CN112094889A - Chemiluminescence sensor and application in detection of bacterial or human methyltransferase - Google Patents
Chemiluminescence sensor and application in detection of bacterial or human methyltransferase Download PDFInfo
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Abstract
The invention discloses a chemiluminescence sensor and application thereof in detecting bacteria or human methyltransferase, wherein the chemiluminescence sensor comprises a dumbbell probe, S-adenosylmethionine, a primer, DNA polymerase, heme, luminol and H2O2(ii) a The stem structure of the dumbbell probe contains two hemimethylated recognition sites, the small-loop structure is rich in a C sequence, when the hemimethylated recognition sites are converted into full methylated sites under the action of DNA methyltransferase and S-adenosylmethionine, the dumbbell probe can perform rolling circle DNA amplification under primers and DNA polymerase, the rolling circle DNA amplification can generate a product rich in a G sequence under the action of the small-loop structure rich in the C sequence, the product rich in the G sequence can be folded and matched with heme to form heme-G-quadruplex DNase, and the heme-G-quadruplex DNase can catalyze H2O2Mediating luminol oxidation results in an enhanced chemiluminescent signal.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and relates to a chemiluminescence sensor and application thereof in detection of bacteria or human methyltransferase.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
DNA methylation is a highly characterized epigenetic modification in the human genome. DNA methyltransferases establish and maintain certain genomic methylation patterns by transferring a methyl group (SAM) from S-adenosylmethionine to an adenine/cytosine residue in a particular DNA sequence. Recent studies have shown that altered DNA methyltransferase activity leads to abnormal DNA methylation patterns.
To the best of the inventors' knowledge, various methods have been developed to date to detect the activity of DNA methyltransferases. The traditional methods are mainly radiolabelling, immunoblotting, High Performance Liquid Chromatography (HPLC) and gel electrophoresis. However, they involve unsafe isotope labeling, lengthy assay procedures, expensive antibodies and instruments, greatly limiting their practical applications. Recently, new methods, including colorimetric, fluorescent and electrochemical methods, have been used for quantitative detection of DNA methyltransferases, but, as a result of research by the inventors, these methods have not been sufficiently sensitive to detect the extremely low abundance of DNA methyltransferases.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a chemiluminescence sensor and application thereof in detecting bacteria or human methyltransferases, which can detect DNA methyltransferases with high sensitivity more simply.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, a chemiluminescent sensor comprises a dumbbell probe, S-adenosylmethionine, a primer, DNA polymerase, heme, luminol, H2O2；
The dumbbell probe is a closed large-ring-shaped DNA, the dumbbell probe is structurally in a dumbbell-shaped structure with a stem in the middle and small rings at two ends, the stem structure is double-stranded DNA, the stem structure contains two hemimethylated recognition sites, the small ring structure is rich in a C sequence, and when the hemimethylated recognition sites are converted into full-methylated sites under the action of DNA methyltransferase and S-adenosylmethionine, the dumbbell probe polymerizes primers and the DNAThe synthase can perform rolling circle DNA amplification (RCA), the rolling circle DNA amplification can generate a product rich in a G sequence under the action of a small circle structure rich in a C sequence, the product rich in the G sequence can be folded into a G-quadruplex secondary structure, the G-quadruplex secondary structure can form heme-G-quadruplex DNase with heme, and the heme-G-quadruplex DNase can catalyze H2O2Mediating luminol oxidation results in an enhanced chemiluminescent signal.
The invention designs a dumbbell type probe with a switchable structure, and the probe integrates target recognition, BssHII endonuclease recognition, RCA amplification and signal transduction into one probe, thereby avoiding the use of a plurality of probes and greatly reducing false positive caused by accidental collision of the plurality of probes. The sensor of the present invention can be simply performed without any labeling and separation procedures. The sensor can sensitively detect the activity of M.SssI MTase, and the detection line reaches 1.004 multiplied by 10-7U/. mu.L, can detect Dnmt1 activity even sensitively.
In another aspect, a chemiluminescent sensor as described above is used to detect bacterial or human methyltransferases.
In a third aspect, a method for detecting bacterial or human methyltransferase is provided, comprising providing the above-described chemiluminescent sensor;
mixing a dumbbell probe, S-adenosylmethionine and a solution to be detected for methylation reaction, adding BssHII endonuclease into a system after methylation reaction for incubation treatment, and then adding exonuclease I (Exo I) and exonuclease III (Exo III) for incubation treatment; then adding a primer and DNA polymerase to carry out a rolling circle DNA amplification reaction, adding luminol and heme to a system obtained after the rolling circle DNA amplification reaction for incubation, and then adding H2O2Performing chemiluminescence detection; the test solution contains M.SssI MTase or Dnmt 1.
In a fourth aspect, the use of the above chemiluminescent sensor in screening for an m.sssi MTase inhibitor or a Dnmt1 inhibitor.
In a fifth aspect, a kit for detecting m.sssi MTase or Dnmt1 comprises the above chemiluminescent sensor and a buffer.
The invention has the beneficial effects that:
1. the chemiluminescence sensor disclosed by the invention is used for sensitively detecting bacteria and human methyltransferases based on the integration of a dumbbell probe and RCA; (1) high amplification efficiency of RCA reaction, (2) high chemiluminescence efficiency of G-quadruplex/heme DNase, (3) use of a multifunctional dumbbell probe without participation of multiple probes, which greatly reduces false positives caused by accidental probe collisions, (4) the dumbbell probe comprises a hemimethylated 5'-GmCGCGC-3' recognition site, thereby enabling construction of a universal sensor for detection of bacterial and human methyltransferases, and the two BssHII endonuclease recognition sites, thereby reducing false positives caused by incomplete cleavage of the dumbbell probe by BssHII endonuclease.
2. The chemiluminescence sensor of the invention can detect the activity of M.SssI MTase with low detection limit of 1.004 multiplied by 10-7U/uL, large dynamic range from 1X 10-7U/muL is 0.05U/muL, which can reach 6 orders of magnitude.
3. The chemiluminescence sensor reaction is carried out under isothermal condition in the whole process, and no marking and separation procedures are needed, so that the chemiluminescence sensor is simple, rapid and convenient to operate.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
Fig. 1 is a schematic diagram of a scheme of a label-free chemiluminescent sensor for detecting m.sssi MTase according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a label-free chemiluminescent sensor for detecting Dnmt1 according to an embodiment of the present invention.
FIG. 3 is a schematic representation of the principle verification result according to the embodiment of the present invention. A is methylation reaction followed by bshii endonuclease-mediated dumbbell probe cleavage and non-denaturing gel electrophoresis monitoring of Exo I and Exo III digestion of cleaved dumbbell probe. Band 1, hairpin probe 1+ hairpin probe 2+ T4DNA ligase; band 2, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + m.sssi MTase; band 3, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + M.SssI MTase + BssHII; band 4, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + BssHII; band 5, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + M.SssI MTase + BssHII + ExoI + ExoIII; band 6, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + BssHII + ExoI + ExoIII. B is 1% agarose gel electrophoresis analysis of the RCA reaction product. Band 1, RCA reaction product in absence of m.sssi MTase; band 2, RCA reaction product in the presence of M.SssI MTase. C is the fluorescence monitoring of the amplification product in the presence (red line) and absence (black line) of m.sssi MTase. SssI MTase was at a concentration of 0.2U/. mu.L.
FIG. 4 is a non-denaturing PAGE analysis of BssHII endonuclease-mediated cleavage of dumbbell probes and digestion of cleaved dumbbell probes by Exo I and III according to examples of the invention. M band, DNA ladder marker; band 1, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + BssHII + Exo I + Exo III; band 2, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + lysis buffer only + bshii + Exo I + Exo III; band 3, hairpin probe 1+ hairpin probe 2+ T4DNA ligase + Dnmt1+ BssHII + Exo I + Exo III. Error bars show the standard deviation of three experiments.
FIG. 5 shows the intensity of chemiluminescence with the concentration of M.SssI MTase at 1X 10 in an example of the present invention-7Change pattern in the range of U/. mu.L to 0.3U/. mu.L. The inset shows the linear dependence between the chemiluminescence intensity and the logarithm of the M.SssI MTase concentration, ranging from 1X 10-7U/muL to 0.05U/muL.
FIG. 6 is a bar graph of specific assays according to embodiments of the invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the problems of poor sensitivity and the like of the existing detection of DNA methyltransferase, the invention provides a chemiluminescence sensor and application thereof in detection of bacteria or human methyltransferase.
In an exemplary embodiment of the present invention, there is provided a chemiluminescent sensor comprising a dumbbell probe, S-adenosylmethionine, a primer, DNA polymerase, heme, luminol, H2O2；
The dumbbell probe is closed large-ring-shaped DNA, the dumbbell probe is structurally in a dumbbell-shaped structure with a stem in the middle and small rings at two ends, the stem structure is double-chain DNA, the stem structure contains two hemimethylated recognition sites, the small ring structure is rich in a C sequence, when the hemimethylated recognition sites are converted into full-methylated sites under the action of DNA methyltransferase and S-adenosylmethionine, the dumbbell probe can perform rolling circle DNA amplification under the action of a primer and DNA polymerase, the rolling circle DNA amplification can generate a product rich in a G sequence under the action of the small ring structure rich in the C sequence, the product rich in the G sequence can be folded into a G-quadruplex secondary structure, the G-quadruplex secondary structure can form heme-G-quadruplex DNase with heme, and the heme-G-quadruplex DNase can catalyze H2O2Mediating luminol oxidation results in an enhanced chemiluminescent signal.
The invention designs a dumbbell type probe with a switchable structure, and the probe integrates target recognition, BssHII endonuclease recognition, RCA amplification and signal transduction into one probe, thereby avoiding the use of a plurality of probes and greatly reducing false positive caused by accidental collision of the plurality of probes. The sensor of the present invention can be simply performed without any labeling and separation procedures. The sensor can sensitively detect the activity of M.SssI MTase, and the detection line reaches 1.004 multiplied by 10-7U/. mu.L, can detect Dnmt1 activity even sensitively.
Some examples of this embodiment include a bshii endonuclease.
Some examples of this embodiment include exonuclease I and exonuclease III.
In some embodiments of this embodiment, the hemimethylated recognition site is 5 '-GmCGCGC-3'.
In some embodiments of this embodiment, the nucleotide sequence of the primer is AAA GCT GTG GGT GGG TGG GT.
In some examples of this embodiment, the dumbbell probes are formed by DNA ligase ligation after complementary hybridization of the overhangs of the two hairpin probes.
In one or more embodiments, a hairpin probe nucleotide sequence is: GAA AAC TGT TAA CTA TGC GCG CTG ACC CAC CCA CCC ACC CAC AGC GCG CAT, respectively;
another hairpin probe nucleotide sequence is: AGT TAA CAG TTT TCA TGC GCG CTG ACC CAC CCA CCC ACC CAC AGC GCG CAT are provided.
In another embodiment of the present invention, there is provided a use of the above chemiluminescent sensor for detecting bacterial or human methyltransferases. Preferably, the use is for the purpose of diagnosis and treatment of non-diseases.
In a third embodiment of the present invention, there is provided a method for detecting bacterial or human methyltransferase, comprising providing the above-described chemiluminescent sensor;
mixing a dumbbell probe, S-adenosylmethionine and a solution to be detected for methylation reaction, adding BssHII endonuclease into a system after methylation reaction for incubation treatment, and then adding exonuclease I and exonuclease III for incubation treatment; then adding a primer and DNA polymerase to carry out a rolling circle DNA amplification reaction, adding luminol and heme to a system obtained after the rolling circle DNA amplification reaction for incubation, and then adding H2O2Performing chemiluminescence detection; the test solution contains M.SssI MTase or Dnmt 1.
Preferably, the detection method is used for diagnosing and treating non-diseases.
In some examples of this embodiment, the methylation reaction conditions are: incubating at 35-40 ℃, and then heating to 60-70 ℃ for inactivation. The incubation time is 0.5-1.5 h. The inactivation time is 10-30 min.
In some examples of this embodiment, the conditions under which the bshii endonuclease is added for the incubation treatment are: incubating at 35-40 ℃, and then heating to 60-70 ℃ for inactivation. The incubation time is 1.0-2.0 h. The inactivation time is 10-30 min.
In some examples of this embodiment, the conditions under which the exonuclease I and exonuclease III are added for the incubation treatment are: incubating at 35-40 ℃, and then heating to 75-85 ℃ for inactivation. The incubation time is 0.5-1.0 h. The inactivation time is 10-30 min.
In some examples of this embodiment, the conditions for the rolling circle DNA amplification reaction are: incubating at 50-60 ℃, and then heating to 75-85 ℃ for inactivation. The incubation time is 0.5-1.0 h. The inactivation time is 10-30 min.
In a fourth embodiment of the invention, there is provided a use of the above chemiluminescent sensor in screening for an m.sssi MTase inhibitor or a Dnmt1 inhibitor.
In a fifth embodiment of the invention, a detection kit of m.sssi MTase or Dnmt1 is provided, comprising the above chemiluminescent sensor and buffer.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
Drugs and materials. All oligonucleotides were synthesized by Biotechnology, Inc. (Shanghai, China) and purified by HPLC. CpG methyltransferase (M.SssI), 10 XNEB buffer 2(500 mM sodium chloride (NaCl), 100mM potassium chloride (KCl), 10mM Dithiothreitol (DTT), 100mM Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), pH 7.9),10 Xhuman DNA (cytosine-5) methyltransferase (Dnmt1) reaction buffer (500 mM Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), 10mM ethylenediamine)Tetraacetic Acid (EDTA), 10mM dibutyleneglycol, 5% glycerol, pH 7.8), BssHII endonuclease, 10 XCUTSmart buffer (500 mM potassium acetate (KAc),200 mM Tris (hydroxymethyl) aminomethane-acetate (Tris-acetate), 100mM magnesium acetate (Mg (Ac))2) 1 mg per ml Bovine Serum Albumin (BSA), pH 7.9), exonuclease I (Exo I), 10 × exonuclease I reaction buffer (670 mmol Glycine-potassium hydroxide (Glycine-KOH), 67 mmol magnesium chloride (MgCl)2) 100 mmol of 2-mercaptoethanol (. beta. -ME), pH 9.5), exonuclease III (Exo III), 10 XNEB buffer 1(100 mmol of 1, 3-Bis (trihydroxymethyl) methylaminopropane-hydrochloric acid (Bis-Tris-Propane-HCl), 100 mmol of magnesium chloride (MgCl)2) 10 mmole Dithiothreitol (DTT) pH 7), DNA adenine Methyltransferase (Dam MTase), 10 XDNA adenine Methyltransferase reaction buffer (500 mmole Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), 50 mmole 2-mercaptoethanol (beta-ME), 100 mmole ethylenediaminetetraacetic acid (EDTA), pH 7.5), AluI Methyltransferase (AluI Methylransferase), HaeIII Methyltransferase (eIII MTase), S-adenosylmethionine (SAM), T4DNA ligase (T4 DNA ligase), 10 XT 4DNA ligase reaction buffer (500 mmole Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl), 100 mmole magnesium chloride (MgCl)2) 10mM Adenosine Triphosphate (ATP),100 mM Dithiothreitol (DTT) pH 7.5, Bst DNA polymerase (large fragment), 10 × ThermoPol reaction buffer (200 mM Tris (hydroxymethyl) aminomethane-HCl (Tris-HCl)), 100mM ammonium sulfate ((NH)4)2SO4) 100 mmole potassium chloride (KCl), 20 mmole magnesium sulfate (MgSO)4) 1% Polyethyleneglycol octylphenyl ether, pH 8.8), deoxyribonucleoside 5 '-triphosphate mixture (dNTPs), protoporphyrin IX (PPIX), SYBR Gold, 5-azacytosine (5-Aza), 5-Aza-2' -deoxycytidine (5-Aza-dC), N-phthaloyl-L-tryptophan (RG108), Bovine Serum Albumin (BSA), Fetal Bovine Serum (FBS), heme (Hemin), Luminol (Luminol), hydrogen peroxide (H)2O2) 4- (2-hydroxyethyl) piperazine 1-ethanesulfonic acid sodium salt (HEPES). All other reagents were analytical grade and used without further purification. Book (I)The nucleotide sequences in the examples are shown in Table 1.
TABLE 1 nucleotide sequencea
aIn hairpin probes 1 and 2, "P" represents a phosphate group, italics represents the recognition site for BssHII, and the underlined base "C" represents C5-methylcytosine.
And (4) preparing the dumbbell probe. The steps of constructing a dumbbell probe of a switchable structure are as follows. All oligonucleotides were diluted with 1 × Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH 8.0) to prepare stock solutions. Mixture of hairpin Probe 1 and hairpin Probe 2 for M.SssI MTase assay (or Dnmt1 assay) in buffer (1.5mM MgCl)210mM Tris-HCl, pH 8.0) to 350nM and incubated at 95 ℃ for 5 minutes, then slowly cooled to room temperature to completely fold the hairpin structure. Subsequently, the hairpin probe obtained was added to a 20. mu.L reaction containing 200U T4DNA ligase and 1 XT 4DNA ligase reaction buffer, followed by incubation overnight at 16 ℃ and inactivation of the reaction at 65 ℃ for 10 minutes. The dumbbell probe obtained was used immediately or stored at-20 ℃ until use.
DNA methylation and BssHII endonuclease cleavage of dumbbell template. The DNA methylation and bshii endonuclease-mediated cleavage process involves three sequential steps. First, 175nM dumbbell probe was added to 20. mu.L of reaction solution containing M.SssI MTase (or Dnmt1), 160. mu.M SAM and 1 XNEBuffer 2 (or Dnmt1 reaction buffer) and incubated at 37 ℃ for 1h, followed by inactivation at 65 ℃ for 20 min. Next, 10. mu.L of the above reaction product was added to 20. mu.L of a reaction system containing 1 XCutSmart buffer and 5U of BssHII endonuclease, and the mixture was incubated at 37 ℃ for 1.5h and then inactivated at 65 ℃ for 20 minutes. Third, 10. mu.L of the above reaction product was added to 20. mu.L of a reaction system comprising 10U of Exo I, 50U of Exo III, 1 XExo I reaction buffer and 1 XNEBuffer 1, and the mixture was incubated at 37 ℃ for 45 minutes and then inactivated at 80 ℃ for 20 minutes.
And (3) RCA reaction process. The RCA reaction was performed in 20. mu.L of a solution containing 14. mu.L of the above reaction product, 100nM primer, 500. mu.M dNTP solution mixture, 4U of Bst DNA polymerase and 1 XThermoPol reaction buffer, and incubated at 55 ℃ for 40 minutes, followed by inactivation at 80 ℃ for 20 minutes.
And (4) detecting chemiluminescence. For chemiluminescent analysis, 20. mu.L of the RCA reaction product was mixed with 0.5mM luminol solution, 750nM heme solution and 1 XHEPES buffer (40mM HEPES, 20mM KCl, 300mM NaCl, pH 8.0) to form a mixture (30. mu.L), which was then incubated in the dark at room temperature for 30 minutes to allow the resulting G-rich polymerization product to fold into an active quadruplex structure. Subsequently, 30 μ L H was added2O2(50mM) was added to the above solution and the chemiluminescent signal was recorded at 1.5s intervals on a GloMax 96 microplate luminometer (Promega, Madison, Wis., USA).
And (4) measuring fluorescence. 10 μ LRCA reaction products were incubated at 95 ℃ for 5 minutes, then slowly cooled to room temperature to completely fold the G-quadruplex structure. Subsequently, the obtained G-quadruplex probe was added to a 20. mu.L reaction system containing 2. mu.MPPIX and 1 XTTris-buffer (10mM Tris-HCl, 1mM EDTA, 100mM NaCl, 20mM KCl, pH 8.0) at room temperature, and incubated at room temperature for 2 hours in the dark. The fluorescence emission spectrum of the G-quadruplex-PPIX complex was scanned in the 550-750nm range with a HitachiF-7000 spectrofluorometer at an excitation wavelength of 410nm, an excitation slit of 5.0nm, an emission gap of 5.0nm, and the fluorescence intensity at the emission wavelength of 634nm for data analysis.
And (4) performing gel electrophoresis. To analyze the products of the DNA methylation and BssHII endonuclease-mediated cleavage process, 12% native polyacrylamide gel electrophoresis (PAGE) was performed in 1 XTBE buffer (9mM boric acid, 0.2mM EDTA, 9mM Tris-HCl, pH 7.9) and placed at room temperature for 55 minutes at 110V constant pressure at room temperature with 1 SYBR Gold as the fluorescence indicator. The gel was imaged by a chemiDoc MP imaging system. To analyze the products of RCA, 1% agarose gel electrophoresis was performed in 1 XTAE buffer (2mM EDTA, 40mM Tris-ethyl acetate, pH 8.0) with 1 SYBR Gold as a fluorescent indicator at room temperature under a constant pressure of 110V for 45 minutes. The gel was imaged by a chemiDoc MP imaging system.
The principle of the SssI MTase assay is shown in FIG. 1. This example designed two hairpin probes (hairpin probes 1 and 2) with phosphorylated 5 'ends and hydroxylated 3' ends. The longer stems of the two hairpin probes can hybridize to each other to ensure that the 5 'and 3' ends can be pulled into adjacent positions and successfully ligated by T4DNA ligase to form a closed dumbbell probe. The dumbbell probe consists of two functional domains: (1) both sssi MTase and bshii endonucleases recognize two hemimethylated 5 '-gmcgc-3'/3 '-CGCGCG-5' sites in the stem sequence, (2) both loops have identical C-rich sequences, can generate complementary G-rich sequences, and generate G-quadruplex secondary structure. Sssi MTase can be considered a DNA methyltransferase in the muramidase spirochete strain MQ1 that can bind to double stranded DNA (dsDNA) of CpG recognition sites, then scan the DNA and catalyze the transfer of a methyl group from the SAM to the carbon 5 position of cytosine in all CpG recognition sites. In the absence of m.sssi MTase, bshii endonuclease can cleave the hemimethylated site in the double-stranded stem sequence of the dumbbell probe, and then Exo I and Exo III can completely digest the cleaved dumbbell probe into single nucleotide, effectively eliminating background signal. In the presence of m.sssi MTase, the hemimethylated sites would be methylated to the fully methylated sites and the bshii endonuclease cleavage would be blocked, the closed dumbbell probe could effectively prevent digestion of Exo I and Exo III. Subsequently, the dumbbell probe can be used as a circular template to initiate RCA amplification by means of a DNA polymerase, primers and dNTP solution mixture to produce long single-stranded DNA (ssDNA) fragments containing a plurality of G-rich sequences in the repeat sequence complementary to the C-rich portion of the dumbbell template, the amplified G-rich products can fold into a G-quadruplex secondary structure, can form heme-G-quadruplex DNase in the presence of cofactor heme, and catalyze H2O2Mediating luminol oxidation results in an enhanced chemiluminescent signal.
The principle of the Dnmt1 assay is shown in FIG. 2. This example designs a reconfigurable halfMethylated dumbbell probes consisting of hairpin probes 1 and 2 with Dnmt1 and a BssHII endonuclease recognition site (hemimethylated 5'-GmCGCGC-3'/3 '-CGCGCG-5'). In the absence of Dnmt1, BssHII endonuclease can cleave the hemimethylated recognition site in the double-stranded stem sequence of the dumbbell probe, and then Exo I and Exo III can completely digest the cleaved dumbbell probe into single nucleotides, effectively eliminating background signals. When Dnmt1 is present, the hemimethylated recognition site will be methylated to the fully methylated sequence and the BssHII endonuclease cleavage of the fully methylated dumbbell template is blocked, effectively preventing digestion of the fully methylated dumbbell probe by Exo I and Exo III. Subsequently, the dumbbell probe can serve as a circular template, initiate RCA amplification by means of a DNA polymerase, primers and dNTP solution mixture, generate long ssDNA fragments with a large number of repeated G-rich sequences, can form heme-G-quadruplex DNase in the presence of cofactor heme, and catalyze H2O2Mediating luminol oxidation results in an enhanced chemiluminescent signal.
Experimental verification of principle
To demonstrate the feasibility of this method, the reaction products of the methylation process, the subsequent bshii endonuclease-mediated cleavage of the dumbbell probe, and the exonuclease I and III-catalyzed digestion of the dumbbell probe were analyzed using 12% native gel electrophoresis using SYBR Gold as the fluorescent indicator (fig. 3A). When hairpin probes 1 and 2 hybridize to each other and are subsequently ligated by T4DNA ligase (FIG. 3A, lane 1), a characteristic band is detected, indicating that the dumbbell probe has been successfully formed. SssI MTase catalyzes the transfer of a methyl group to a cytosine in a dumbbell probe (FIG. 3A, lane 2), and then BssHII endonuclease cleaves the fully methylated dumbbell probe (FIG. 3A, lane 3), and the position of the characteristic band is the same as that of the dumbbell probe (FIG. 3A, lane 1); however, in the absence of m.sssi MTase, bshii endonuclease cleaved the dumbbell probe and a new shorter signature band was detected (fig. 3A, lane 4), indicating that the substrate of the dumbbell probe could be cleaved by bshii endonuclease. To further investigate whether bsshi endonuclease can cleave the dumbbell probe in the presence or absence of m.sssi MTase, this example used Exo I and Exo III to digest the dumbbell probe cleaved by bsshi endonuclease, and when m.sssi MTase was present (fig. 3A, lane 5), characteristic bands of the dumbbell probe were detected, indicating that bshi endonuclease was not able to cleave the fully methylated dumbbell probe and that Exo I and Exo III were not able to digest the blocked fully methylated dumbbell probe. In the absence of M.SssI MTase, no characteristic band was detected for the dumbbell probe (FIG. 3A, lane 6), indicating that the dumbbell probe can be cleaved by BssHII endonuclease and that the cleaved dumbbell probe can be digested by Exo I and Exo III.
This example further uses 1% agarose gel electrophoresis to verify the RCA reaction. In the absence of m.sssi MTase, no significant DNA product was observed (fig. 3B, lane 1), indicating that bshii endonuclease cleaved dumbbell probe products could be digested by Exo I and Exo III, and no RCA reaction occurred. In contrast, a distinct dispersion band was observed in the presence of M.SssI MTase (FIG. 3B, lane 2), indicating that BssHII-mediated endonuclease cleavage was successfully blocked when M.SssI MTase catalyzes the dumbbell probe to a fully methylated dumbbell probe (note: BssHII endonuclease cannot cleave the fully methylated site), and thus an RCA reaction occurred. This example further investigated the feasibility of such a sensor using fluorescence spectroscopy with PPIX as the fluorescence indicator. In aqueous solution, PPIX has a low fluorescence intensity, but when it binds to G-quadruplexes, the fluorescence intensity is greatly enhanced. A significant PPIX fluorescence signal was observed in the presence of m.sssi MTase (fig. 3C), indicating that m.sssi MTase can successfully prevent bsshi endonuclease-mediated cleavage of dumbbell probes because m.sssi MTase methylates hemimethylated recognition sites in dumbbell probes to fully methylated sequences, whereas bshii endonuclease cannot cleave fully methylated sites, and then the intact dumbbell probes can initiate subsequent RCA amplification to generate large amounts of G-quadruplex sequences. However, in the absence of m.sssi MTase, a low fluorescence signal due to PPIX autofluorescence was observed (fig. 3C), indicating that no RCA reaction occurred and no G-quadruplex product was produced. These results demonstrate the feasibility of the sensor for m.sssi MTase activity assays.
To evaluate DNA methylation and bshii endonuclease-mediated cleavage of Dnmt1, the product was analyzed using 12% native PAGE (fig. 4). When Dnmt1 was present, a characteristic band for the dumbbell probe was detected (fig. 4, lane 3), indicating that bshii endonuclease was unable to cleave the fully methylated dumbbell probe and Exo I and Exo III were unable to digest the blocked fully methylated dumbbell probe. In the presence of lysis buffer (FIG. 4, lane 2) or ultrapure water (FIG. 4, lane 1) alone, no characteristic band of the dumbbell probe was detected, indicating that the hemimethylated dumbbell probe could be cleaved by BssHII endonuclease and that Exo I and Exo III could effectively digest the dumbbell probe cleaved by BssHII endonuclease.
Sensitivity test
To evaluate the sensitivity of the sensor, this example measured the chemiluminescence intensity of different concentrations of m.sssi MTase under optimal reaction conditions. As shown in FIG. 5, the chemiluminescent signal varied from 1X 10 with M.SssI MTase concentration- 7The U/. mu.L is gradually improved by increasing to 0.3U/. mu.L, and the concentration is 1X 10-7The chemiluminescence intensity is in linear relation with the logarithm of the concentration of M.SssI MTase within the dynamic range of 6 orders of magnitude from U/muL to 0.05U/muL. The correlation equation is 7.33E6+9.3E5 log10C(R20.9961) where I is the chemiluminescence intensity and C is the m.sssi MTase concentration (U/. mu.l) (fig. 5 inset). The detection limit calculated by evaluating the mean signal of the control group plus three times the standard deviation was 1.004X 10-7U/. mu.L. Notably, the sensitivity of this sensor was improved by 3 orders of magnitude compared to the electrochemical sensor (0.12U/mL), by 3 orders of magnitude compared to the surface plasmon resonance-based method (0.12U/mL), and by 690-fold compared to the colorimetric method (0.069U/mL). The high sensitivity of this sensor is due to the following factors: (1) high amplification efficiency of RCA reaction, (2) high efficiency of chemiluminescence G-quadruplex/hemin DNAzyme; (3) the multifunctional dumbbell-shaped probe eliminates the participation of a plurality of probes, and greatly reduces false positives caused by accidental collision of the probes; (4) two BssHII endonucleases in dumbbell type probeThe design of enzyme recognition sites can reduce false positives due to incomplete cleavage of dumbbell probes by BssHII endonuclease. Since once one of the two bshii recognition sites in the dumbbell probe was recognized by bshii, the dumbbell probe could be digested by Exo I and Exo III, effectively blocking RCA, thereby eliminating non-specific amplification.
Experiment of specificity
To evaluate the detection specificity of this method, three members of the DNA methyltransferase family, including AluI MTase, HaeIII MTase and Dam MTase, were used as the interfering enzymes. Theoretically, none of these interferents could recognize and fully methylate the 5'-GmCGCGC-3' sequence to prevent BssHII endonuclease from cleaving the dumbbell-shaped probe. As shown in fig. 6, a unique chemiluminescent signal was detected in the presence of m.sssi MTase (0.2U/. mu.l) (fig. 6). In contrast, in the presence of AluI MTase (0.2U/. mu.L) (FIG. 6), HaeIII MTase (0.2U/. mu.L) (FIG. 6), Dam MTase (0.2U/. mu.L) (FIG. 6) and a control in the presence of reaction buffer only (FIG. 6), very low chemiluminescent signals were observed, indicating that only M.SssI MTase was able to specifically permethylate the 5 '-GmCGCGCGC-3' sequence to prevent BssHII endonuclease from cleaving the dumbbell probe and subsequently initiating RCA amplification. These results indicate that this chemiluminescent sensor can distinguish m.sssi MTase from other DNA methyltransferase members with high specificity.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> chemiluminescence sensor and application in detection of bacteria or human methyltransferase
<130>
<160> 3
<170> PatentIn version 3.3
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gaaaactgtt aactatgcgc gctgacccac ccacccaccc acagcgcgca t 51
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agttaacagt tttcatgcgc gctgacccac ccacccaccc acagcgcgca t 51
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Claims (10)
1. A chemiluminescence sensor is characterized by comprising a dumbbell probe, S-adenosylmethionine, a primer, DNA polymerase, heme, luminol and H2O2；
The dumbbell probe is closed large-ring-shaped DNA, the dumbbell probe is structurally in a dumbbell-shaped structure with a stem in the middle and small rings at two ends, the stem structure is double-chain DNA, the stem structure contains two hemimethylated recognition sites, the small ring structure is rich in a C sequence, when the hemimethylated recognition sites are converted into full-methylated sites under the action of DNA methyltransferase and S-adenosylmethionine, the dumbbell probe can perform rolling circle DNA amplification under the action of a primer and DNA polymerase, the rolling circle DNA amplification can generate a product rich in a G sequence under the action of the small ring structure rich in the C sequence, the product rich in the G sequence can be folded into a G-quadruplex secondary structure, the G-quadruplex secondary structure can form heme-G-quadruplex DNase with heme, and the heme-G-quadruplex DNase can catalyze H2O2Mediating luminol oxidation productionEnhanced chemiluminescent signal.
2. The chemiluminescent sensor of claim 1 comprising a bshii endonuclease;
or, comprises exonuclease I and exonuclease III.
3. The chemiluminescent sensor of claim 1 wherein the hemimethylated recognition site is 5 '-GmCGCGC-3'.
4. The chemiluminescent sensor of claim 1 wherein the dumbbell probes are formed by DNA ligase ligation after complementary hybridization of the overhangs of the two hairpin probes.
5. Use of a chemiluminescent sensor of any one of claims 1 to 4 for detecting bacterial or human methyltransferases.
6. A method for detecting a bacterial or human methyltransferase, comprising providing the chemiluminescent sensor of any one of claims 1 to 4;
mixing a dumbbell probe, S-adenosylmethionine and a solution to be detected for methylation reaction, adding BssHII endonuclease into a system after methylation reaction for incubation treatment, and then adding exonuclease I and exonuclease III for incubation treatment; then adding a primer and DNA polymerase to carry out a rolling circle DNA amplification reaction, adding luminol and heme to a system obtained after the rolling circle DNA amplification reaction for incubation, and then adding H2O2Performing chemiluminescence detection; the solution to be tested contains M.SssIMTase or Dnmt 1.
7. The method for detecting bacterial or human methyltransferase of claim 6 wherein the methylation reaction is carried out under the following conditions: incubating at 35-40 ℃, and then heating to 60-70 ℃ for inactivation;
or, the incubation conditions with BssHII endonuclease were as follows: incubating at 35-40 ℃, and then heating to 60-70 ℃ for inactivation.
8. The method for detecting bacterial or human methyltransferase of claim 6 wherein the incubation conditions for adding exonuclease I and exonuclease III are as follows: incubating at 35-40 ℃, and then heating to 75-85 ℃ for inactivation;
or, the conditions of the rolling circle DNA amplification reaction are as follows: incubating at 50-60 ℃, and then heating to 75-85 ℃ for inactivation.
9. Use of a chemiluminescent sensor of any one of claims 1 to 4 for screening for an m.ss imtase inhibitor or a Dnmt1 inhibitor.
10. A kit for detecting M.SssIMTase or Dnmt1, comprising the chemiluminescent sensor of any one of claims 1 to 4 and a buffer.
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