenetic modification and is one of the earliest discovered and most intensively studied epigenetic regulatory mechanisms. Broadly defined, DNA methylation refers to the chemical modification process in which specific bases on the DNA sequence obtain a methyl group through covalent bonding under the catalytic action of DNA methyltransferase (DNMT), with S-adenosylmethionine (SAM) as the methyl donor. This DNA methylation modification can occur at sites such as the C-5 position of cytosine, the N-6 position of adenine, and the G-7 position of guanine. DNA methylation is an important genomic epigenetic behavior in both eukaryotes and prokaryotes. As a relatively stable modification state, DNA methylation can be inherited to newly generated daughter DNA during DNA replication under the action of DNA methyltransferase and is an important epigenetic mechanism. Dam methyltransferase is a methylase encoded by the Escherichia coli chromosome and is the product of the Dam gene. Its main principle of action is to methylate the N6 site of specific adenine (A) in the DNA sequence. Specifically, Dam methyltransferase can recognize the adenine in the sequence GATC and convert it into 6-methyladenine. For decades, 6mA-methyl deoxyadenine has been considered to be widely present in prokaryotes and regulates DNA replication, repair, and transcription. Abnormal DNA methylation status is related to the interruption of DNA methyltransferase activity, and abnormal enzyme activity is an important biochemical index for certain diseases, which can lead to the occurrence of genetic diseases and cancers, etc. Therefore, DNA methyltransferase is considered to be a key biomarker for early clinical diagnosis. DamMTase has become a potential target for new anticancer therapies and antibacterial drugs. Therefore, it is crucial to develop a rapid, convenient, sensitive, and selective method for monitoring the activity of DaM MTase. Ultra-sensitive detection of DNA methyltransferase (Dam MTase) is of great significance for biomedical research and clinical diagnosis due to its profound impact on gene regulation. Traditional methods for detecting Dam MTase mainly include high-performance liquid chromatography, radioactivity analysis, electrophoresis, and RT-PCR, etc. However, these methods all have the disadvantages of complicated experimental operations, high detection costs, and long time consumption. Therefore, the design of a rapid, simple, and ultra-sensitive detection method has emerged. DNA tetrahedron nanostructures (TDN) have good programmability and significant cell and tissue permeability. TDN is a nanomaterial with high mechanical stiffness, stability, and four-corner Toeholds with functional modification sites. Therefore, TDN has shown good application prospects in biological detection, imaging, drug delivery, and as a gene carrier, etc.It is reported that as a gene vector, TDN can be used as a loading vector for nucleic acid molecules and has been proven to be of great value in delivering various bioactive molecules. Due to the base complementary pairing rule, TDN presents a typical three-dimensional structure. Compared with two-dimensional single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), DTN carries signaling substances and is an important medium for efficient fluorescence generation, providing an important vector function for the reaction of Cas12a enzyme. The clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins of Cas12a, it is an RNA-guided nuclease in the type V CRISPR-Cas system and a powerful gene editing tool. In 1987, Japanese researchers discovered tandem repeat spacer sequences near the alkaline phosphatase gene of Escherichia coli K19, and later found that it was widely present in bacteria. This sequence was named "clustered regularly interspaced short palindromic repeats" (CRISPR) in 2002.

[0003] The CRISPR-Cas12a system forms a complex with crRNA that has DNA cleavage ability and exerts its trans-cleavage activity by recognizing the PAM site on the target pathogen gene. After amplifying the pathogenic gene, display materials such as fluorescent probes are added to the detection system. At the same time, the CRISPR system has the advantages of rapid detection and high sensitivity, and can diagnose pathogenic bacteria faster and more accurately. The mechanism of the CRISPR-Cas system programmed by CRISPR guide RNA (CRISPR guide RNA, abbreviated as crRNA) can not only cleave the target nucleic acid, but also cleave all adjacent nucleic acids indiscriminately. This target-triggered collateral activity of the CRISPR-Cas system has stimulated the development of CRISPR-based biosensing technologies, which have unique advantages such as simple fabrication, ultra-high sensitivity, high specificity for single-base mutations, and good POC diagnostic capabilities. The protocol for constructing a fluorescent biosensor for detecting DNA methyltransferase based on the binding of DNA tetrahedron to the CRISPR / Cas system was reported for the first time. The construction of such sensors with rapid detection and high sensitivity, based on other methods such as electrochemical biosensors, colorimetry, chemiluminescence, etc., avoids the disadvantages of detection instability and high false positive rate. This protocol can greatly improve the detection efficiency. In this protocol, each apex of the DNA tetrahedron designed by us contains dumbbell strands. The 5' end of each long strand of the tetrahedron is modified with P, and the 3' end is still OH. Under the catalysis of T4 DNA ligase, the two ends are combined. Under certain reaction conditions, the formation of the tetrahedron and the structure of the dumbbell are both very stable. The dumbbell has the recognition site 5'-GATC-3' of Dam methyltransferase (Dam MTase). After this structure is formed, in the presence of different concentrations of Dam MTase, the site is methylated. Under the recognition of Dpn I specific cleavage enzyme, the binding part of the reaction crRNA of CRISPR / Cas12a is exposed. After the entire reaction system is formed, in the presence of FQ-ssDNA, it emits fluorescence under the cleavage of CRISPR / Cas12a, and corresponding fluorescence signals are generated according to the concentration of Dam MTase. When Dam MTase is absent, almost no fluorescence is generated, and the matrix effect of this reaction is also very weak. This protocol also verified the inhibitory effect of 5-FU on Dam MTase. Finally, the detection of this enzyme in human serum was achieved. The implementation of this protocol not only provides important reference value for clinical drug treatment, but also provides great help for early detection, early diagnosis and early treatment in the clinical aspect. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system. Another objective of the invention is to provide the application of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system in detecting DNA methyltransferase activity. The third objective of the present invention is to provide the application of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system in screening candidate methylation inhibitors.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] 1. A highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system, which includes a DNA tetrahedron with dumbbell loops at its four corners, T4 DNA ligase, S-adenosylmethionine and a methylation reaction buffer, a Dpn I restriction endonuclease reaction system, a CRISPR / Cas12a reaction system containing Cas12a enzyme and crRNA, and a fluorescent reporter strand.

[0007] Preferably, the DNA tetrahedron with dumbbell loops at its four corners is formed by sequences modified with a phosphate P at the 5'-end as shown in SEQ ID NOs. 2 to 5.

[0008] Preferably, the conditions for forming the DNA tetrahedron with dumbbell loops at its four corners are to mix the sequences modified with a phosphate P at the 5'-end as shown in SEQ ID NOs. 2 to 5 with TM buffer, react at 95°C for 5 minutes, 4°C for 30 minutes, then 37°C for 30 minutes to form the dumbbells, and finally react at 4°C for 30 minutes to obtain.

[0009] Preferably, 160 μM SAM and 5 μL of methylation reaction buffer are added to the methylation reaction. The components of the methylation reaction buffer are as follows: 50 mM Tris-HCl; 5 mM β-ME; 10 mM EDTA; pH 7.5 @ 25°C. The methylation reaction conditions are to react at 37°C for 45 minutes.

[0010] Preferably, the Dpn I restriction endonuclease reaction is to react with 9 U of Dpn I restriction endonuclease and 5 μL of the corresponding buffer at 37°C for 40 minutes.

[0011] Preferably, the sequence of the crRNA is as shown in SEQ ID NO. 10, and the fluorescent reporter strand is FAM-TTATT-BHQ1.

[0012] 2. Use of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system in detecting DNA methyltransferase activity.

[0013] Preferably, the present invention relates to the use of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system in screening for candidate methylation inhibitors.

[0014] 3. Method for detecting DNA methyltransferase activity using the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system: Mix 1 μM of DNA tetrahedron with dumbbell loops at the four corners, 400 U of T4 DNA ligase, and react at 16 °C for 1 hour, then react at 70 °C for 10 minutes to inactivate the enzyme. Adjust the concentration of the DNA tetrahedron with dumbbell loops at the four corners to 0.75 μM, add 100 U / mL of methyltransferase, 160 μM of SAM, and 5 μL of methylation reaction buffer, and react at 37 °C for 45 minutes. Next, adjust the DNA tetrahedron concentration to 0.5 μM, add 9 U of DpnⅠ restriction endonuclease and 5 μL of digestion buffer, and react at 37 °C for 40 minutes. Finally, adjust the DNA tetrahedron concentration to 0.4 μM, prepare a reaction system with a Cas12a concentration of 0.25 μM, 0.25 μM of crRNA, and 0.1 μM of ss-DNA fluorescent strand, react at 37 °C for 35 minutes, and detect the fluorescence intensity.

[0015] The beneficial effects of the present invention are as follows: The present invention provides a highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system. By designing a DNA tetrahedron with dumbbell loops that can be formed at the four corners, when Dam MTase is present, the Dam methyltransferase modifies the adenine residue (N6) in the GATC sequence. After being recognized and cleaved by Dpn I and combined with the CRISPR / Cas12a system, only when the recognition site is methylated can DpnI recognize and cleave this site, and the exposed single strand can bind to the CRISPR / Cas system to produce an enzymatic cleavage effect, cutting the fluorescent single strand to produce fluorescence. According to the fluorescence intensity, the concentration of Dam MTase can be known. This scheme can achieve the detection in human serum and the screening of the enzyme inhibitor 5-FU, with ultra-high sensitivity and specificity, and has certain clinical significance in the early diagnosis of diseases such as cancer. The successful implementation of this scheme and the candidate target DamMTase suggest an important research direction for many researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1For the synthesis verification of TDN and bTDN (A: 8% Native PAGE of TDN; from left to right: lane M, 1000bp DNA marker; lanes 1 to 4, single-strands S1, S2, S3 and S4; lanes 6 to 9, S123, S124, S134 and S234; lane 5, S1234; B: Atomic force microscopy image of bTDN).

[0018] Figure 2 For feasibility verification (A: 8% Native PAGE of the complete fluorescence reaction; from left to right: lane 1, separate DNA methylation reaction; lane 2, Dpn I digestion reaction after DNA methylation reaction; lane 3, fluorescence reaction generated by bTDNs binding to CRISPR / Cas12a; lanes 4 to 7, single-strands S1, S2, S3 and S4; lane 8, bTDNs; B: Fluorescence reaction generated by the binding of DNA strands in 8% Native PAGE to CRISPR / Cas12a; from right to left: half dumbbell strand, dumbbell strand, crRNA, single-strand fluorescent DNA; lane 3, the dumbbell loop is methylated; lane 2, Dpn I digestion reaction of the dumbbell strand, and the cleavage product is the half dumbbell strand; lane 1, the dumbbell loop undergoes Dpn I digestion reaction after DNA methylation, and binds to CRISPR / Cas12a and crRNA for fluorescence reaction; lane 4, single-strand fluorescent DNA; the single-strand fluorescent DNA has too few bases to be obvious; C: Verification of feasibility by fluorescence reaction; as shown in the following reaction curves: (a) Fluorescence reaction of bTDN binding to CRISPR / Cas12a to detect Dam MTase, (b) Fluorescence reaction of the dumbbell loop binding to CRISPR / Cas12a to detect Dam MTase, (c) Fluorescence reaction of the half dumbbell strand binding to CRISPR / Cas12a to detect Dam MTase, (d) Fluorescence reaction of the single-strand dumbbell binding to CRISPR / Cas12a to detect Dam MTase, (e) Single-strand fluorescent DNA; D: Fluorescence intensities of different numbers of bTDN long strands: (a) Fluorescence intensity when only S4' is a short strand, (b) Fluorescence intensity when only S3' and S4' are short strands, (c) Fluorescence intensity when only S2', S3' and S4' are short strands, (d) Fluorescence intensity when S1', S2', S3' and S4' are short strands).

[0019] Figure 3 For the ultrasensitive detection scheme of Dam MTase based on the DNA tetrahedron multi-Toeholds dumbbell binding CRISPR / Cas12a system (Part A: Formation of bTDN; Part B: DNA methylation process; Part C: Fluorescence generated by the binding of bTDN to CRISPR / Cas12a).

[0020] Figure 4For experimental condition optimization (A: optimize the bTDN concentration; B: optimize the DpnⅠ concentration; C: optimize the Dam methylation time; D: optimize the DpnⅠ reaction time; E: optimize the CRISPR / Cas12a reaction time).

[0021] Figure 5 For linear verification (A: fluorescence intensities corresponding to different concentrations: 0 U / mL, 0.001 U / mL, 0.002 U / mL, 0.004 U / mL, 0.006 U / mL, 0.008 U / mL, 0.01 U / mL, 0.02 U / mL, 0.06 U / mL, 0.1 U / mL, 0.5 U / mL, 1 U / mL, 10 U / mL, 50 U / mL; B: scatter plots of fluorescence signals corresponding to different concentrations; C: linear curve of fluorescence intensity corresponding to 0.01 - 1 U / mL).

[0022] Figure 6 For specificity verification, compare the methylation effects of Dam MTase and M.SssI MTase.

[0023] Figure 7 For stability verification.

[0024] Figure 8 For the inhibitory effects of different concentrations of 5 - FU on 50 U / mL Dam MTase. Specific embodiments

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0026] All DNA oligonucleotides and crRNAs used in this study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and purified by HPLC. The detailed sequences of these oligonucleotides are shown in Table 1. EnGen Lba Cas12a (Cpf1), DamMTase, T4 DNA ligase, Dpn I methylated restriction endonuclease, M.Sss I MTase, and the corresponding buffers were ordered from NEB Biolabs (Beijing, China). 5-Fluorouracil was purchased from Solarbio Life Sciences. TE buffer, Tris (tris(hydroxymethyl)aminomethane) powder, magnesium chloride hexahydrate (MgCl2·6H2O) reagent, ammonium persulfate (APS), acrylamide / methylene bisacrylamide 30% solution (29:1), N,N,N',N'-tetramethylethylenediamine (TEMED), 5×TBE buffer were all purchased from Sangon Biotech Co., Ltd. The third-generation Gelred nucleic acid stain was purchased from Beijing Polymer Beauty Biotechnology Co., Ltd. RNase inhibitor, 20bp DNA Ladder, 1000bp DNA marker, and 6×loading buffer were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All DNA oligonucleotides were dissolved in TE buffer, and crRNAs were dissolved in DEPC water and stored at -20°C for subsequent experiments. All reagents were analytical grade reagents and were not further purified. RNase-free water was used throughout the experiment.

[0027] Table 1. Oligonucleotide sequences used in this work

[0028]

[0029]

[0030] All fluorescence spectral results were obtained from an F-4700 fluorescence spectrophotometer (Hitachi, Japan), using a quartz fluorescence cuvette with an optical path length of 1.0 cm. The detection parameters were set as follows: excitation wavelength was 490 nm, emission wavelength was 500 - 600 nm, and both the excitation slit and emission slit were 5 nm. DNA tetrahedron, dumbbell, and DNA methylation enzyme digestion reactions were all formed in a B1000 thermal cycler (Biorad, USA). Native polyacrylamide gel electrophoresis (Native-PAGE) was carried out on an electrophoresis analyzer (Liuyi Biology, Beijing), and the results were obtained on an imager (Saiwei'er, Wuhan). The dry powder of the oligonucleotide was pre-centrifuged at high speed at 4°C (Thermo Fisher, USA). Atomic force microscopy (AFM) imaging was performed using a Dimension Icon instrument (Bruker Nano Inc., Berlin, Germany).

[0031] Example 1. Synthesis of DNA tetrahedron (TDN)

[0032] The successful synthesis of the TDN structure was characterized and verified by 8% native PAGE and atomic force microscopy (AFM). The synthesis process is as follows: First, the four strands (dry powder) of tetrahedrons S1’, S2’, S3’ and S4’ were centrifuged at 12,000 g for 10 minutes in a pre-cooled 4°C high-speed centrifuge and then placed on ice. A certain amount of TE buffer was accurately added and vortexed repeatedly 3 times to fully dissolve it to 100 μM. It was set in a 95°C B1000 thermal cycler to heat for 5 minutes first and then quickly cooled to 4°C for 30 minutes to form the tetrahedron, abbreviated as TDN (dumbbell-free tetrahedron).

[0033] The formation conditions of the tetrahedron with a hairpin (bTDN) are as follows: React at 95°C for 5 minutes, then react at 37°C for 30 minutes in a timely manner, and finally react at 4°C for 30 minutes to form it. The reaction products are all stored at 2 μM at -20°C for later use. The DNA tetrahedron is completed in TM buffer (20 mM Tris and 50 mM MgCl2·6H2O). Each single strand, S1, S2, S3, S1, S2, S4, S1, S3, S4, S2, S3, S4 and S1, S2, S3, S4 (concentration of 0.5 μM) were mixed with 2 μL of 6× loading buffer and added to the electrophoresis gel, and then run at 100 V for 35 minutes. The gel was removed and stained in 50 mL of 1× TBE buffer containing 2 μL of nucleic acid stain for 50 minutes, and finally imaged in a gel imager.

[0034] To verify the feasibility of the reaction, the synthesis of the tetrahedron with a hairpin (bTDN) was carried out in TM buffer and verified by 8% non-denaturing polyacrylamide gel electrophoresis. The results are as Figure 1 shown in A. The results show that from left to right are 1000 bp DNA marker, the four single strands of bTDN, lane 5 is S1234 TDN, and lanes 6 to 9 are S123, S124, S134, S234 TDN. It can be seen that the synthesis effect of TDN is very good, increasing the feasibility of the experiment. The detection concentration of each is 0.2 μM. Then, AFM was used to verify bTDN (the synthesis method is as described above for the bTDN synthesis method, and the detection concentration was diluted to 1 μM). The results are as Figure 1 shown in B.

[0035] Next, the entire reaction process was verified by 8% non-denaturing polyacrylamide gel electrophoresis, as Figure 2As shown in A, from left to right, lane 1 is the product after bTDN methylation, lane 2 is the product after bTDN methylation and then addition of DpnI enzyme for digestion reaction, lane 8 is pure bTDN, lane 3 is the product after bTDN methylation, digestion reaction, and then addition of Cas12a for fluorescence detection, lanes 4 - 7 are the four long chains of bTDN. In this electrophoresis, the concentration of S1 - S4 is 1 μM, while the product concentrations in other lanes are the initial 2 μM bTDN, the bTDN after addition of T4 DNA ligase is 1 μM, the bTDN after methylation is 0.75 μM, the bTDN concentration after addition of Dpn I enzyme for digestion reaction is 0.5 μM, and the product concentration after addition of Cas12a for fluorescence detection is 0.4 μM. Figure 2 In B, simply verifying the reaction of the dumbbell part with CRISPR / Cas12a, from right to left are the half - dumbbell chain, dumbbell chain, crRNA, ss - fluorescent DNA chain. Lane 3 is the product after methylation of the dumbbell loop. It can be seen that the movement of the methylated dumbbell starts to slow down. Lane 2 is the product after digestion reaction after addition of Dpn I, and the digested product is the half - dumbbell. Finally, lane 1 is the product after DNA methylation and then digestion reaction of the dumbbell loop, and finally combined with Cas12a and crRNA for fluorescence reaction. Lane 4 is the ssDNA fluorescent single - strand, which is not very obvious due to too few bases in the strand. All products in this electrophoresis are 2 μM. Figure 2 In C, the reaction of various chains binding to Cas12a was verified. First, curve a is a complete fluorescence reaction, and the strongest fluorescence signal can be seen. Curve b is the signal of the fluorescence reaction after DNA methylation restriction endonuclease reaction of the dumbbell loop. Curves c, d, and e are the fluorescence reactions of the half - dumbbell chain binding fluorescence, the dumbbell single - strand binding fluorescence, and the simple detection of the fluorescence of ss - DNA (the fluorescence is quenched), respectively. According to the experimental sequence, the complete digestion effect of the CRISPR / Cas12a system on any single - strand was fully verified, resulting in fluorescence of ss - DNA before it could be digested by ss - DNA, further verifying the principle of the CRISPR / Cas12a digestion of the scheme and the feasibility of the scheme. Figure 2In D, the fluorescence intensities of the complete DNA methylation reaction and the CRISPR / Cas12a cleavage reaction involving different numbers of bTDN long chains (S1 - S4) were verified respectively. From top to bottom, they were the fluorescence when only S4' was a short chain without a dumbbell, the fluorescence when S3'S4' were short chains without a dumbbell, the fluorescence when S2'3'4' were short chains without a dumbbell, and the fluorescence when S1'2'3'4' were all short chains without a dumbbell. It can be seen that the fluorescence signal was still relatively strong even when only 1 chain was not involved in the fluorescence reaction, and the impact on the reaction fluorescence was not significant. For the verification of the above feasibility, the initial concentration of each TDN was set to 2 μM. When adding T4 DNA ligase, the concentration of bTDN after T4 DNA ligase was 1 μM. After bTDN methylation, the concentration was 0.75 μM. After adding Dpn I enzyme for the cleavage reaction, the concentration of bTDN was 0.5 μM. After adding Cas12a for fluorescence detection, the concentration of the product was 0.4 μM. Finally, 50 μL of enzyme-free water was added to the detection concentration to make the detection volume 100 μL, and the detection concentration was 0.2 μM.

[0036] Example 2, Dam MTase Detection Strategy

[0037] The scheme for detecting Dam MTase based on bTDN binding to CRISPR / Cas12a is shown in Figure 3 , such as Figure 3 Part A in. Under corresponding conditions, bTDN is stably formed. The dumbbells at the four corners of bTDN are single-stranded with 40 bases and can form dumbbells under certain conditions. The Toehold of bTDN is the dumbbell part with exactly the same structure. The four long chains (S1 - S4) of bTDN, the tetrahedral part has 55 bases, and every 17 bases are complementary to the bases of other chains. The dumbbell part has 40 bases and is composed of 2 identical 20-base parts. The 5'-ends of S1 - S4 are phosphorylated, and the 3'-ends are hydroxyl groups. After the action of T4 DNA ligase, a closed loop is formed. After the dumbbell is formed, DNA methylation reaction and cleavage are carried out. Since the number of bases maintaining the dumbbell is small, two identical single-strands are formed, which are exactly used for the subsequent CRISPR / Cas12a cleavage reaction to shear the fluorescent single-strand to produce fluorescence, and the fluorescence intensity is positively correlated with the Dam MTase concentration. Verification of the effective synthesis of bTDN Figure 3 In Part B, the results show that after bTDN is stably formed, the most important part of this scheme is carried out. T4 DNA ligase is added to connect the 5'-P and 3'-OH of each chain of the tetrahedron. Next, the DNA methylation reaction is carried out. Dam recognizes the 5'-GATC-3' site and binds to the methyl group in SAM. After the formation of the four-corner dumbbell ring of TDN, methylation forms the cleavage site of Dpn I, exposing the reaction chain of CRISPR / Cas12a. Finally, as Figure 3As can be seen in Figure C, after CRISPR / Cas12a binds to crRNA, a trans-cleavage reaction occurs, cleaving the fluorescent single-stranded DNA. The 5' end of the ss-DNA binds to the FAM fluorescent group, and the 3' end binds to the BHQ1 quenching group. After cleaving the ss-DNA, the groups separate and release fluorescence. This method has a high signal-to-noise ratio for detecting fluorescence signals, laying a solid foundation for subsequent serological detection and inhibition experiment verification.

[0038] Example 3: DNA Methylation Reaction

[0039] The entire DNA methylation reaction begins with the synthesis of dumbbell-shaped DNA tetrahedron (bTDN). For feasibility verification, we diluted the bTDN concentration to 2 μM. After dissolving the dry powder single-stranded S1 - S4, the concentration was 100 μM. Take 1 μL each of S1 - S4 and 46 μL of TM buffer, heat at 95°C for 5 minutes on a B1000 thermal cycler, cool to 4°C for 30 minutes, then react at 37°C for 30 minutes to form the dumbbell, and finally react at 4°C for 30 minutes and store at 4°C in the refrigerator for later use. The T4 DNA ligase reaction stabilizes the formation of the dumbbell. The 5' ends of the single strands of the DNA tetrahedron are all modified with phosphate P. After adding T4, the 5' end is ligated to the 3'-OH, making the bTDN concentration 1 μM at this step. Add 400 U of T4 and react at 16°C for 1 hour, then react at 70°C for 10 minutes to inactivate the enzyme, enabling the reaction to proceed fully. The next and most crucial DNA methylation reaction makes the bTDN concentration 0.75 μM. Add 100 U / mL Dam MTase, 160 μM SAM, and 5 μL of the special buffer for methylation reaction. The entire reaction system is 50 μL and react at 37°C for 45 minutes on the thermal cycler. Next, the reaction makes the bTDN concentration 0.5 μM. Add 9 U of DpnⅠ restriction endonuclease and 5 μL of the corresponding buffer. The reaction system is 50 μL and react at 37°C for 40 minutes. Finally, for the fluorescence reaction, make the bTDN concentration 0.4 μM, and the reaction system is also 50 μL. Prepare a reaction system with a Cas12a concentration of 0.25 μM, 0.25 μM crRNA (RNase inhibitor has been pre-added), and 0.1 μM ss-DNA fluorescent strand, react at 37°C for 35 minutes (operate in the dark), and perform detection on a Hitachi F-4700 fluorescence detector. When detecting, make up to 100 μL with enzyme-free water. When detecting, the bTDN is 0.2 μM, and the entire reaction feasibility is completed.

[0040] Example 4: Experimental Condition Optimization

[0041] To detect Dam MTase with maximum efficiency, various experimental conditions were optimized. In this part, we statistically analyzed the direct fluorescence signals detected. For each part of the experiment, the blank signal without the target was verified. The signal-to-noise ratio F / F0 trend of the experiment was basically the same as F.

[0042] First, the concentration of bTDN was verified. A certain concentration of bTDN plays a crucial role in the smooth progress of subsequent experiments. As Figure 4 shown in A of Figure 4 , as the concentration of bTDN increases, the fluorescence signal gradually increases and reaches the highest fluorescence signal at 2 μM. When the bTDN concentration continues to increase subsequently, it can be seen that the fluorescence signal decreases. Therefore, the bTDN concentration of 2 μM was set as the reaction concentration in the experiment.

[0043] The influence of DpnⅠ concentration is as shown in Figure 4 B of Figure 4 . The results show that during the reaction, the enzymatic digestion effect of DpnⅠ on the dumbbell part of bTDN after methylation by Dam MTase also increases with the increase of enzyme concentration, and the fluorescence signal gradually increases. However, when the concentration keeps increasing, it instead inhibits the experiment. When the added concentration reaches 12 U, the fluorescence signal reaches the highest. But when DpnⅠ continues to increase, the fluorescence signal decreases instead. Therefore, 12 U of DpnⅠ was set as the optimal reaction concentration.

[0044] The time of DNA methylation and the time of methylation enzymatic digestion also have certain variations on the effective progress of the experiment. Therefore, the reaction times of Dam MTase and DpnⅠ were optimized respectively. The results are Figure 4 shown in C and D of Figure 4 . The results show that during the reaction, as time increases, the enzyme continues to be consumed, which has a certain impact on maintaining the stability of the experiment. As the reaction time of Dam MTase increases, the fluorescence intensity increases and reaches the best fluorescence signal at 45 minutes. When the reaction time continues to increase, the signal continues to slightly decline. Therefore, the reaction time of 45 minutes was taken as the optimal reaction time for DNA methylation. And as shown in Figure 4 D of Figure 4 , as the reaction time of DpnⅠ gradually increases, when the reaction time reaches 40 minutes, the fluorescence intensity reaches the strongest. When the time continues to increase, the fluorescence signal decreases. Therefore, 40 minutes was selected as the optimal reaction time for methylation enzymatic digestion. In the last step of the reaction, the fluorescence reaction time of the CRISPR / Cas12a system is crucial for detection. After stably adding crRNA and fluorescent single-stranded DNA in the reaction, the reaction proceeds for a certain time, and the fluorescence signal will change. As the fluorescence enzymatic digestion reaction proceeds, the fluorescence signal gradually increases. If the reaction at 37 °C continues for too long, the fluorescence signal will decrease to a certain extent, and it should be taken out in time and stored in a refrigerator at 0 - 4 °C in the dark. As shown in Figure 4 E of Figure 4 , 35 minutes was selected as the optimal reaction time and detected in time.

[0045] Example 5. Linear Detection

[0046] Under the optimized conditions, linear detection of Dam MTase was carried out, and the detection was carried out from 0 U / mL to 50 U / mL. As shown in Figure 5Figure A shows the fluorescence detection of Dam MTase at the following different dilution concentrations (concentrations are 0 U / mL, 0.001 U / mL, 0.002 U / mL, 0.004 U / mL, 0.006 U / mL, 0.008 U / mL, 0.01 U / mL, 0.02 U / mL, 0.06 U / mL, 0.1 U / mL, 0.5 U / mL, 1 U / mL, 10 U / mL, 50 U / mL). Obviously, the fluorescence intensity increases with the increase of the target concentration, and Figure 5 Figure B shows the positive curve change trend of fluorescence and target concentration, as Figure 5 Figure C shows a proportional change trend in the concentration range of 0.01 - 1 U / mL, and the linear curve is described as y = 831.67 + 280.77x, and R 2 is 0.99715, showing a good correlation. The lowest detection limit is calculated according to the 3δ / S rule (the standard deviation of the blank value multiplied by 3, plus the average value of the blank signal, S is the slope), and the lowest detection limit is 0.001 U / mL. In the published research, the detection of Dam MTase concentration (Table 2) shows that our detection method has relatively good stability and high sensitivity.

[0047] Table 2. Comparison of the methodology of the highly sensitive fluorescence detection scheme for Dam MTase

[0048]

[0049]

[0050] Example 6. Specificity and stability verification

[0051] To verify the specificity of Dam MTase detection, under the same conditions, the fluorescence signals of M.SssⅠ MTase and the fluorescence signals when Dam MTase and M.SssⅠ MTase are detected together were detected, as Figure 6 . The recognition site of Dam methyltransferase is 5'-GACT-3', and the function of CpG methyltransferase (M.SssI) is to methylate all cytosine residues (C5) in the double-stranded dinucleotide recognition sequence 5′-CG-3′. The enzyme concentration is 50 U / mL for both. It can be seen that only when Dam MTase recognizes the corresponding site on the dumbbell can the subsequent fluorescence detection proceed smoothly.

[0052] The verification results of the stability of the fluorescence reaction are as Figure 7 shown. The results show that CRISPR / Cas12a has a certain time effect on the stability of the reaction. As time goes on, from the detection on the first day to the detection on the fifth day, the fluorescence gradually decreases. Therefore, the experiment needs to be configured and detected immediately to maintain the effectiveness of the detection.

[0053] Example 7, Human Serological Detection

[0054] Similar to the previous feasibility verification step, in the DNA methylation step, 1% human serum (randomly selected from Jiulongpo District People's Hospital) was added to Dam MTase. Three groups of Dam MTase experiments with concentrations of 0.01 U / mL, 0.1 U / mL, and 1 U / mL were added for the calculation of the replication rate. 160 μM SAM was also added. The following formula was used for calculation: Recovery ratio (R): R = C2 / C1 × 100%, where C1 is the concentration added after dilution, and C2 is the concentration calculated by substituting the detection into the linear curve. Each concentration was detected in three parallel experiments, and the respective recovery rates were 99%, 103%, and 110%. According to the RSD%, it can be seen that the experiment has good precision, which are 12%, 0.99%, and 0.88% respectively.

[0055] Table 3, Detection Effect of Dam Methyltransferase in Serum

[0056]

[0057] Example 8, 5-FU Inhibition Experiment

[0058] For the drug screening of disease treatment, we verified the enzyme inhibition effect of 5-fluorouracil (5-FU). Any abnormal changes in the DNA methylation pattern will promote the occurrence of various diseases and malignancies including cancer. The selection of Dam MTase enzyme inhibitors in the research of antibiotics can effectively control the abnormal occurrence of DNA methylation inhibited by Dam MTase. Therefore, we diluted 5-FU at 5 different concentrations, namely 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM, and judged the enzyme inhibition situation by observing the fluorescence weakening situation ( Figure 8) It is judged according to the relative activity (RA): RA = (F2–F0) / (F1–F0), where the fluorescence readings corresponding to F0, F1, and F2 are as follows: F0 is the concentration when there is no Dam MTase and 5-fluorouracil is not added to the reaction; F2 is the fluorescence intensity when 50 U / mL Dam MTase and different concentrations of 5-fluorouracil inhibitor are present; F1 is the fluorescence intensity when 50 U / mL Dam MTase is present but 5-fluorouracil is absent. The average value of F1 and the reaction blank value F0 from 3 parallel experiments is taken for subsequent calculations. When the concentration gradually increases to 100 μM, the fluorescence signal approaches the blank and no longer changes. Therefore, this concentration is the optimal concentration for enzyme inhibition. 10.9 μM is calculated as the IC50 of 5-fluorouracil, that is, the concentration of 5-fluorouracil has a 50% inhibitory effect on the activity of Dam MTase. The above inhibitory effect of 5-fluorouracil indicates that the proposed strategy can be used to screen candidate MTase inhibitors, as well as tools for clinical treatment and diagnosis.

[0059] A sensitive DNA methyltransferase (MTase) activity assay method combined with the CRISPR-Cas system was designed. In this protocol, a DNA tetrahedron (bTDN) with stable structure was first designed. The Toehold of bTDN is a dumbbell part with exactly the same structure. The four long chains (S1-S4) of bTDN have phosphate modifications at the 5' ends and hydroxyl groups at the 3' ends. After the action of T4 DNA ligase, a closed loop is formed. When the DNA methylation reaction process occurs, combined with the CRISPR / Cas system, a fluorescence is generated by the cleavage reaction. The fluorescence intensity is positively correlated with the Dam MTase concentration. Therefore, after optimizing each experimental condition, the concentrations of different Dam MTase concentrations (from 0 U / mL, 0.001 U / mL, 0.002 U / mL, 0.004 U / mL, 0.006 U / mL, 0.008 U / mL, 0.01 U / mL, 0.02 U / mL, 0.06 U / mL, 0.1 U / mL, 0.5 U / mL, 1 U / mL, 10 U / mL, 50 U / mL) were well detected, and a linear detection in the concentration range of 0.01 - 1 U / mL was achieved, R 2It is 0.99715, with a very good linear trend and a minimum detection limit of 0.001U / mL. Compared with the minimum detection limit achieved by the same fluorescence detection scheme in recent years, this scheme has the characteristics of ultra-high sensitivity, high specificity, and fast detection time. Compared with electrochemical and colorimetric detection methods, this scheme has the advantages of low false positive rate and stability. In addition, this scheme realizes the detection of human serum and enzyme inhibitor 5-FU. Any abnormal changes in DNA methylation patterns will promote the occurrence of various diseases and malignant tumors including cancer. The selection of Dam MTase enzyme inhibitors in the study of antibiotics can effectively control Dam MTase and inhibit the abnormal occurrence of DNA methylation, which has important reference value for clinical diagnosis and treatment. It has certain clinical significance for the early diagnosis of diseases such as cancer. The smooth implementation of this scheme and the candidate target Dam MTase suggest to many researchers that it is an important research direction.

[0060] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system, characterized in that: The system includes a DNA tetrahedron with dumbbell rings at four corners, T4 DNA ligase, S-adenosylmethionine and methylation reaction buffer, a Dpn I restriction endonuclease reaction system, a CRISPR / Cas12a reaction system containing Cas12a enzyme and crRNA, and a fluorescent reporter chain.

2. The highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to claim 1, characterized in that: The DNA tetrahedron with dumbbell rings at its four corners is formed by sequences shown in SEQ ID NOs. 2 to 5 with phosphate P modified at the 5' end.

3. The highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to claim 2, characterized in that: The conditions for forming the DNA tetrahedron with dumbbell rings at the four corners are as follows: the sequences shown in SEQ ID NO. 2 to 5 with phosphate P modified at the 5' end are mixed with TM buffer, heated at 95°C for 5 minutes, 4°C for 30 minutes, and 37°C for 30 minutes to form dumbbells, and finally reacted at 4°C for 30 minutes.

4. The highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to claim 1, characterized in that: The methylation reaction was performed by adding 160 μM SAM and 5 μL methylation reaction buffer, wherein the components of the methylation reaction buffer were as follows: 50 mM Tris-HCl; 5 mM β-ME; 10 mM EDTA; pH 7.5@25° C. The methylation reaction was performed at 37° C. for 45 minutes.

5. The highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to claim 1, characterized in that: The Dpn I restriction endonuclease reaction was carried out at 37° C. for 40 minutes in the presence of 9 U of Dpn I restriction endonuclease and 5 μL of the corresponding buffer.

6. The highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to claim 1, characterized in that: The sequence of the crRNA is shown in SEQ ID NO.10, and the fluorescent reporter chain is FAM-TTATT-BHQ1.

7. Use of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to any one of claims 1 to 6 in detecting DNA methyltransferase activity.

8. Use of the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to any one of claims 1 to 6 in screening candidate methylation inhibitors.

9. A method for detecting DNA methyltransferase activity using the highly sensitive DNA methyltransferase activity assay system combined with the CRISPR-Cas system according to any one of claims 1 to 6, characterized in that: 1 μM DNA tetrahedron containing dumbbell rings at four corners and 400U T4 DNA ligase were reacted at 16°C for 1 hour, and then reacted at 70°C for 10 minutes to inactivate the enzyme, and the concentration of the DNA tetrahedron containing dumbbell rings at four corners was adjusted to 0.75 μM, 100U / mL methyltransferase, 160μM SAM and 5μL methylation reaction buffer were added to react at 37°C for 45 minutes, and then the DNA tetrahedron concentration was made to be 0.5 μM, DpnⅠ restriction endonuclease 9U and 5μL enzyme digestion buffer were added, and the reaction was carried out at 37°C for 40 minutes, and finally the DNA tetrahedron concentration was made to be 0.4 μM, and a reaction system with a Cas12a concentration of 0.25 μM, 0.25 μM crRNA, and 0.1 μM ss-DNA fluorescent chain was prepared, and the reaction was carried out at 37°C for 35 minutes, and the fluorescence intensity was detected.

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