CN111088324B - Single quantum dot nano sensor for detecting methyltransferase and detection method and application thereof - Google Patents
Single quantum dot nano sensor for detecting methyltransferase and detection method and application thereof Download PDFInfo
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Abstract
The invention provides a single quantum dot nano sensor for detecting methyltransferase as well as a detection method and application thereof, belonging to the technical field of molecular detection. The nanosensor includes at least: a double-stranded DNA probe, quantum dots and adenosine triphosphate deoxynucleotides marked with fluorescent molecules; the double-stranded DNA probe is designed with a recognition site of CpG methyltransferase. The invention develops a nano-sensor with CpG methyl cytosine recognition site performance based on single quantum dot. The CpG methyltransferase detected by the sensor has lower detection limit which can be one to two orders of magnitude lower than that of the current method, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a single quantum dot nano sensor for detecting methyltransferase as well as a detection method and application thereof.
Background
The information disclosed 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.
CpG island sequence methylation is an important epigenetic marker and plays an essential role in development and differentiation, genomic stability, genomic markers, X chromosome inactivation. Aberrant CpG island sequence methylation is associated with many diseases, such as cancer. DNA methylation is largely dependent on methyltransferase activity, and therefore, detection of CpG methyltransferases is critical for early diagnosis of related diseases.
Conventional CpG methyltransferase assays are dominated by radiolabeled methylation and bisulfite, but have limitations: the participation of radioactive substances limits the widespread use of the former, bisulfite being used to identify cytosines, rather than methylcytosine, in DNA; this method can lead to sample DNA degradation and false positive rates due to incomplete conversion from cytosine to uracil. In addition, several fluorescence methods and electrochemical immunoreaction methods have also been used for the detection of CpG methyltransferases, such as methylation sensitive endonuclease HpaII-based fluorescence: the HpaII endonuclease is used for cutting a specific DNA sequence with a cytosine site and cannot cut corresponding DNA containing methylcytosine; these methods also suffer from false positive rates due to incomplete cleavage of specific DNA sequences by restriction endonucleases. The electrochemical immune reaction method has the defects of specific antibody requirement, poor sensitivity and the like. Therefore, it is important to develop a method for detecting CpG methyltransferase accurately and with high sensitivity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a single quantum dot nano sensor for detecting methyltransferase, and a detection method and application thereof. The invention develops a nano sensor with CpG methyl cytosine recognition site performance based on single quantum dot. The CpG methyltransferases detected by the sensor have lower detection limits, which can be one to two orders of magnitude lower than the current methods. Therefore, it has good practical application value.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a single quantum dot nanosensor for detecting methyltransferases, the nanosensor comprising at least: a double-stranded DNA probe, quantum dots and adenosine triphosphate deoxynucleotide marked with fluorescent molecules.
Wherein, the double-stranded DNA probe is designed with a recognition site (5 '-G-C-G-mC-3'/3 '-mC-G-mC-G-5') of CpG methyltransferase;
preferably, biotin (biotin) is designed on the 5' end of the double-stranded DNA probe so as to connect quantum dots coated by streptavidin; design of PO at 3' end 4 To prevent non-specific amplification of terminal transferase (TDT);
the quantum dots are 605QDs; 605QDs used in the present invention are streptavidin-coated CdSe/ZnS QDs, and the surface of the quantum dot is coated with streptavidin.
Although various fluorescent dye molecules capable of generating fluorescence resonance energy transfer with quantum dots, such as TAMRA/Cy3/Texas Red/rhodamine, and the like, exist in the prior art, the Fluorescence Resonance Energy Transfer (FRET) efficiency of the pair of 605QDs/Cy5 combination in the method of the invention is higher through comparative experimental research. And in the present invention, a plurality of Cy5 receptors are bound to one DNA molecule, and then, a plurality of Cy 5-labeled double-stranded DNAs are assembled to one 605 quantum dot, thereby further significantly improving the fluorescence resonance energy transfer efficiency.
In a second aspect of the present invention, there is provided a use of the above-described nanosensor for detecting DNA methyltransferase; the DNA methyltransferase is a CpG methyltransferase.
In a third aspect of the present invention, there is provided a method for detecting CpG methyltransferase, the method comprising:
(1) Carrying out methylation reaction on a sample to be detected and the double-stranded DNA probe to obtain a methylation product;
(2) Adding the methylated product into GlaI endonuclease to perform enzyme digestion reaction;
(3) Mixing the cut product with terminal transferase and adenosine triphosphate deoxynucleotide marked with fluorescent molecules to obtain a reaction product;
(4) And mixing the reaction product with the quantum dot to obtain the structure of the quantum dot/probe/Cy 5.
The detection method also comprises the step of measuring a fluorescence signal by using a fluorescence imaging system, so that the quantitative detection of the CpG methyltransferase is realized.
Specifically, the method for measuring the fluorescence signal comprises the following steps: the QDs were excited at 405nm and the signals at 609.8nm (605 QDs) and 670nm (Cy 5) were collected respectively.
In a fourth aspect of the present invention, there is provided the use of the above-described nanosensor and/or detection method for detecting DNA methyltransferase activity and/or screening DNA methyltransferase drugs.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) The invention does not need radioactive labeling substance and specific antibody, thus the method reduces the detection cost. Compared with a cytosine recognition site method with a false positive rate, the method has the property of a methylcytosine recognition site.
(2) A plurality of biotin and Cy 5-labeled double-stranded DNAs are assembled on a single 605 quantum dot, so that the fluorescence resonance energy transfer efficiency is remarkably improved.
(3) Has high sensitivity and the lowest detection limit can reach 2.1 multiplied by 10 -7 U/. Mu.L, one to two orders of magnitude lower than the prior art.
Drawings
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 included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a diagram of the detection mechanism of the nanosensor of the invention.
FIG. 2 (A) is a graph of native polyacrylamide gel analysis after methylation of dsDNA-1 substrate by CpG methyltransferase and cleavage by GlaI endonuclease. Lane 1, dsdna-1 substrate + CpG methyltransferase + GlaI endonuclease; lane 2, dsDNA-1 substrate + GlaI endonuclease; lane 3, dsDNA-1 substrate + CpG methyltransferase; lane 4, dsDNA-1 substrate. FIG. 2 (B) is a 605QD and Cy5 fluorescence spectra in the presence and absence of CpG methyltransferase.
FIG. 3 is a single molecule fluorescence image of 605QD and Cy5 in the absence (A-C) and presence (D-F) of CpG methyltransferase. The fluorescence signal of 605 quantum dots is green (a, D), and the fluorescence signal of Cy5 is red (B, E). Yellow fluorescence signal indicated that 605QD and Cy5 are present together (C, F).
FIG. 4 is a graph showing the change in Cy5 intensity with the concentration of CpG methyltransferase.
FIG. 5 is a graph showing the change in the number of Cy5 molecules depending on the concentration of CpG methyltransferase. The inset shows that the number of Cy5 is logarithmically linearly related to the concentration of CpG methyltransferase.
FIG. 6 shows the measured numbers of Cy5, respectively: cpG methyltransferase, aluI methyltransferase, haeIII methyltransferase, dam methyltransferase and a control containing only reaction buffer.
FIG. 7 is a graph showing the inhibitory effect of 5-azacytidine and 5-azacytidine-2-deoxycytidine on CpG methyltransferase activity, in which A is 5-azacytidine and B is 5-azacytidine-2-deoxycytidine.
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. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, the existing CpG methyltransferase detection method has the defects of complex operation, false positive, poor sensitivity and the like.
In view of the above, in one embodiment of the present invention, there is provided a single quantum dot nanosensor for detecting methyltransferase, the nanosensor comprising at least: a double-stranded DNA probe, quantum dots and adenosine triphosphate deoxynucleotide marked with fluorescent molecules.
In still another embodiment of the present invention, the double-stranded DNA probe is designed with a recognition site for CpG methyltransferase (5 '-G-C-G-mC-3'/3 '-mC-G-mC-G-5');
in another embodiment of the present invention, the double-stranded DNA probe is designed with biotin (biotin) at the 5' end to link quantum dots coated with streptavidin; design of PO at 3' end 4 To prevent non-specific amplification of terminal transferase (TDT);
in another embodiment of the present invention, the double-stranded DNA probe has the following base sequence:
5’-biotin-GAC TAC TGT GCG m CTT CAT GAT C-PO 4 -3’(SEQ ID NO.1)；
5’-biotin-GAT CAT GAA G m CG m CAC AGT AGT C-PO 4 -3’(SEQ ID NO.2)；
wherein the content of the first and second substances, m c is methylcytosine.
In yet another embodiment of the present invention, the quantum dots are 605QDs; 605QDs used in the present invention is a streptavidin-coated CdSe/ZnS QDs having a maximum emission wavelength of 605nm.
In another embodiment of the present invention, the fluorescent molecule is cyanine 5 (Cy 5).
In still another embodiment of the present invention, the nanosensor further comprises S-adenosylmethionine, glaI endonuclease, and terminal transferase.
In still another embodiment of the present invention, there is provided a use of the above-described nanosensor for detecting DNA methyltransferase; the DNA methyltransferase is a CpG methyltransferase.
In still another embodiment of the present invention, there is provided a method for detecting CpG methyltransferase, the method comprising:
(1) Carrying out methylation reaction on a sample to be detected and the double-stranded DNA probe to obtain a methylation product;
(2) Adding the methylated product into GlaI endonuclease to perform enzyme digestion reaction;
(3) Mixing the cut product with terminal transferase and adenosine triphosphate deoxynucleotide marked with fluorescent molecules to obtain a reaction product;
(4) And mixing the reaction product with the quantum dot to obtain the structure of the quantum dot/probe/Cy 5.
In still another embodiment of the present invention, in the step (1), a coenzyme factor participating in the methyl transfer reaction, i.e., S-adenosylmethionine, is added to promote the methylation reaction; the methylation reaction is carried out under the specific conditions of 30-150min (preferably 120 min) at 37 ℃;
in another embodiment of the present invention, in the step (2), the enzyme digestion reaction specifically includes: reacting at 30 deg.C for 30-150min (preferably 120 min), and then reacting at 80 deg.C for 20min to terminate the reaction;
in still another embodiment of the present invention, in the step (3), the reaction conditions are specifically a reaction at 37 ℃ for 30-180min (preferably 120 min), followed by a reaction at 80 ℃ for 20min to stop the reaction;
in still another embodiment of the present invention, in the step (4), the reaction condition is specifically a reaction at room temperature for 15 to 30 minutes (preferably 20 minutes).
In another embodiment of the present invention, the detection method further comprises measuring a fluorescence signal using a fluorescence imaging system, thereby achieving quantitative detection of CpG methyltransferase.
In another embodiment of the present invention, the method for measuring fluorescence signal comprises: the QDs were excited at 405nm and the signals at 609.8nm (605 QDs) and 670nm (Cy 5) were collected, respectively.
In another embodiment of the present invention, there is provided a use of the above-mentioned nanosensor and/or detection method for detecting DNA methyltransferase activity and/or screening DNA methyltransferase drugs.
The DNA methyltransferase drugs include, but are not limited to, DNA methyltransferase promoters and DNA methyltransferase drug inhibitors.
From the above, the present invention provides a nanosensor based on single quantum dots (605 QD) with properties of recognizing methylcytosine sites: double-stranded DNA (dsDNA-1) is methylated in the presence of CpG methyltransferase and S-adenosyl methionine. Subsequently, the GlaI endonuclease recognizes the corresponding site of the methylated product, and the cleavage reaction is performed. Under the action of terminal transferase (TDT), multiple Cy5-dATP structures are continuously connected to the free 3' -hydroxyl in the enzyme digestion product, so that the biotin/Cy5 labeled double-stranded DNA is obtained. After it is mixed with streptavidin-coated 605QDs, the streptavidin-coated biotin is linked to form quantum dot/probe/Cy 5 nanosensor. In the presence of an excitation wavelength of 405nm, fluorescence signals of 605 quantum dots and Cy5 can be observed simultaneously due to fluorescence resonance energy transfer from the quantum dots to Cy 5. Subsequently, the emission signals of Cy5 can be counted.
According to the invention, terminal transferase (TDT) is introduced for assisted polymerization, a plurality of Cy5 receptors can be combined on one DNA molecule, and then a plurality of Cy 5-labeled double-stranded DNAs are assembled on one 605 quantum dot, so that the fluorescence resonance energy transfer efficiency is remarkably improved. Meanwhile, due to the specific recognition between CpG methyltransferase induced methylation reaction and subsequent GlaI endonuclease assisted methylation and non-methylation cytosine, and then, a plurality of biotin and Cy5 labeled dsDNAs are assembled on a single 605 quantum dot, the fluorescence resonance energy transfer is obviously improved, and in addition, the advantages of high signal-to-noise ratio and low background of single molecule detection are fully utilized, so that the invention has good selectivity and high sensitivity.
It should be noted that, based on the inventive concept of the present invention, it is also within the scope of the present invention to detect other DNA methyltransferases.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Formation of double stranded DNA Structure (dsDNA-1): 5 micromoles per liter of Probe 1 (biotin-GAC TAC TGT GCG) m CTT CAT GAT C-PO 4 ， m C is methylcytosine) and 5. Mu. Mol per liter of Probe 2 (biotin-GAT CAT GAA G) m CG m CAC AGT AGT C-PO 4 ) Adding a methylated buffer solution (50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L magnesium chloride, 1 mmol dithiothreitol, pH 7.4) to the mixture, 9Reacting at 5 ℃ for 5 minutes, slowly cooling to room temperature, and placing the obtained annealing product at 4 ℃ for later use.
Determination of CpG methyltransferase: (1) Methylation reactions were carried out in 10. Mu.l systems containing methylation buffer, 0.64 mmoles per liter S-adenosyl methionine, 1. Mu. Moles per liter annealing product and various concentrations of CpG methyltransferase. The mixture is reacted at 37 ℃ for 30-150min (preferably 120 min). (2) The cleavage reaction is also carried out in a 10. Mu.l system comprising 5. Mu.l of the methylated product, 0.05-0.3 units per. Mu.l (preferably 0.2 units per. Mu.l) of the GlaI endonuclease, a 1 XGlaI endonuclease reaction buffer solution (33 mmoles per liter of Tris-acetic acid, 10 mmoles per liter of magnesium acetate, 66 mmoles per liter of potassium acetate, 1 mmole per liter of dithiothreitol, pH 7.9), which system is reacted at 30 ℃ for 30-150min (preferably 120 min) and subsequently at 80 ℃ for 20min to terminate the reaction. (3) 4.8. Mu.l of the cleavage product, 2 to 12 units (preferably 10 units) of terminal transferase (TDT), 6 to 18. Mu. Mol (preferably 16. Mu. Mol) per liter of deoxyadenosine triphosphate (Cy 5-dATP) labeled with a Cy5 fluorescent molecule and 0.25. Mu. Mol per liter of cobalt chloride were added to a 15. Mu.l reaction system, reacted at 37 ℃ for 30 to 180min (preferably 120 min), and then reacted at 80 ℃ for 20min to stop the reaction. (4) The reaction product of the above step 15. Mu.l and 605. Mu. Mol/l quantum dots were added to 80. Mu.l of reaction buffer solution (100 mmol/l tris-hcl, 10 mmol/l ammonium sulfate, 3 mmol/l magnesium chloride, pH 8.0) and reacted at room temperature for 20 minutes to form a quantum dot/probe/Cy 5 structure.
Gel electrophoresis imaging: the products after methylation and cleavage were electrophoresed on 12% native polyacrylamide gel at room temperature for 45 min at 110 v. Subsequently, the cleavage reaction product was subjected to image analysis.
Fluorescence detection: fluorescence spectra were recorded using a fluorescence spectrometer, QDs excited at 405nm and signals at 609.8nm (605 QDs) and 670nm (Cy 5) were collected for subsequent image and data analysis processing.
Single molecule assay and data analysis: the reaction product was diluted 200-fold with imaging buffer (100 mmol/l tris-hcl, 10 mmol/l ammonium sulfate, 3 mmol/l magnesium chloride, pH 8.0), 10 μ l of sample was placed on a glass slide and single molecule imaging was performed using a total internal reflection fluorescence microscope. 605QD were excited with a 405nm laser source and then Cy5 dots in a 500 x 500 pixel area were counted and 10 images were summed.
Specificity and inhibitor assays: three methyltransferases HaeIII, aluI, dam were used as interfering enzymes to test the specificity of the technique. The reaction procedure is the same as the CpG methyltransferase assay. 5-azacytidine or 5-azacytidine-2-deoxycytidine at various concentrations was added to the methylation reaction system, and the reaction steps followed this technique were otherwise followed.
The principle of the assay is as follows (FIG. 1): the dsDNA-1 contains 5'-G-C-G-mC-3'/3'-mC-G-mC-G-5' site as recognition site of CpG methyltransferase. We designed a biotin on the 5 'end of the dsDNA-1 structure to link the streptavidin-coated 605 quantum dots, and a PO on the 3' end 4 To prevent non-specific amplification of terminal transferase (TDT). In the presence of CpG methyltransferase and S-adenosyl methionine, dsDNA-1 is methylated. Subsequently, the GlaI endonuclease recognizes the corresponding site of the methylated product, and the cleavage reaction is carried out. Under the action of terminal transferase (TDT), multiple Cy5-dATP structures are continuously connected to the free 3' -hydroxyl in the enzyme digestion product, so that the biotin/Cy5 labeled double-stranded DNA is obtained. After it is mixed with streptavidin-coated 605QDs, biotin coated with specific streptavidin is linked to form quantum dot/probe/Cy 5 nanosensor. In the presence of an excitation wavelength of 405nm, the fluorescence signals of 605 quantum dots and Cy5 can be observed simultaneously due to fluorescence resonance energy transfer from the quantum dots to Cy 5. Subsequently, the emission signals of Cy5 can be counted. However, unmethylated dsDNA-1 has no corresponding sites to cleave in the absence of CpG methyltransferase. Since the probe, which remains intact, has no free 3' -hydroxyl group, terminal transferase (TDT) cannot attach the Cy5-dATP structure thereto. Thus, there was no Cy5 assemblyNo fluorescence resonance energy transfer occurs between the quantum dots to 605.
(1) Feasibility verification
First, in order to verify whether the methylation reaction and the cleavage reaction of the probe (dsDNA-1) were effective, we analyzed the cleaved products using gel electrophoresis. As shown in FIG. 2A, when only dsDNA-1 is present, there is a 22bp oligopeptide nucleotide band (lane 4); when either CpG methyltransferase or GlaI endonuclease is present, there is only one 22bp oligopeptide nucleotide band (lane 2 or lane 3), indicating that no methylation reaction or cleavage reaction occurs; in contrast, when both CpG methyltransferase and GlaI endonuclease were present, a new 11bp oligopeptide nucleotide band was generated (lane 1), demonstrating that CpG methyltransferase can recognize and initiate methylation reactions and that GlaI endonuclease cleaves almost all of the methylated products. Thus, methylation reactions and enzymatic reactions are feasible.
Subsequently, we verified the fluorescence resonance energy transfer between 605 quantum dots and Cy5 using fluorescence spectroscopy. As shown in fig. 2B: cy5-dATP was free of fluorescent signal in the presence of an excitation wavelength of 405 nm; if no CpG methyltransferase exists, the fluorescence signal of Cy5 is zero, which indicates that fluorescence resonance energy transfer does not occur between the 605 quantum dot and the Cy 5; in the presence of CpG methyltransferase, the fluorescence signal of 605 quantum dots is weakened, the fluorescence signal of Cy5 is strengthened, which can indicate that the quantum dot/probe/Cy 5 nano structure is formed, and the 605 quantum dots and Cy5 have fluorescence resonance energy transfer. The transfer efficiency was calculated to be 67%.
Finally, in order to further verify the feasibility of the technology, a single-molecule fluorescence image is adopted to visually observe the fluorescence resonance energy transfer condition between the 605 quantum dot and the Cy 5. As shown in fig. 3: in the absence of CpG methyltransferase, only 605 quantum dots (3A) and Cy5 (3B) can be observed; in the presence of CpG methyltransferase, fluorescence signals of 605 quantum dots (3D) and Cy5 (3E) were observed simultaneously, and a yellow signal further indicated that the 605 quantum dots and Cy5 signals were present simultaneously (3F). In summary, this technique is feasible.
(2) Sensitivity detection
To examine the sensitivity of this technique, we recorded the change in fluorescence intensity of Cy5 for different CpG methyltransferase concentrations. As shown in fig. 4: the fluorescence intensity of Cy5 increases with increasing concentration of CpG methyltransferase. Furthermore, cy5 fluorescence intensity was logarithmically linearly related to CpG methyltransferase concentration. The correlation equation is delta I =276.2+51.4log 10 C(R 2 = 0.987). By further calculation, the detection limit of the technology is 9.7 multiplied by 10 -6 U/μL。
We further recorded the variation in Cy5 number for different CpG methyltransferase concentrations. As shown in fig. 5: the number of Cy5 increases with increasing concentration of CpG methyltransferase. Furthermore, the number of Cy5 was logarithmically linearly related to the CpG methyltransferase concentration. The correlation equation is N =7648.4+1069.4log 10 C(R 2 = 0.992). By further calculation, the detection limit of the technique is 2.1 × 10 -7 U/. Mu.L. The detection limit is about 2 orders of magnitude (5 multiplied by 10) higher than that of the electrochemical immunoreaction method -5 U/μL)。
(3) Specificity detection
To verify the specificity of this technique, we performed specific experiments with Dam methyltransferases, haeIII methyltransferase and AluI methyltransferase as potentially interfering enzymes, and obtained the following results (see fig. 6): only CpG methylases caused a significant increase in Cy5 signal, in contrast Dam methyltransferase, haeIII methyltransferase, aluI methyltransferase did not cause an increase in Cy5 signal. These results clearly demonstrate the high selectivity of this assay for CpG methylases.
(4) Inhibitor assay
In order to verify the experimental feasibility of the inhibitor of the technology, two inhibitors, namely 5-azacytidine and 5-azacytidine-2-deoxycytidine, are selected. Both inhibitors had no inhibitory effect on GlaI endonuclease and terminal transferase (TDT). In contrast, the inhibitory effect on CpG methyltransferase increased with increasing concentrations of 5-azacytidine or 5-azacytidine-2-deoxycytidine (see FIG. 7). In addition, we also separately calculated the semi-inhibitionsSystem of concentration (IC) 50 ) Namely: IC of 5-azacytidine 50 IC of 5-azacytidine-2-deoxycytidine at 3.29 micromoles per liter 50 At 1.15 micromoles per liter. This result also reflects that 5-azacytidine-2-deoxycytidine is superior in inhibitory effect to 5-azacytidine.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
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<120> single quantum dot nano sensor for detecting methyltransferase, detection method and application thereof
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Claims (11)
1. A single quantum dot nanosensor for detection of methyltransferase, comprising at least: a double-stranded DNA probe, quantum dots and adenosine triphosphate deoxynucleotides marked with fluorescent molecules;
the double-stranded DNA probe is designed with a recognition site of CpG methyltransferase;
the above-mentionedBiotin is designed at the 5' end of the double-stranded DNA probe; design of PO at 3' end 4 ；
The base sequence of the double-stranded DNA probe is as follows:
5’-biotin-GAC TAC TGT GCG m CTT CAT GAT C-PO 4 -3’(SEQ ID NO.1)；
5’-biotin-GAT CAT GAA G m CG m CAC AGT AGT C-PO 4 -3’(SEQ ID NO.2)；
wherein, the first and the second end of the pipe are connected with each other, m c is methylcytosine.
2. The nanosensor of claim 1, wherein said quantum dots are 605QDs, and wherein said quantum dots are surface coated with streptavidin.
3. The nanosensor of claim 1, wherein said fluorescent molecule is cyanine 5.
4. The nanosensor of claim 1, further comprising S-adenosylmethionine, glaI endonuclease, and terminal transferase.
5. Use of the nanosensor of any of claims 1-4 for detecting a DNA methyltransferase; the DNA methyltransferase is a CpG methyltransferase and is not used for diagnostic and therapeutic purposes.
6. A method for detecting CpG methyltransferase without the purpose of disease diagnosis and treatment using the single quantum dot nanosensor for detecting methyltransferase of claim 1, comprising:
(1) Carrying out methylation reaction on a sample to be detected and the double-stranded DNA probe to obtain a methylation product;
(2) Adding the methylated product into GlaI endonuclease to perform enzyme digestion reaction;
(3) Mixing the cut product with terminal transferase and adenosine triphosphate deoxynucleotide marked with fluorescent molecules to obtain a reaction product;
(4) And mixing the reaction product with the quantum dot to obtain the structure of the quantum dot/probe/Cy 5.
7. The detection method according to claim 6,
in the step (1), a coenzyme factor participating in the methyl transfer reaction, namely S-adenosyl methionine is added to promote the methylation reaction; the methylation reaction is carried out for 30-150min at 37 ℃;
in the step (2), the specific conditions of the enzyme digestion reaction are as follows: reacting at 30 ℃ for 30-150min, and then reacting at 80 ℃ for 20min to terminate the reaction;
in the step (3), the reaction conditions are specifically reaction at 37 ℃ for 30-180min, and then reaction at 80 ℃ for 20min is stopped;
in the step (4), the reaction condition is specifically that the reaction is carried out for 15 to 30 minutes at room temperature.
8. The detection method according to claim 7,
in the step (1), a coenzyme factor participating in the methyl transfer reaction, namely S-adenosyl methionine is added to promote the methylation reaction; the methylation reaction is carried out for 120min at 37 ℃;
in the step (2), the specific conditions of the enzyme digestion reaction are as follows: the reaction was terminated at 30 ℃ for 120min and then at 80 ℃ for 20min;
in the step (3), the reaction conditions are specifically reaction at 37 ℃ for 120min, and then reaction at 80 ℃ for 20min is stopped;
in the step (4), the reaction conditions are specifically reaction at room temperature for 20 minutes.
9. The assay of any one of claims 6-8, further comprising measuring the fluorescent signal using a fluorescence imaging system to achieve quantitative detection of CpG methyltransferase;
the method for measuring the fluorescence signal comprises the following steps: the QDs were excited at 405nm and the signals at 609.8nm and 670nm were collected, respectively.
10. Use of a nanosensor according to any of claims 1-4 and/or a detection method according to any of claims 6-9 for the detection of DNA methyltransferase activity and/or for the screening of DNA methyltransferase drugs.
11. The use of claim 10 wherein the DNA methyltransferase drug comprises a DNA methyltransferase promoter and a DNA methyltransferase drug inhibitor.
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| CN113151401A (en) * | 2020-09-07 | 2021-07-23 | 山东师范大学 | Fluorescence sensor based on quantum dots and three-dimensional hybridization structure and preparation and application thereof |
| CN114381498A (en) * | 2021-12-03 | 2022-04-22 | 山东师范大学 | Chemiluminescence sensor based on cooperative in-situ assembly of G-quadruplex DNAzyme nanowires and application thereof |
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